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WO2024216211A2 - Methods of inhibiting tumor progression and metastasis by inhibition of enpp1 - Google Patents

Methods of inhibiting tumor progression and metastasis by inhibition of enpp1 Download PDF

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Publication number
WO2024216211A2
WO2024216211A2 PCT/US2024/024497 US2024024497W WO2024216211A2 WO 2024216211 A2 WO2024216211 A2 WO 2024216211A2 US 2024024497 W US2024024497 W US 2024024497W WO 2024216211 A2 WO2024216211 A2 WO 2024216211A2
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enpp1
subject
breast cancer
inhibitor
mrna expression
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PCT/US2024/024497
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French (fr)
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WO2024216211A3 (en
Inventor
Songnan WANG
Lingyin Li
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The Board Of Trustees Of The Leland Stanford Junior University
Arc Institute
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Publication of WO2024216211A2 publication Critical patent/WO2024216211A2/en
Publication of WO2024216211A3 publication Critical patent/WO2024216211A3/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/42—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving phosphatase
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    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/44—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving esterase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/156—Polymorphic or mutational markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • ICB resistance can occur through a variety of mechanisms, one of which is insufficient lymphocyte infiltration into the tumor, which is determined by our innate immune system’s ability to detect and communicate the presence of cancer.
  • Cancer cells have a variety of strategies for suppressing innate immunity; therefore, a deep understanding of innate immune checkpoints holds the potential to unlock the full power of immunotherapy against immunologically “cold” tumors.
  • cytosolic double-stranded DNA (dsDNA)-sensing STING pathway is a key innate immune pathway that detects and responds to chromosomal instability (CIN) and extrachromosomal DNA present in cancer cells (Harding et al., 2017; MacKenzie et al., 2017; Turner et al., 2017; Bakhoum et al., 2018). Cytosolic dsDNA is detected by the enzyme cyclic- GMP-AMP synthase (cGAS), which synthesizes the cyclic dinucleotide cGAMP.
  • cGAS cyclic- GMP-AMP synthase
  • cGAMP is secreted into the extracellular matrix and taken up by surrounding host cells, where it binds and activates the STING pathway, leading to production of type I interferons (IFNs) and downstream immune cell infiltration in a paracrine fashion (Marcus et al., 2018; Luteijn et al., 2019; Ritchie et al., 2019; Jacqueline A. Carozza, Böhnert, et al., 2020; Lahey et al., 2020; Cordova et al., 2021; Maltbaek, Snyder and Stetson, 2021). Previous work has demonstrated that STING activation is required for the efficacy of ICB therapies (PMID: 25517615).
  • ENPP1 inhibitors have shown efficacy in murine models of breast and pancreatic cancers as a single agent and in combination with IR (Jacqueline A. Carozza, Böhnert, et al., 2020; Jacqueline A. Carozza, Brown, et al., 2020).
  • IR Jacqueline A. Carozza, Böhnert, et al., 2020
  • Jacqueline A. Carozza, Brown, et al., 2020 Jacqueline A. Carozza, Brown, et al.
  • ENPP1 expression has been linked to cancer stem cell populations in breast cancer, lung cancer, and glioblastoma (Takahashi et al., 2015; Hu et al., 2019; Bageritz et al., 2014).
  • ENPP1 has also been shown to produce immunosuppressive adenosine as a downstream metabolite of cGAMP hydrolysis, which has been linked to metastasis of murine CIN-high breast cancers (Li 2021) and self-seeding of circulating breast tumor cells (Cordoba 2021). Therefore, numerous questions remain surrounding the mechanisms of ENPP1’s tumorigenic activity, ENPP1’s contribution to different stages of tumor development including initiation, progression and metastasis, how ENPP1 shape the local and distant tumor immune microenvironment (TIME), and how ENPP1 genetic variants present across the human population may contribute to cancer risk.
  • TIME tumor immune microenvironment
  • ENPP1 inhibition can synergize with existing cancer therapies that are upstream of cGAMP production, such as PARP inhibitors, or with existing therapies that are downstream of cGAMP production, such as anti-PD1 or anti-PDL1 therapies.
  • the invention also provides methods for treating cancer, including breast cancer, by administering to subjects having high levels of ENPP1 expression (or simply positive for ENPP1 protein expression when examining a tumor biopsy with immunohistochemistry) with both an ENPP1 inhibitor and either a PARP inhibitor, an anti- PD1 therapy, and/or an anti-PDL1 therapy.
  • methods for treating cancer are for subjects who are positive for ENPP1 expression and whose tumors were not responsive to PARP inhibitor, anti-PD1, or anti-PDL1 therapy.
  • the present disclosure relates to ENPP1 expression as a prognostic biomarker for anti-PD1 and PARP inhibitors.
  • FIGS. 1A-1F show ENPP1 contributions to tumor progression and metastasis in humans and mice.
  • FIG. 1G shows ENPP1 degradation activity in 4T1 and its derived cell lines as assessed by TLC (Thin Layer Chromatography).
  • Figure 1H shows ENPP1-GFP expression (top) and ENPP1 degradation activity (bottom) of ENPP1 WT-OE 4T1 and ENPP1 T238A-OE 4T1 clones as assessed by western blotting and TLC.
  • FIG. 1I shows ENPP1-GFP degradation activity (left) and ENPP1 expression (right) of ENPP1 WT-OE 4T1 and ENPP1 T238A-OE 4T1 pooled clones as assessed by TLC and western blotting.
  • Figure 1K shows ENPP1 expression in patients with stage 1-4 breast cancer. Shown as box plots of median and interquartile levels. P value was determined by the nonparametric Mann-Whitney U test.
  • Figure 1O shows UMAP plots of the annotated clusters of ENPP1 T238A-OE and ENPP1 WT- OE 4T1 primary tumors and metastasis colonized lungs.
  • Figure 1P shows barplots comparing immune cell compositions (containing C08-C18) between ENPP1 T238A-OE and ENPP1 WT-OE 4T1 primary tumors and metastasis colonized lungs. *P ⁇ 0.05.; P value is shown if it is between 0.05 - 0.15.
  • Figure 1Q shows scRNA-seq analysis of 4T1 murine primary tumors and lung metastases.
  • the bubble heatmap shows expression of selected marker genes for cluster annotation. Dot size indicates fraction of expressing cells, colored based on average expression levels.
  • Figure 1R shows a UMAP plot of the annotated clusters of ENPP1 T238A-OE and ENPP1 WT-OE 4T1 primary tumors and metastasis colonized lungs.
  • Figure 1S shows barplots comparing non-immune cell compositions (containing C01- C05) between ENPP1 T238A-OE and ENPP1 WT-OE 4T1 primary tumors and metastasis colonized lungs.
  • FIGS 2A-2C show that ENPP1 catalytic activity promotes immune suppression in primary tumors and lung metastases.
  • A Arg1 in macrophages in primary tumors and lung metastases.
  • B Itgae and H2-Ab1 in cDC1s in primary tumors.
  • C Cd69, Ilr2a, Pdcd1, and Tox in T cells in primary tumors and lung metastases. P values were determined by the nonparametric Mann-Whitney U test.
  • cDC1 stands for conventional dendritic cell type 1.
  • Figures 2D-2E show Violin plots of indicated transcripts of indicated cell types comparing between ENPP1 T238A-OE and ENPP1 WT-OE 4T1 tumors or metastases.
  • D Arg1 in monocytes in primary tumors and lung metastases.
  • E Itgae and H2-Ab1 in cDC1s in lung metastases (E). P values were determined by nonparametric Mann-Whitney U test.
  • cDC1 stands for conventional dendritic cell type 1.
  • Figure 2F shows the UMAP plot of the annotated subclusters of macrophages.
  • Figure 2G shows a bubble heatmap showing expression of marker genes for macrophage subcluster annotation and functional markers indicating M1-like versus M2-like macrophage cell phenotypes. Dot size indicates fraction of expressing cells, colored based on average expression levels.
  • Figure 2H shows barplots comparing macrophage subcluster compositions (containing Ma1-Ma4) between ENPP1 T238A-OE and ENPP1 WT-OE 4T1 primary tumors and metastasis colonized lungs.
  • Figure 2I shows the UMAP plot of the annotated subclusters of T cells.
  • Figure 2J shows a bubble heatmap showing expression of marker genes for T cell subcluster annotation.
  • FIG. 1 shows barplots comparing T cell subcluster compositions (containing T01- T11) between ENPP1 T238A-OE and ENPP1 WT-OE 4T1 primary tumors and metastasis colonized lungs. P values were determined by unpaired t test. *P ⁇ 0.05.; P value is shown if it is between 0.05 - 0.15.
  • Figures 3A-3L shows ENPP1 expressed on cancer and responder cells blocks paracrine cGAMP-STING activation. P values were determined by nonparametric Mann-Whitney U test unless otherwise noted.
  • Figure 3A shows bar graphs of Cgas and Sting1 expression across the annotated clusters.
  • Figure 3B shows Violin plots of Lrrc8c, Lrrc8a, Lrrc8e and Ifitm2 across the annotated clusters. Schematic of cGAMP responder cells and their putative transporters: LRRC8A:C in endothelial cells, TAM and cDC; LRRC8A:E in fibroblasts.
  • Figure 3C shows bar graphs of indicated transcripts in indicated cell types comparing between ENPP1 T238A-OE and ENPP1 WT-OE 4T1 tumors or metastases.
  • Figure 3D shows Ifitms in macrophages in primary tumors and metastases.
  • Figure 3E shows Ifitms in myCAFs in primary tumors and metastases.
  • Figure 3F shows Enpp1 in myCAFs in primary tumors and metastases.
  • Figure 3G shows bar graphs of Enpp1 expression across the annotated clusters.
  • Figure 3H shows bar graphs of Enpp1 and Ifitms across the annotated macrophage subclusters. P values were determined by ordinary one-way ANOVA test.
  • Figure 3I shows differentially expressed genes in Enpp1-high vs.
  • Figure 3L shows bar graphs of indicated transcripts in indicated cell types comparing between ENPP1 T238A-OE and ENPP1 WT-OE 4T1 tumors or metastases. Lrrc8c in macrophages (C10) in primary tumors and lung metastases. Shown as mean ⁇ SEM. P values were determined by nonparametric Mann-Whitney U test.
  • Figure 3M shows the contribution of the eADO pathway and HP secretion in ENPP1 overexpression.
  • Figure 4E shows the percentage of Ly6G-Ly6C low cells out of MHC-II + CD11b + CD11c- macrophages. Data were plotted as mean ⁇ SD.
  • Figure 4F shows the geometric mean of pIRF3 of F40/80 + CD206- M1-like macrophages.
  • Figure 4G shows the percentage of MHC-II hi CD103 + cells out of CD11b-CD11c + cells.
  • Figure 4H shows the geometric mean of CD69 (H) of CD3 + CD4-CD8 + cytotoxic T lymphocytes (CTLs).
  • Figure 4I shows the geometric mean of CD25 (I) of CD3 + CD4-CD8 + cytotoxic T lymphocytes (CTLs).
  • Figure 4J shows the geometric mean of Foxp3 of CD45 + CD3 hi CD4 + CD8- regulatory T cells (Tregs).
  • Figures 5A-5G show selective disruption of ENPP1’s cGAMP hydrolysis activity delays breast cancer onset, growth, and metastasis. Data are shown as mean. P values comparing mice with lung metastasis % were determined by chi-squared test. P values comparing number of colonies were determined by nonparametric Mann-Whitney U test. P value for Kaplan-Meier curves were determined by log-rank Mantel-Cox test. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001. not significant (ns).
  • (C) shows a schematic of the experiment.
  • (D) shows representative images of WT, Enpp1 H362A , Enpp1 -/- .
  • E shows quantification of lung metastatic colonies of WT, Enpp1 H362A , Enpp1 -/- .
  • F shows individual tumor growth curves in WT and Enpp1H362A mice in (A).
  • FIG. 6A-6Q shows individual tumor growth curves in Sting-/-, and Enpp1H362A x Sting1-/- in (A).
  • Figures 6A-6Q shows that a common human ENPP1 variant, K173Q, exhibits increased cGAMP hydrolysis activity at the cell membrane.
  • a – Q Bars represent mean SD. P values were determined by unpaired t test. *P 0.05; not significant (ns).
  • PBMC stands for peripheral blood mononuclear cells.
  • hENPP1 stands for human ENPP1.
  • TM stands for transmembrane.
  • SMB stands for somatomedin B-like.
  • (A) shows domain structure of hENPP1 and allele frequency of the K173Q variant reported by gnomAD (website: gnomad.broadinstitute.org/).
  • (G) shows the percentage of dimer hENPP1 in lysates of 293T cGAS ENPP1-/- cells overexpressing WT or K173Q hENPP1 proteins assessed by non-reducing western blot.
  • (I) shows the proposed mechanism of how the K173Q could affect ENPP1’s cGAMP and ATP hydrolysis activity. Structure of hENPP1 dimers (PDB:6weu). The K173 residue (red) is in the SMB domains (yellow) proximal to cell membrane and away from the catalytic domain (light purple). The catalytic pocket (dark purple) is determined by side chains of residues within 5 ⁇ of Zn2+ (violet).
  • K173 could interact with other transmembrane proteins and/or the cell membrane to impact activity.
  • J shows the representative images of DNA sequences of donors with the A/A reference sequence (Lys) or the variant C/C sequence (Gln) at residue 173.
  • (M) shows the schematic of transfection of WT or mutant ENPP1 expressing plamids into 293T cGAS ENPP1-/-, followed by isolation of memENPP1 in lysate and secENPP1 in supernatant.
  • FIG. 7A-7M shows that transmembrane and secreted ENPP1 isoforms contribute to primary tumor growth and metastasis.
  • A shows the domain structure of memENPP1 and secENPP1.
  • B shows the schematic of levels of transmembrane versus secreted fraction of WT, A84S, and T238A ENPP1. Structure of mouse ENPP1 monomer (PDB:6XKD) and dimer (PDB: 4B56).
  • (C) shows the Ex vivo relative cGAMP hydrolysis activity calculated from kinetic analysis by tumor lysates (left) and sera (right) at physiological condition (pH 7.4, 37oC).
  • D shows tumor volumes in experiment C plotted as mean +/- SEM with some error bars too small to visualize.
  • (J and K) show the representative images (J) and quantification (K) of cGAMP degradation in 4 hours by lysate and supernatant of ENPP1WT-OE, ENPP1A84S-OE, and ENPP1T238A-OE cells.
  • (L) shows a repeat experiment of (C).
  • (M) shows the primary tumor sizes at the time of experimental endpoints in experiment (C).
  • FIG. 8A-8V shows ENPP1 drives distinct long-term immuno-suppressive programs in primary and metastatic niche.
  • Figures 9A-9K shows that ENPP1 expression is inducible in stromal cells.
  • Figure 10A shows ENPP1 expression in Immune+ vs. immune- patients across 10 treatment arms in the I-SPY2 breast cancer clinical trial. P value for Kaplan-Meier curves were determined by log-rank Mantel-Cox test. *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001. not significant (ns). Immune+ stands for immune-positive; immune- stands for immune-negative; pCR stands for pathological complete response.
  • FIG. 10B shows ENPP1 expression in pCR vs. no pCR patients across 10 treatment arms in the I-SPY2 breast cancer clinical trial.
  • Figure 10C shows that all of ENPP1-low patients (ENPP1 ⁇ 8.34) remained free of distant metastasis nearly seven years after Paclitaxel + Pembrolizumab neoadjuvant therapy followed by surgical resection, whereas 12% ENPP1-hig patients (ENPP1 > 8.34) developed distant metastasis within 5 years after Paclitaxel + Pembrolizumab neoadjuvant therapy followed by surgical resection.
  • Figure 10D shows a proposed model of mechanism of action in ENPP1 inhibition.
  • Figure 10E shows pre-treatment ENPP1 mRNA expression in pCR vs. no pCR patients in Paclitaxel + Pembrolizumab treatment arm in the I-SPY2 breast cancer clinical trial.
  • the ENPP1 mRNA threshold of 6.39 categorizes patients who likely have pCR (ENPP1 ⁇ 6.39) versus who have a chance of not having pCR (ENPP1 > 6.39) following Paclitaxel + Pembrolizumab neoadjuvant therapy.
  • Figure 10F shows pre-treatment ENPP1 mRNA expression in pCR vs. no pCR patients in Paclitaxel + Veliparib + Carboplatin treatment arm in the I-SPY2 breast cancer clinical trial.
  • ENPP1 mRNA threshold of 6.78 categorizes patients who likely have pCR (ENPP1 ⁇ 6.78) versus who have a chance of not having pCR (ENPP1 > 6.78) following Paclitaxel + Veliparib + Carboplatin neoadjuvant therapy.
  • DETAILED DESCRIPTION OF THE DISCLOSURE [0076] The present disclosure relates generally to detection and regulation of ENPP1 for the purposes of the treatment and prevention of metastatic cancer as well as the diagnosis of cancer in a subject. Without being bound by theory, provided herein is evidence that ENPP1 promotes breast cancer progression and metastasis in a STING-dependent manner by dampening the innate-adaptive immune crosstalk in the tumor microenvironment (TME).
  • Both tumor- and tissue-derived ENPP1 anchored on the cell surface can be important for its cancer-promoting activity, while soluble ENPP1 released into the serum can play a minor role.
  • tumors can induce expression of ENPP1 in the TME.
  • a potential solid tumor biopsy diagnostic is to assay for increased expression of membrane bound ENPP1 on tumor cells and/or in the surrounding TME (as compared to a reference, normal subject), such as with immunohistocytochemistry.
  • soluble ENPP1 might play a minor functional role
  • increased levels of soluble ENPP1 in the serum can be used as a diagnostic or prognostic for tumor onset and growth, primary progression, and/or metastasis.
  • ENPP1 is an innate immune checkpoint restraining STING pathway activation in the TME, and that therapeutically targeting ENPP1 catalytic activity could boost anti-tumor immunity in ways that could synergize with adaptive ICB therapy.
  • administration refers to the delivery of a bioactive composition or formulation by an administration route comprising, but not limited to, intranasal, transdermal, intravenous, intra-arterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration, or combinations thereof.
  • administration route comprising, but is not limited to, intranasal, transdermal, intravenous, intra-arterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration, or combinations thereof.
  • administration route comprising, but not limited to, intranasal, transdermal, intravenous, intra-arterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration, or combinations thereof.
  • the term includes, but is not limited to, administering by a medical professional and self-administering.
  • a composition of the disclosure e.g., nucleic acid constructs, srRNAs, recombinant cells, and/or pharmaceutical compositions
  • a composition of the disclosure generally refers to an amount sufficient for the composition to accomplish a stated purpose relative to the absence of the composition (e.g., achieve the effect for which it is administered, stimulate an immune response, prevent or treat a disease, or reduce one or more symptoms of a disease, disorder, infection, or health condition).
  • an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.”
  • a “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s).
  • the term “about” indicates the designated value ⁇ up to 10%, up to ⁇ 5%, or up to ⁇ 1%.
  • pharmaceutically acceptable excipient refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administration of a compound(s) of interest to a subject.
  • pharmaceutically acceptable excipient can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers.
  • the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a health condition of interest (e.g., rabies infection) and/or one or more symptoms of the health condition.
  • the subject can also be an individual who is diagnosed with a risk of the health condition of interest at the time of diagnosis or later.
  • non-human animals includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, non-human primates, and other mammals, such as e.g., sheep, dogs, cows, chickens, and non-mammals, such as amphibians, reptiles, etc.
  • a solid tumor sample can be obtained by any means available to those skilled in the art.
  • the solid tumor sample is obtained from a biopsy, such as a needle biopsy, for example, a core needle biopsy (CNB).
  • the solid tumor sample is obtained from a percutaneous core needle biopsy.
  • the solid tumor sample is obtained from a surgical biopsy. Core biopsy can be done guided by imaging or palpation (freehand). Routinely, stereotactic biopsy (needle biopsy guided by mammography done in 2 planes and analyzed by computer to produce a 3-dimensional image) or ultrasound- guided biopsy can be used to improve accuracy. Clips are placed at the biopsy site to identify it.
  • the solid tumor sample is obtained through surgery, such as surgical resection or surgical removal of the solid tumor sample.
  • the solid tumor can be classified or predicted to be invasive recurrent or indolent based on analysis of the features identified herein. The determination of the aggressiveness phenotype of the solid tumor sample can be used to develop a treatment plan for the subject with the solid tumor and to treat the patient accordingly.
  • pCR is defined as the absence of residual invasive cancer and in situ cancer on hematoxylin and eosin evaluation of the complete resected breast specimen and all sampled regional lymph nodes following completion of neoadjuvant systemic therapy (i.e. ypT0 ypN0 in the current AJCC staging system.)
  • genes or gene products from a particular species are intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates.
  • genes or gene products disclosed herein which in some embodiments relate to mammalian nucleic acid and amino acid sequences, are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds.
  • the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides and proteins are human.
  • the term “gene” is also intended to include variants thereof.
  • aspects of the disclosure include methods that involve administering to a subject with cancer a therapeutically effective amount of an ENPP1 inhibitor to prevent or treat the subject for metastasis.
  • the subject is one who is diagnosed with or suspected of having cancer.
  • the subject is one who is diagnosed with metastatic cancer.
  • the methods of treatment are not directed to non-metastatic cancer, for example the cancers described in WO2019051269.
  • metastasis includes the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location.
  • a “metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures.
  • Any convenient ENPP1 inhibitors can be used in the subject methods of treating cancer.
  • Exemplary ENPP1 inhibitors include, without limitation, those described in WO2019051269.
  • the ENPP1 inhibitor is compound 1 from WO2019051269.
  • the ENPP1 inhibitor is STF-1623 (Carozza, Brown, et al., 2020).
  • the ENPP1 inhibitor is a member selected from the group consisting of:
  • the ENPP1 inhibitor is a member selected from the following table: Table A
  • the ENPP1 inhibitor is a member selected from the following table B:
  • the ENPP1 inhibitor is described in WO2020/160333. In certain embodiments, the ENPP1 inhibitor is described in Table 1 in WO2020/160333. In certain embodiments, the ENPP1 inhibitor is described in Table 2 in WO2020/160333. In certain embodiments, the ENPP1 inhibitor is described in Table 3 in WO2020/160333. In certain embodiments, the ENPP1 inhibitor is described in Table 3a in WO2020/160333. [0099] In certain embodiments, the ENPP1 inhibitor is described in WO2022/125614, WO2022/125613, WO2022/119928, WO2020/190912, WO2020/028724, WO2021/158829, or WO2019/023635.
  • the ENPP1 inhibitor is described in WO2022/212488, WO2022/197734, WO2020/210649, or WO2020/140001.
  • the subject is mammalian. In some embodiments, the subject is human. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees, and monkeys). The subject may be in need of treatment for cancer.
  • domestic pets e.g., dogs and cats
  • livestock e.g., cows, pigs, goats, horses, and the like
  • rodents e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease
  • non-human primates e.g.,
  • the subject methods include diagnosing cancer, including any one of the cancers described herein.
  • the compound is administered as a pharmaceutical preparation.
  • the ENPP1 inhibitors are administered to a subject, e.g., a subject having, suspected of having, or at risk of developing cancer selected from, but not limited to, a pancreatic cancer, an endometrial cancer, a non-small cell lung cancer (NSCLC), a renal cell carcinoma ((RCC), e.g. clear cell RCC, non-clear cell RCC), a urothelial cancer, a head and neck cancer (e.g.
  • a melanoma e.g., advanced melanoma such as Stage III-IV high- risk melanoma, unresectable or metastatic melanoma
  • a bladder cancer e.g., a hepatocellular carcinoma, a breast cancer (e.g., triple negative breast cancer, ER + /HER2 ⁇ breast cancer), an ovarian cancer, a gastric cancer (e.g.
  • PMBCL primary mediastinal B-cell lymphoma
  • cervical cancer an advanced or refractory solid tumor
  • a small cell lung cancer e.g., stage IV non-small cell lung cancer
  • a non-squamous non-small cell lung cancer desmoplastic melanoma
  • pediatric advanced solid tumor or lymphoma a mesothelin-positive pleural mesothelioma
  • pNET neuroectodermal tumor
  • a "therapeutically effective amount” is an amount of a subject inhibitor that, when administered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to prevent metastasis of a primary tumor in a subject.
  • the ENPP1 inhibitor can be administered in combination with a chemotherapeutic agent selected from the group consisting of alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, steroid hormones, taxanes, nucleoside analogs, steroids, anthracyclines, thyroid hormone replacement drugs, thymidylate- targeted drugs, Chimeric Antigen Receptor T cell therapies, Chimeric Antigen Receptor/NK cell therapies, apoptosis regulator inhibitors (e.g., B cell CLL/lymphoma 2 (BCL-2) BCL-2-like 1 (BCL- XL) inhibitors), CARP-1/CCAR1 (Cell division cycle and apoptosis regulator 1) inhibitors, colony- stimulating factor-1 receptor (CSF1R) inhibitors, CD47 inhibitors, cancer vaccine (e.g., a Thl7- inducing dendritic cell vaccine, or a genetically
  • chemotherapeutic agents of interest include, but are not limited to, Gemcitabine, Docetaxel, Bleomycin, Erlotinib, Gefitinib, Lapatinib, Imatinib, Dasatinib, Nilotinib, Bosutinib, Crizotinib, Ceritinib, Trametinib, Bevacizumab, Sunitinib, Sorafenib, Trastuzumab, Ado- trastuzumab emtansine, Rituximab, Ipilimumab, Rapamycin, Temsirolimus, Everolimus, Methotrexate, Doxorubicin, Abraxane, Folfirinox, Cisplatin, Carboplatin, 5-fluoro uracil, Teysumo, Paclitaxel, Prednisone, Levothyroxine, Pemetrexed, navitoclax, and ABT-
  • Cancer chemotherapeutic agents of interest include, but are not limited to, dolastatin and active analogs and derivatives thereof; and auristatin and active analogs and derivatives thereof (e.g., Monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). See, e.g., WO 96/33212, WO 96/14856, and U.S.6,323,315. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad.
  • the ENPP1 inhibitors can be administered to a subject alone or in combination with an additional, i.e., second, active agent. Combination therapeutic methods where the ENPP1 inhibitors may be used in combination with a second active agent or an additional therapy, e.g., radiation therapy.
  • additional therapy e.g., radiation therapy.
  • ENPP1 inhibitors can be administered alone or in conjunction with one or more other drugs, such as drugs employed in the treatment of diseases of interest, including but not limited to, immunomodulatory diseases and conditions and cancer.
  • the subject method further includes coadministering concomitantly or in sequence a second agent, e.g., a small molecule, a chemotherapeutic, an antibody, an antibody fragment, an antibody-drug conjugate, an aptamer, a protein, or a checkpoint inhibitor.
  • the method further includes performing radiation therapy on the subject.
  • co-administration and “in combination with” include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially within no specific time limits.
  • Conscomitant administration of a known therapeutic drug or additional therapy with a pharmaceutical composition of the present disclosure means administration of the compound and second agent or additional therapy at such time that both the known drug and the composition of the present invention will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug with respect to the administration of a subject compound. Routes of administration of the two agents may vary, where representative routes of administration are described in greater detail below. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs or therapies and compounds of the present disclosure.
  • the therapeutic agents can be administered by any suitable means including, but not limited to, for example, oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal), intravesical or injection into an affected organ.
  • the ENPP1 inhibitor may be administered in a unit dosage form and may be prepared by any methods well known in the art. Such methods include combining the subject compound with a pharmaceutically acceptable carrier or diluent which constitutes one or more accessory ingredients.
  • a pharmaceutically acceptable carrier is selected on the basis of the chosen route of administration and standard pharmaceutical practice.
  • Each carrier must be “pharmaceutically acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject.
  • This carrier can be a solid or liquid and the type is generally chosen based on the type of administration being used.
  • suitable solid carriers include lactose, sucrose, gelatin, agar and bulk powders.
  • suitable liquid carriers include water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions, and solution and or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules.
  • Such liquid carriers may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents.
  • Preferred carriers are edible oils, for example, corn or canola oils.
  • Polyethylene glycols, e.g. PEG, are also good carriers.
  • the method includes obtaining a biopsy from a subject.
  • a biopsy can include a biological sample obtained from a subject.
  • a biopsy may be used for analysis (e.g., diagnosis) to determine the presence or status of disease (e.g., type of disease, severity of disease, or cause of disease).
  • a biopsy may be used to direct disease treatment or provide a prognosis.
  • a liquid biopsy is obtained from a subject.
  • a liquid biopsy is a simple and non-invasive alternative to surgical biopsies which enables doctors to discover a range of information about a tissue (e.g., a tumor) through a simple blood sample.
  • Liquid biopsy is a type of technique for sampling and analyzing of non-solid biological tissues, mainly used in disease diagnosis.
  • a liquid biopsy obtained from a subject with cancer may identify the subject as having heterozygous or homozygous lysine to glutamine mutation at position 173 (K173Q) of ENPP1.
  • the biopsy sample is assayed using methods including, but not limited to, sequencing (e.g., next-generation sequencing methods), real-time PCR, digital droplet PCR, and other known PCR and sequencing methods of analyzing DNA.
  • the next generation sequencing method comprises a method selected from the group consisting of Ion Torrent, Illumina, SOLiD, 454; Massively Parallel Signature Sequencing, solid phase reversible dye terminator sequencing; and DNA nanoball sequencing.
  • “next generation sequencing” refers to the speeds that were not possible with conventional sequencing methods (e.g., Sanger sequencing) by reading thousands of millions of sequencing reactions simultaneously.
  • Next generation sequencing techniques and sequencing primer designs are well known in the art (e.g., Shendure, et al., “Next-generation DNA sequencing,” Nature, 2008, vol.26, No.10, 1135-1145; Mardis, “The impact of next- generation sequencing technology on genetics, “Trends in Genetics, 2007, vol.24, No.3, pp 133-141; Su, et al., “Next generation sequencing and its applications in molecular diagnostics,” Expert Rev Mol Diagn, 2011, 11 (3): 333-43; Zhang et al., “The impact of next- generation sequencing on genomics,” J Genet Genomics, 2011, 38 (3): 95-109; (Nyren, P. et al.
  • the invention provides a method for treating breast cancer in a subject, comprising: a) obtaining a sample from the subject; b) measuring ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in the sample from the subject; c) classifying the sample, wherein the sample has a high classification when its ENPP1 levels are increased; and d) if the sample has the high classification, then administering to the subject: i) an ENPP1 inhibitor; and ii) a PD1 inhibitor, wherein the breast cancer is responsive to a PD1 inhibitor, thereby treating the breast cancer in the subject.
  • ENPP1 ectonucleotide pyrophosphatase/phosphodiesterase 1
  • the method further comprises assessing that the breast cancer is responsive to a PD1 inhibitor.
  • the treating is achieving a pathological complete response (pCR).
  • the breast cancer is triple negative, microsatellite instability-high (MSI- H), or mismatch repair deficient (dMMR).
  • the step d) further comprises iii) administering chemotherapy.
  • the chemotherapy is paclitaxel.
  • the sample is a breast cancer tissue sample.
  • the measuring is selected from the group consisting of ENPP1 mRNA expression, ENPP1 immunohistochemical levels, ENPP1 gene amplification levels, and ENPP1 K173Q homozygosity.
  • the measuring is ENPP1 mRNA expression, and the high classification occurs when the breast cancer tissue sample ENPP1 mRNA expression has, relative to a reference, a value selected from the group consisting of 6.2 or above, 6.39 or above, 6.4 or above, 7.0 or above, 7.4 or above, 7.8 or above, 8.0 or above, 8.9 or above, 9.2 or above, 9.4 or above, 9.6 or above, 9.8 or above, 10.0 or above.
  • the reference is the ENPP1 mRNA expression in a population of breast cancer tissue samples.
  • the measuring is ENPP1 immunohistochemical levels, and the high classification occurs when a percentage of cells positive for ENPP1 expression in the breast cancer tissue sample is 0.1 or above, 0.5 or above, 0.75 or above, 0.9 or above, 1 or above, 10 or above, or 50 or above.
  • the measuring is ENPP1 immunohistochemical levels, and the high classification occurs when a percentage of cells positive for ENPP1 expression in the breast cancer tissue sample is 1 or above.
  • the measuring is ENPP1 gene amplification levels, and the high classification occurs when ENPP1/CEP6 probe ratio > 2, when ENPP1 copy number > 6, or when ENPP1 copies are > 6 if a single probe is used.
  • the measuring is ENPP1 K173Q homozygosity, and the high classification occurs when the breast cancer tissue sample is homozygous for the K173Q mutation.
  • the sample is a blood sample.
  • the measuring is of ENPP1 activity through plasma ENPP1 cGAMP degradation half-life, and the high classification is when the cGAMP degradation half-life is less than 60 minutes, or less than 50 minutes or less than 70 minutes.
  • the measuring is of ENPP1 plasma protein levels
  • the high classification is when the ENPP1 plasma protein level is > 4.4 ⁇ g/L, > 3.4 ⁇ g/L, or > 5.4 ⁇ g/L.
  • the PD1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, and toripalimab.
  • the PD1 inhibitor is pembrolizumab.
  • the ENPP1 inhibitor is described herein.
  • the invention provides a method for treating breast cancer in a subject, comprising: a) obtaining a sample from the subject; b) measuring ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in the sample from the subject; c) classifying the sample, wherein the sample has a high classification when its ENPP1 levels are increased; and d) if the sample has the high classification, then administering to the subject: i) an ENPP1 inhibitor; and ii) a PDL1 inhibitor, wherein the breast cancer is responsive to a PDL1 inhibitor, thereby treating the breast cancer in the subject.
  • ENPP1 ectonucleotide pyrophosphatase/phosphodiesterase 1
  • the method further comprises assessing that the breast cancer is responsive to a PDL1 inhibitor.
  • the treating is achieving a pathological complete response (pCR).
  • the breast cancer is triple negative, microsatellite instability-high (MSI- H), or mismatch repair deficient (dMMR).
  • the step d) further comprises iii) administering chemotherapy.
  • the chemotherapy is paclitaxel.
  • the sample is a breast cancer tissue sample.
  • the invention provides a method for treating breast cancer in a subject, comprising: a) obtaining a sample from the subject; b) measuring ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in the sample from the subject; c) classifying the sample, wherein the sample has a high classification when its ENPP1 levels are increased; and d) if the sample has the high classification, then administering to the subject: i) an ENPP1 inhibitor; and ii) a PARP inhibitor, wherein the breast cancer is responsive to a PARP inhibitor, thereby treating the breast cancer in the subject.
  • the method further comprises assessing that the breast cancer is responsive to a PARP inhibitor.
  • the treating is achieving a pathological complete response (pCR).
  • the breast cancer has a BRCA1 mutation, a BRCA2 mutation, is HER2 negative, is locally advanced, or is metastatic.
  • the step d) further comprises administering chemotherapy.
  • the chemotherapy is paclitaxel.
  • the sample is a breast cancer tissue sample.
  • the measuring is selected from the group consisting of ENPP1 mRNA expression, ENPP1 immunohistochemical levels, ENPP1 gene amplification levels, and ENPP1 K173Q homozygosity.
  • the measuring is ENPP1 mRNA expression
  • the high classification occurs when the breast cancer tissue sample ENPP1 mRNA expression has, relative to a reference, a value selected from the group consisting of 6.4 or above, 6.78 or above, 7.0 or above, 7.4 or above, 7.8 or above, 8.0 or above, 8.9 or above, 9.2 or above, 9.4 or above, 9.6 or above, 9.8 or above, 10.0 or above, 10.2 or above, 10.4 or above, and 10.5 or above.
  • the reference is the ENPP1 mRNA expression in a population of breast cancer tissue samples.
  • the measuring is ENPP1 immunohistochemical levels, and the high classification occurs when a percentage of cells positive for ENPP1 expression in the breast cancer tissue sample is 0.1 or above, 0.5 or above, 0.75 or above, 0.9 or above, 1 or above, 10 or above, or 50 or above.
  • the measuring is ENPP1 immunohistochemical levels, and the high classification occurs when a percentage of cells positive for ENPP1 expression in the breast cancer tissue sample is 1 or above.
  • the measuring is ENPP1 gene amplification levels, and the high classification occurs when ENPP1/CEP6 probe ratio > 2, when ENPP1 copy number > 6, or when ENPP1 copies are > 6 if a single probe is used.
  • the measuring is ENPP1 K173Q homozygosity, and the high classification occurs when the breast cancer tissue sample is homozygous for the K173Q mutation.
  • the sample is a blood sample.
  • the measuring is of ENPP1 activity through plasma ENPP1 cGAMP degradation half-life, and the high classification is when the cGAMP degradation half-life is less than 60 minutes, or less than 50 minutes or less than 70 minutes.
  • the measuring is of ENPP1 plasma protein levels, and the high classification is when the ENPP1 plasma protein level is > 4.4 ⁇ g/L, > 3.4 ⁇ g/L, or > 5.4 ⁇ g/L.
  • the PARP inhibitor is selected from the group consisting of veliparib, olaparib, rucaparib, niraparib, and talazoparib. In an exemplary embodiment, the PARP inhibitor is veliparib. In an exemplary embodiment, the step d) further comprises administering a platinum complex selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin. In an exemplary embodiment, the platinum complex is carboplatin. In an exemplary embodiment, the ENPP1 inhibitor is described herein.
  • C57BL/6J (strain #000664), BALB/cJ (strain #000651), BALB/cJ- Enpp1 asj-2J/GrsrJ (referred to as Enpp1-/-, strain #019107), C57BL/6J-Sting gt /J (referred to as Sting-/-, strain #017537), C57BL/6J-Enpp1 asj/GrsrJ (strain #012810), and FVB/N-Tg(MMTV- PyVT)634Mul/J (referred to as MMTV-PyMT, strain #002374) mice were purchased from the Jackson Laboratory.
  • C57BL/6J-Enpp1 H362A and C57BL/6J-Enpp1 H362A x Sting1 -/- mice were and characterized in house (Carozza et al., 2021).
  • FVB/N-Tg(MMTV-PyVT) 634Mul/J mice were bred with C57BL/6J-WT or C57BL/6J-Enpp1 H362A to generate B6;FVB-MMTV and B6;FVB- Enpp1 H362A x MMTV respectively.
  • MMTV spontaneous tumor model female mice from the second generation of both B6;FVB-MMTV and B6;FVB-Enpp1 H362A x MMTV genotypes were used for experiment.
  • mice between 6-15 weeks old were used for tumor experiments. Mice were maintained at Stanford University in compliance with the Stanford University Institutional Animal Care and Use Committee (IACUC) regulations. All procedures were approved by the Stanford University Administrative Panel on Laboratory Animal Care (APLAC).
  • ALAC Stanford University Administrative Panel on Laboratory Animal Care
  • Mammalian cell lines and primary cells.4T1, E0771.lmb, MDA-MB-231, NMuMG, MCF-7, Neuro-2a, mouse embryonic fibroblast (MEF), U937, L-929, and EMT6 cells were procured from ATCC. E0771 cells were procured from CH3 BioSystems. Panc02 were procured from the DTP/DCTD/NCI Tumor Repository.
  • Blood was dispensed into a 15-ml conical tube containing 10 mL 1x HBSS (Gibco), centrifuged at 1500 rpm at room temperature for 5 minutes before plated. dLNs were teased apart by forcing through a 100 ⁇ M strainer before plated. All other organs were minced with scissors and forces and digested in collagenase from Clostridium histolyticum (Sigma-Aldrich) at various conditions: 4°C for 75 minutes for lungs, 37°C for 30 minutes for livers, and 37°C for 120 minutes for brains. Digested organs were passed through a 100 ⁇ M cell strainer, washed twice with 1x HBSS (Gibco) before plating.
  • MCF-7 and NMuMG cells were maintained in DMEM (Corning Cellgro) supplemented with 10% FBS (R&D Systems), 10 ⁇ g/mL bovine insulin (Sigma-Aldrich) and 1% penicillin-streptomycin (Thermo Fisher).
  • U937 and PBMC cells were maintained in RPMI (Corning Cellgro) supplemented with 10% heat-inactivated FBS (R&D Systems) and 1% penicillin-streptomycin (Thermo Fisher).
  • HUVEC cells were maintained in EMG-2 supplemented growth media (Lonza).
  • APC anti-FoxP3 (1:100) was purchased from Tonbo Biosciences.
  • BUV 395 anti-CD103 (1:400), BUV 563 anti-CD115 (1:200), BUV 563 anti-Ly-6G (1:200), and BUV 805 anti-CD4 (1:200) were purchased from BD Biosciences.
  • eFluor 450 anti-CD25 (1:200), PE anti-EOMES (1:100), and PerCP-eFluor 710 anti-CD3e (1:200) are purchased from eBioscience Invitrogen.
  • VioGreen anti-Ly-6C (1:400) was purchased from Miltenyi Biotec.
  • Rabbit anti-phospho-STING (1:200) was purchased from Cell Signaling Technology.
  • mice anti-tubulin (1:2000), rabbit anti-DYKDDDDK (FLAG, 1:1000), and rabbit anti-GFP (1:1000) were purchased from Cell Signaling.
  • IRDye 800CW goat anti-rabbit (1:15,000) and IRDye 680RD goat anti-mouse (1:15,000) were purchased from LI-COR Biosciences.
  • Synthesis and purification of cGAMP and [ 32 P] cGAMP were to enzymatically synthesize cGAMP (Ritchie et al., 2019), 1 ⁇ M purified sscGAS was incubated testis DNA (Sigma) for 24 h.
  • cGAMP was purified from the reaction mixture using a PLRP-S polymeric reversed phase preparatory column (100 ⁇ , 8 ⁇ m, 300 x 25 mm; Agilent Technologies) on a preparatory HPLC (1260 Infinity LC system; Agilent Technologies) connected to UV-vis detector (ProStar; Agilent Technologies) and fraction collector (440-LC; Agilent Technologies).
  • the flow rate was set to 25 mL/min.
  • the mobile phase consisted of 10 mM triethylammonium acetate in water and acetonitrile. The mobile phase started as 2% acetonitrile for the first 5 min.
  • Acetonitrile was then ramped up to 30% from 5-20 min, then to 90% from 20-22 min, maintained at 90% from 22-25 min, and then ramped down to 2% from 25-28 min.
  • Fractions containing cGAMP were lyophilized and resuspended in water. The concentration was determined by measuring absorbance at 280 nm.
  • Neutralizing STING (WT) or non-binding STING (R237A) were expressed and purified using previously published methods (Jacqueline A. Carozza, Böhnert, et al., 2020).
  • pTB146 His-SUMO-mSTING was expressed in Rosetta (DE3) pLysS competent cells (Sigma-Aldrich). Cells were grown in 2xYT medium with 100 ⁇ g/mL ampicillin until they reached an OD600 of 1. They were then induced with 0.75 mM IPTG at 16°C overnight.
  • the resin-bound protein was washed with 50 column volumes of 50 mM Tris pH 7.5, 150 mM NaCl, 2% triton X-114; 50 column volumes of 50 mM Tris pH 7.5, 1 M NaCl; and 20 column volumes of 50 mM Tris pH 7.5, 150 mM NaCl. Protein was eluted from resin with 600 mM imidazole in 50 mM Tris pH 7.5, 159 mM NaCl. Fractions containing His-SUMO-STING were pooled, concentrated, and dialyzed against 50 mM Tris pH 7.5, 150 mM NaCl while incubating with SUMOlase His-ULP1 to remove the His-SUMO tag overnight.
  • mENPP1-WT sequence was amplified from pcDNA3-mENPP1-FLAG (synthesized by Genscript) using pLenti_mENPP1_fwd and pLenti_mENPP1_rev primers in Table S1 and inserted into the XbaI- BamHI sites of pLenti-CMV-GFP-Puro (Addgene).
  • mENPP1-WT sequence was amplified from pcDNA3-mENPP1-FLAG using pLenti_mENPP1_fwd and pLenti_mENPP1_rev primers in Table S1 and inserted into the XbaI- BamHI sites of pLenti-CMV-GFP-Puro (Addgene).
  • T238A and A84S point mutants were first introduced into pcDNA3-mENPP1-FLAG using QuikChange mutagenesis (Agilent).
  • mENPP1-T238A and mENPP1-A84S sequences were then introduced into the XbaI-BamHI sites of pLenti-CMV-GFP-Puro (Addgene).
  • the pcDNA3- humanENPP1(hENPP1)-FLAG plasmid was synthesized by Genscript.
  • 4T1 underwent transient transfection with Lipofectamine 3000 of the following pairs of sgRNAs targeting mouse Enpp1: PX458-mENPP1_sgRNA1-GFP and PX458- mENPP1_sgRNA2-mCherry; PX458- mENPP1_sgRNA3-GFP and PX458- mENPP1_sgRNA1- mCherry. Double GFP and mCherry positive cells were sorted with FACS (SONY) and underwent single-cell cloning.
  • Sequence knockout was confirmed with PCR using mENPP1_sgRNA12_seq_fwd and mENPP1_sgRNA12_seq_rev, or mENPP1_sgRNA34_seq_fwd and mENPP1_sgRNA34_seq_rev primer pairs. Functional knockout was confirmed with activity assay (commercial antibodies are not sensitive enough for verification of protein expression). Multiple clean knockout clones were pooled to generate the 4T1 Enpp1 -/- cell line. [0143] Generation of stable expression cell lines.
  • 4T1 Enpp1 -/- cells were virally transfected to stably express WT, T238A, or A84S mouse ENPP1, giving rise to ENPP1WT-OE, ENPP1T238A-OE, ENPP1A84S-OE cell lines.
  • lentiviral packaging plasmids (pHDM-G, pHDM-Hgmp2, pHDM-tat1b, and pRC.CMV-rev1b) were purchased from Harvard Medical School.500 ng of pLenti-CMV-mENPP1-GFP-Puro, pLenti-CMV-mENPP1-T238A-GFP-Puro, or pLenti-CMV-mENPP1-A84S-GFP-Puro plasmid, and 500 ng of each of the packaging plasmids were transfected into 293T cells using FuGENE 6 transfection reagent (Promega).
  • the viral media was exchanged after 24 h, harvested after 48 h and passed through a 0.45 ⁇ m filter, and used to transduce 4T1 Enpp1 -/- cells (4T1 cells are used as 4T1-luc cells already carry puromycin resistance which will interfere with subsequent drug selection process).48 h post- transduction cells were selected with 1–2 ⁇ g/ml puromycin and single-cell cloned, and multiple clones were pooled after verification by activity assay.
  • E0771.LMB cells were virally transfected with empty pLenti-TetONE-FLAG-Puro vector to stably carry puromycin resistance following the previously described lentiviral transfection protocol, giving rise to the E0771.LMB.Puro cell line.
  • Lysate and supernatant collection from cell lines are plated in 6 well plate.24 h before collection and when cells reach around 60-70% confluency, media is replaced with serum-free media consisting of DMEM or PRMI, 1%Insulin-Transferrin-Selenium (ThermoFisher), and 1% penicillin-streptomycin (Thermo Fisher).
  • serum-free media consisting of DMEM or PRMI, 1%Insulin-Transferrin-Selenium (ThermoFisher), and 1% penicillin-streptomycin (Thermo Fisher).
  • WT or mutant mouse or human pcDNA-ENPP1-FLAG plasmids were transiently transfected into 293T cGAS ENPP1 -/- cells with polyethylenimine (PEI) at ratio of 1 ⁇ g plasmid to 3 ⁇ g PEI per well of a 6 well plate.
  • PEI polyethylenimine
  • Cell lysate preparation for cGAMP degradation activity assay is as following: cells from a confluent well in a 6 well plate was collected in 1 mL of PBS, centrifuged at 1000 x g for 3 minutes, lysed in 50-100 ⁇ L of lysis buffer (10 mM Tris pH 9, 150 mM NaCl, 10 ⁇ M ZnCl2, 1% NP-40), and stored in -20°C until use. For western blotting, cells were lysed on the plate in 100-250 ⁇ L of Laemmli sample buffer, boiled at 95°C for 5 minutes and sonicated.
  • Mouse tumor lysate (100 mg/mL) for cGAMP degradation activity assay were generated by lysing tissues in 10 mM Tris pH 7.5, 150 mM NaCl, 10 ⁇ M ZnCl2, 1.5% NP-40, and freshly added protease inhibitors (cOmplete, EDTA-free protease inhibitor cocktail, Sigma- Aldrich). Organ lysates were then homogenized with a bead homogenizer (Omni International) and stored at -20°C until use. [0146] Human PBMC and plasma isolation. Buffy coat (obtained de-identified from the Stanford Blood Center) was diluted 1:3 with PBS.
  • PBMCs Diluted buffy coat was layered on top of 50% Percoll (GE Healthcare) containing 140 mM NaCl and centrifuged at 600 x g for 30 min. The separated plasma and PBMC layer were collected. PBMCs were washed once with PBS and once with RPMI. Human PBMCs were then lysed in 10 mM Tris pH 7.5, 150 mM NaCl, and 1% NP-40 to generate 2.5-5x10 6 cells/ ⁇ L lysates. Lysates were stored at -80°C. Genomic DNA (gDNA) of human PBMCs were isolated using DNeasy Blood & Tissue Kit (Qiagen).
  • hENPP1 exon 4 region was amplified from donor gDNA templates and sequenced with hENPP1_K173Q_seq_fwd and hENPP1_K173Q_seq_rev primers (Table S1).
  • cDNA complementary DNA
  • cells were lysed with 0.5-0.75 mL TRIzol (Invitrogen) and total RNA from cells were extracted using Direct-zol RNA Kit (Zymo).
  • cGAMP activity assays (20-30 ⁇ l total) were composed of the following: cell lysate, organ lysate, mouse serum, or human plasma (50-75%), cGAMP (1 to 5 ⁇ M, with trace [ 32 P] cGAMP spiked in), and buffer (standard assay buffer unless otherwise noted was 50 mM Tris pH 9 or pH 7.5, 250 mM NaCl, 0.5 mM CaCl 2 , 1 ⁇ M ZnCl 2 ). At indicated times, 1 ⁇ l aliquots of the reaction were quenched by spotting on HP-TLC silica gel plates (Millipore).
  • TLC plates were run in mobile phase (85% ethanol, 5 mM NH4HCO3) and exposed to a phosphor screen (GE BAS-IP MS). Screens were imaged on a Typhoon 9400 scanner and the 32 P signal was quantified using ImageJ. For cell lysates and supernatants, assays were run in standard buffer listed above at pH 9.0 at room temperature with 1 ⁇ M cGAMP spkied with [ 32 P] cGAMP.
  • cGAMP degradation activity assay (20 ⁇ l) for mouse serum containing 50% serum, 5 ⁇ M cGAMP and physiological ENPP1 activity buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1.5 mM CaCl 2 , 10 ⁇ M ZnCl 2 ) took place in 37°C. At indicated times, 1 ⁇ l aliquots of the reaction were quenched by spotting on HP-TLC silica gel plates (Millipore). The TLC plates were run in mobile phase (85% ethanol, 5 mM NH4HCO3) and exposed to a phosphor screen (GE BAS-IP MS). Screens were imaged on a Typhoon 9400 scanner and the 32 P signal was quantified using ImageJ.
  • IRDye 800CW goat anti-rabbit (1:15,000) and IRDye 680RD goat anti-mouse (1:15,000) were purchased from Li-COR Biosciences and added for 1 h at room temperature, followed by three additional washes in TBS-T. Blots were imaged in IR using a LI-COR Odyssey Blot Imager. Bands were quantified using ImageJ. [0151] Mouse primary tumor models (4T1, 4T1-luc, E0771).
  • tumors were either irradiated or not irradiated with 12 Gy using a 225 kVp cabinet X-ray irradiator filtered with 0.5 mm Cu (IC-250, Kimtron Inc., CT).
  • Mice were anesthetized with a mixture of 80 mg/kg ketamine (VetaKet) and 5 mg/kg xylazine (AnaSed) prior to irradiation.
  • Anaesthetized animals were shielded with a 3.2 mm lead shield with a 15 x 20 mm aperture where the tumor was placed.
  • mice were intratumorally injected with 100 ⁇ L of 100 ⁇ M neutralizing or non-binding STING and every other day following that up ting day 15. Mice were euthanized on day 15 and tumors were extracted for FACS analysis.
  • E0771 model six- to fifteen-week-old female WT or Enpp1H362A C57BL/6J mice were inoculated with 2.5 x 10 4 E0771 cells suspended in 50 ⁇ L of PBS into the fifth mammary fat pad.
  • mice tumor metastasis models (4T1 and E0771.LMB).
  • 4T1 metastasis from orthotopic tumors we implanted 2.5 x 10 4 WT or Enpp1 KO 4T1 into WT or Enpp1 KO BALB/cJ respectively following protocols above.
  • day 33 we sacrificed the animals and collected blood, draining inguinal lymph nodes, primary tumors, lungs, livers, and brains.
  • Blood is dispensed into a 15-ml conical tube containing 10 mL 1x HBSS, centrifuged at 1500 rpm at room temperature for 5 minutes and plated into IMDM supplemented with 60 ⁇ M 6-MP, 10% FBS and 1% penicillin-streptomycin. Organs are cut into small pieces with scissors and forces, except for lymph nodes which were teased apart by forcing through a 100 ⁇ M strainer. Organs are digested in 1 mg/mL collagenase IV (Sigma, C5138): 37C for 30 minutes for primary tumor and liver, 4C for 75 minutes for lung, and 37C for 30 minutes for liver.
  • 1 mg/mL collagenase IV Sigma, C5138
  • Dissociated organs are washed twice with 1x HBSS buffer before plated into 60 ⁇ M 6-MP containing IMDM media. Cells are cultured for 6-12 days without disturbance. Then clonogenic metastases are fixed and visualized with 0.03% (w/v) methylene blue). Metastatic colonies were quantified with Fuji Image J.
  • 4T1 metastasis from tail vein injections we injected 2.5 x 10 4 WT or Enpp1 KO 4T1 into WT or Enpp1 KO BALB/cJ directly into the tail vein and monitored the mice weights until any mouse’s weight starts to fall for more than 10%, around day 20-30. We collected lungs and quantified metastatic burden following process described above.
  • E0771.LMB models E0771.LMB cells were transfected with an empty vector containing the puromycin resistant gene (denoted as E0771.LMB.PuroR), so that we could selectively culture cancer cells from lung explants ex vivo.
  • tumors were palpable with an average tumor volume of 100 ⁇ 20 mm 3 (determined by length 2 x width / 2), 10-12 days after cell inoculation, tumors were irradiated with 12 Gy using a 225 kVp cabinet X-ray irradiator filtered with 0.5 mm Cu (IC-250, Kimtron Inc., CT) following previously described procedures (Carozza, Böhnert, et al., 2020).
  • IC-250 225 kVp cabinet X-ray irradiator filtered with 0.5 mm Cu (IC-250, Kimtron Inc., CT) following previously described procedures (Carozza, Böhnert, et al., 2020).
  • E0771 murine breast tumor models For primary tumor experiment in Figure 5A, we orthotopically injected 2.5 x 10 4 E0771 cells into the 4 th MFP of WT, Enpp1 H362A , Sting1 -/- , or Enpp1 H362A x Sting1 -/- C57BL/6J mice. We measured animal survival by the time it took for the tumors to reach 1000 mm 3 .
  • MMTV-PyMT mice (JAX, 002374) on an FVB/NJ background were obtained from The Jackson Laboratories. Homozygous C57BL/6J-Enpp1H362A mice were generated as described (Carozza et al., 2022). FVB/N- Tg(MMTV-PyVT) 634Mul/J mice were bred with C57BL/6J-WT or C57BL/6J-Enpp1H362A to generate B6;FVB-MMTV and B6;FVB-Enpp1H362A x MMTV respectively.
  • mice from the second generation of both B6;FVB-MMTV and B6;FVB-Enpp1H362A x MMTV genotypes were used for the experiment. Mice were fed a standard rodent chow diet and were housed in a special pathogen-free facility. Mice were maintained at Stanford University in compliance with the Stanford University Institutional Animal Care and Use Committee regulations. All procedures were approved by the Stanford University Administrative Panel on Laboratory Animal Care. Mice were euthanized by CO2 inhalation. Mice were monitored and assessed for tumor onset. Tumors were measured in two dimensions used digital calipers and tumor volume was calculated by the modified ellipsoidal formula , where length is the longest diameter and width is the shortest diameter.
  • Tumor onset was defined by the date on which a palpable tumor (tumor volume, V > 0.5 mm 3 ) was observed. Tumor-free survival was plotted in a Kaplan-Meier curve with GraphPad Prism 9.5, and statistical significance was assessed by log-rank Mantel-Cox test. [0156] Flow cytometry analysis of tumors. In certain experiments, seven- to twelve-week-old female WT or Enpp1 KO BALB/cJ mice were inoculated with 5 x 10 4 WT or Enpp1 KO 4T1-luc cells, respectively, suspended in 50 ⁇ L of PBS into the fifth mammary fat pad.
  • tumors were irradiated with 12 Gy and excised four days later.
  • 100 ⁇ L of 100 ⁇ M neutralizing STING (WT) or non-binding STING (R237A) were injected intratumorally into the right fifth mammary fat pad every other day until the end of the experiment on day 15.
  • WT or Enpp1 -/- 4T1-luc cells were orthotopically injected into WT or Enpp1 -/- BALB/cJ mice respectively.
  • mice were intratumorally injected with 100 ⁇ L of 100 ⁇ M neutralizing (WT) or non-binding (R237A) STING every other day up to day 13.
  • WT neutralizing
  • R237A non-binding
  • tumors were extracted and incubated in RPMI + 10% FBS with 20 ⁇ g/ml DNase I type IV (Sigma-Aldrich) and 1 mg/ml Collagenase from Clostridium histolyticum (Sigma-Aldrich) at 37 °C for 30 min.
  • Tumors were passed through a 100 ⁇ m cell strainer (Fisher Scientific) and red blood cells were lysed using red blood cell lysis buffer (155 mM NH4Cl, 12 mM NaHCO 3 , 0.1 mM EDTA) for 5 min at room temperature. Cells were stained with Live/Dead fixable nearIR or blue dead cell staining kit (ThermoFisher). Samples were then fixed and permeabilized with eBioscience Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), Fc-blocked for 10 min using TruStain fcX (BioLegend) and subsequently antibody- stained with antibodies.
  • red blood cell lysis buffer 155 mM NH4Cl, 12 mM NaHCO 3 , 0.1 mM EDTA
  • RNA-seq of mouse primary tumors and lungs were analyzed using a Symphony (BD Biosciences), or an Aurora analyzer (Cytek). Data was analyzed using FlowJo V10 software (BD) and Prism 9.1.0 software (Graphpad) for statistical analysis and statistical significance was assessed using the unpaired t test with Welch’s correction.
  • BD FlowJo V10 software
  • Prism 9.1.0 software Graphpad
  • Cells were handled gently with wide-bore tips from this point up until 10x GEM generation. Cells were washed with PBS and 0.04% BSA, passed through 40 ⁇ m Flowmi cell strainer (Bel-Art, 974-25244) before measuring concentration and viability with an automated cell counter. Viability for tumor samples were between 72-87% and for lung samples were between 91-96%. Cells were resuspended in PBS + 0.04% BSA to 1000 cells/ ⁇ L. Single- cell suspensions were processed using the 10x Gemonics Single Cell 3’v3 RNA-seq kit. Gene expression libraries were prepared according to the manufacturer’s protocol with targeted cell recovery of 6000. Final 12 libraries were pooled and sequenced on the NextSeq 2000 platform (Illumina).
  • Raw sequencing reads from the gene expression libraries were processed using CellRanger v3.0.2, aligning reads to the mm10 build of the mouse genome. Main processing, quality control, and analyses were performed with Cellenics platform (website: scp.biomage.net/data-management).
  • Cellenics platform website: scp.biomage.net/data-management.
  • Mammary fat pad primary tumors and lungs containing metastases were harvested from two ENPP1 WT-OE or ENPP1 T238A-OE tumor bearing mice when primary tumors reached 1000 mm 3 ( Figure 1). Cryopreserved single cell suspensions were thawed into warm RPMI media with 10% HI-FBS.
  • Cells were washed with PBS and 0.04% w/v BSA, passed through 40 ⁇ m Flowmi cell strainer (Bel-Art, 974-25244), and assessed for concentration and viability with an automated cell counter. Cells were resuspended in PBS and 0.04% w/v BSA to 1000 cells/ ⁇ L.
  • Single-cell suspensions were processed with a Chromium Controller microfluidic device (10X Genomics), using the Chromium Next GEM Single Cell 3’ HT Reagent Kits v3.1 (Dual Index). Library preparation was performed according to the manufacturer's instructions (single cell 3’ HT reagent kits v3.1 protocol, Rev D, 10x Genomics).
  • fibroblasts a unique cluster enriched in tumors overexpresses myofibroblast marker Acta2, as well as Tgfb1 and Tgfb2 and are known as myofibroblastic cancer-associated fibroblasts (myCAF) and overtly immunosuppressive (Mhaidly and Mechta-Grigoriou, 2020; Mao et al., 2021).
  • myCAF myofibroblastic cancer-associated fibroblasts
  • the ISPY-2 is an ongoing multicenter, Phase II neoadjuvant (therapies administered before surgery) platform trial for high-risk, early- stage breast cancer designed to rapidly identify new treatments and treatment combinations with increased efficacy compared to standard-of-care (sequential weekly paclitaxel followed by doxorubicin/cyclophosphamide [T-AC] chemotherapy (Wolf et al., 2022).
  • T-AC doxorubicin/cyclophosphamide
  • Multiple investigational treatment regimens are simultaneously and adaptively randomized against the shared control arm.
  • the primary efficacy end point is pathologic complete response (pCR).
  • pCR is a known prognostic biomarker for long-term outcomes; achieving pCR after neoadjuvant therapy implies approximately 80% reduction in recurrence rate (Yee et al., 2020).
  • Gene expression arrays were one of the two primary biomarker platforms assayed pre-treatment to identify biomarkers predictive of pCR and long-term outcomes of experimental neoadjuvant therapies and combinations. Details on conducting the gene expression array is listed in the section herein entitled “Molecular profiling of ENPP1 level using tissue mRNA expression”.
  • Immune+ status was estimated based on the average dendritic cell and STAT1 signatures (Wolf et al., 2022). Additionally, distant-metastasis free survival (DMFS) of ENPP1-high versus ENPP1-low group was compared. The threshold for ENPP1-high versus ENPP1-low was set so that the P-value was the smallest. Specifically, “surv_cutpoint” function from the survminer package (www.sthda.com/english/wiki/survminer-0-2-4#determine-the-optimal-cutpoint-for-continuous- variables) in RStudio was used. [0162] Quantification and Statistical Analysis.
  • CNB was typically performed under local anesthesia. CNB of 16-gauge were taken from the primary breast tumor before treatment. Collected tissue samples were immediately frozen in O.C.T. embedding media and then stored in -80°C until further processing. An 8 ⁇ M section was stained with hematoxylin and eosin (H&E) and pathologic evaluation performed to confirm the tissue contained at least 30% tumor.
  • H&E hematoxylin and eosin
  • a tissue sample meeting the 30% tumor requirement was further cryosectioned at 30 ⁇ M for downstream analysis.
  • Molecular profiling of ENPP1 level using tissue mRNA expression Gene expression arrays of breast sections described in the section herein entitled “Breast biopsy processing” were performed following published protocol (Wolf et al., 2022).
  • ENPP1 mRNA level was used to predict pCR of the following neoadjuvant therapies in the I-SPY2 trial. More details are described in Example 13. 1.
  • Paclitaxel + Pembrolizumab i. ENPP1 mRNA ⁇ 6.0, will have pCR ii. ENPP1 mRNA > 6.0, have a chance of not having pCR 1.
  • ENPP1 mRNA > 8.9 have a heightened chance of not being pCR 2.
  • Paclitaxel + ABT888 + Carboplatin i. ENPP1 mRNA ⁇ 6.5, will be pCR ii. ENPP1 mRNA > 6.5, have a chance of not being pCR 1.
  • ENPP1 mRNA level was used to predict DMFS of Paclitaxel + Pembrolizumab neoadjuvant therapy in the I-SPY2 trial. More details are described in Example 13. 1.
  • Paclitaxel + Pembrolizumab i. ENPP1 mRNA ⁇ 8.3, 0% chance of developing distant metastasis up to 7 years.
  • IHC of ENPP1 can be performed following previously published protocol (Li et al., 2021). Briefly, one to five sections obtained as described in the section herein entitled “Breast biopsy processing” can undergo processing. The antigen is retrieved using sodium citrate pH6 buffer for 30 min. Following blockage with Background Buster (Innovex), the slides are incubated with 2.5 ⁇ g/ml anti-ENPP1 antibody (Abcam ab4003 at 1:200) for 4 hr, and then incubated with the biotinylated secondary antibody for 30 minutes. The Streptavidin-HRP D (DABMap kit, Ventana Medical Systems) and the DAB detection kit (Ventana Medical Systems) are used to detect the signal according to the manufacturer instructions.
  • ENPP1 IHC level can be used to predict ENPP1 status: Any staining above 1% cells positive for ENPP1 expression per slide is considered positive for ENPP1.
  • FFPE formalin-fixed paraffin-embedded
  • Each test section will be stained with the ENPP1 probes designed by Integrated DNA Technologies (Table below) and centromeric chromosome 6 control probe (CEP6, Empire Genomics). Slides will be visualized following the fluorescence microscope manufacturer protocol. A total of 20 cells of invasive carcinoma with optimal nuclear signals were randomly selected in 2–4 separate fields for evaluation. Signals of ENPP1 and CEP6 can be counted manually according to the specification of the kit. ENPP1 gene amplification status is defined below. In cases with equivocal results, a repeat counting of additional 20 cells in another 2–4 separate fields will be performed. Table. Top 5 ENPP1 FISH Probes (Integrated DNA Technologies).
  • ENPP1 FISH result can be used to predict ENPP1 status: Dual probe (ENPP1 and chromosome 6 control probes) 1. Positive: ENPP1/control ratio ⁇ 2, OR ENPP1 copy number ⁇ 6 regardless of ratio by ISH 2. Equivocal: ENPP1/control ratio ⁇ 2, AND ENPP1 copy number ⁇ 4 but ⁇ 6 3. Negative: ENPP1/control ratio ⁇ 2, AND ENPP1 copy number ⁇ 4 Single probe (ENPP1) 1. Positive: ⁇ 6 ENPP1 copies 2. Equivocal: ⁇ 4 but ⁇ 6 ENPP1 copies 3. Negative: ⁇ 4 ENPP1 copies [0172] ENPP1 K173Q mutation sequencing: tissue. One tissue section collected as described in the section herein entitled “Breast biopsy processing” will be used.
  • Genomic DNA (gDNA) of human PBMCs were isolated using DNeasy Blood & Tissue Kit (Qiagen).
  • hENPP1 exon 4 region was amplified from donor gDNA templates and sequenced with hENPP1_K173Q_seq_fwd and hENPP1_K173Q_seq_rev primers (Table S1).
  • cDNA complementary DNA
  • cells were lysed with 0.5-0.75 mL TRIzol (Invitrogen) and total RNA from cells were extracted using Direct-zol RNA Kit (Zymo).
  • ENPP1 K173Q mutation sequencing blood. Collect 10 mL whole blood. Dilute whole blood with sterile PBS at a 1:1 v/v ratio. Prepare a tube with Ficoll density gradient medium (DGM) as per the manufacturer's instructions e.g., a 50 mL tube with 15 ml DGM.
  • DGM Ficoll density gradient medium
  • Molecular profiling of circulating ENPP1 level Collect at least 200uL blood sample into commercially available anticoagulant-treated tubes, e.g., citrate-treated (light blue tops) or heparin-treated (green tops) (ThermoFisher). Avoid using ethylenediaminetetraacetic acid (EDTA) coated tubes for blood collection as it interferes with ENPP1 activity assay. Centrifugation for 15 minutes at 2,000 x g depletes platelets in the plasma sample. Immediately transfer the liquid component (plasma) into a clean polypropylene tube. The samples should be maintained at 2–8°C while handling.
  • EDTA ethylenediaminetetraacetic acid
  • Plasma ENPP1 level can be analyzed using either activity-based or protein-based assays. Details of an ENPP1 activity-based assay are described in the section herein entitled “ENPP1 activity assay by [ 32 P] cGAMP degradation thin-layer chromatography assays”. Briefly, 22 ⁇ L fresh serum or plasma was reacted with 5 ⁇ M 32 P- cGAMP spiked cGAMP at 37 °C and pH 7.4. cGAMP degradation is measured at 0, 10, 30, 60, 90, 120 minutes.
  • ENPP1 protein levels will be validated using commercially available ENPP1 ELISA kits (MyBioSource.com #MBS451862; American Research Products Inc. #ARP-E1282L; Aviva Systems Biology #OKCD08756; Biomatik #EKF59219).
  • Circulating ENPP1 levels in a breast cancer patient compared with normal populations can help infer if the patient’s breast cancer overexpresses ENPP1.
  • the threshold is set as following: 1.
  • ENPP1 activity assay plasma ENPP1 cGAMP degradation half-life less than 60 minutes (the average degradation half-time in healthy donors, Figure 9K) in a breast cancer patient indicates the tumor overexpresses ENPP1 and sheds into circulation and is thus considered positive for ENPP1.
  • ENPP1 protein levels Plasma ENPP1 levels is > 4.4 ⁇ M/L (the average plasma ENPP1 concentration in healthy population, ProteinAtlas) in a breast cancer patient indicates the tumor overexpresses ENPP1 and sheds into circulation and is thus considered positive for ENPP1.
  • ENPP1 expression level has been shown to correlate with poor prognosis in several cancer types, and we confirmed the same relationship in the context of breast cancer ( Figure 1A).
  • Patients in the METABRIC database with breast tumors expressing high levels of ENPP1 mRNA have significantly worse disease-free survival rate, despite exhibiting a similar distribution across disease stages as the ENPP1-low group ( Figure 1A).
  • This observation hints at the potential involvement of ENPP1 in tumorigenesis, and prompted us to ask whether host tissue, tumor, or both sources of ENPP1 expression are important for ENPP1’s tumorigenic role, using breast cancer as a model.
  • ENPP1 catalytic activity drives breast tumor growth and metastasis by restricting adaptive immune infiltration
  • ENPP1 expression levels have been shown to correlate with poor prognosis in several cancer types. Patients in the METABRIC database with breast tumors expressing high levels of ENPP1 mRNA have a significantly worse disease-free survival rate, despite exhibiting a similar distribution across disease stages as the ENPP1-low group ( Figure 1A). Furthermore, patients with stage IV metastatic disease have significantly higher ENPP1 RNA expression than patients with stage III disease ( Figure 1K, 9E).
  • ENPP1 WT-OE 4T1 cells exhibited faster primary tumor growth and more lung metastases (Figure 1L) without affecting cell proliferation ( Figure 1J), implicating a non-tumor cell intrinsic mechanism of enhanced tumor growth and metastasis.
  • TAM tumor-associated macrophage
  • EXAMPLE 4 ENPP1 overexpression in cancer cells inhibits STING signaling to suppress anti-tumor immunity [0183]
  • Cgas expression is the highest in a distinct cancer cell cluster annotated for overexpressing Kif2c, which has been reported to drive CIN and metastasis (Bakhoum et al., 2018a).
  • Sting1 is expressed at relatively high levels in endothelial cells, fibroblasts, macrophages, and DCs ( Figure 3A).
  • cGAS and STING supports our model that cancers produce and secrete cGAMP, which is then detected by surrounding host cells (Carozza, Böhnert, et al., 2020).
  • cGAMP responder cells as those with low expression of Cgas and high expression of Sting1 and interferon- stimulated genes (ISGs) Ifitm1, Ifitm2, and Ifitm3 ( Figure 3B, 3J), which matched well with our previous report of cell types that respond to extracellular cGAMP (Cordova et al., 2021).
  • Adora2b the eADO receptor
  • Tgfb1 and Il10rb downstream of eADO signaling
  • Hp a gene that is transcriptionally upregulated by adenosine signaling
  • Hptoglobin (HP) secretion by cancer cells upon of eADO signaling has been reported to recruit polymorphonuclear MDSCs (PMN-MDSCs) and promote self-seeding of ENPP1-high circulating tumor cells (CTCs) (De Córdoba et al., 2022). While we did not observe increased Hp expression in ENPP1 WT-OE cancer cells, we found that neutrophils and monocytes in the ENPP1 WT-OE metastatic niche had the biggest increase and the highest overall expression of Hp, suggesting that they are the potential source of HP production that facilitate metastasis (Figure 3O).
  • Enpp1 H362A mice retarded E0771 tumor growth to a similar degree as Enpp1-/- mice compared to WT mice (Carozza, Böhnert, et al., 2020), suggesting that ENPP1’s tumorigenic phenotype in the E0771 model is mediated mainly through cGAMP hydrolysis.
  • cGAMP hydrolysis To exclude potential STING-independent roles of cGAMP in this context, we compared orthotopic E0771 breast tumors in Sting-/- and Enpp1 H362A x Sting-/- mice.
  • E0771.lmb a derived metastatic cell line from the parental E0771 cells (Johnstone et al., 2015) into WT, Enpp1 H362A , Enpp1-/-, Sting-/-, and Enpp1 H362A x Sting-/- mice (Figure 5C). While 50% of WT mice had lung metastasis, none of the Enpp1 H362A mice or Enpp1-/- had metastasis, suggesting that endogenous ENPP1 promotes metastasis mainly, if not exclusively, through extracellular cGAMP hydrolysis (Figure 5D, 5E).
  • ENPP1 selective inhibition of ENPP1’s cGAMP hydrolysis activity abolishes breast cancer metastasis a STING-dependent manner
  • ENPP1 Apart from its cGAMP hydrolysis activity, ENPP1 is also known to degrade extracellular ATP (Carozza et al., 2022), and generate immunomodulatory adenosine as byproduct (Li et al., 2021). While our experiments above support a model in which extracellular cGAMP signaling is at least partially responsible for the effects of ENPP1 on tumorigenesis, we wanted to formally test the sufficiency of cGAMP hydrolysis to explain ENPP1’s pro-tumorigenic phenotypes.
  • Enpp1 H362A mice retarded E0771 tumor growth to a similar degree as Enpp1 -/- mice, as compared to WT mice (Carozza, Böhnert, et al., 2020), and the tumor slowing effects in Enpp1 H362A mice were completely abolished in the Sting1 knockout background (Figure 5A).
  • Figure 5A [0199] Furthermore, we noticed a 41% increase in the tumor-free rate in Enpp1 H362A compared with WT mice after E0771 implantation (4/32 [12.5%] vs.2/27 [7.4%]) (Figure 5A). Therefore, we hypothesized that Enpp1 depletion also disfavors primary tumor onset.
  • E0771.lmb.PuroR a derived metastatic cell line from the parental E0771 cells (Johnstone et al., 2015) engineered with puromycin resistance to allow for ex vivo selection
  • the anti-metastatic effect of blocking ENPP1’s cGAMP hydrolysis activity in the Enpp1 H362A mice is completely STING-dependent (Figure 5C).
  • ENPP1 is a type II transmembrane protein with a short N-terminal cytosolic tail, a transmembrane (TM) domain, and an extracellular region containing the extracellular somatomedin B-like (SMB) domains and the catalytic domain (Figure 6A).
  • TM transmembrane
  • SMB somatomedin B-like domains and the catalytic domain
  • Figure 6A In addition to its full-length membrane-anchored form (memENPP1), an ENPP1 isoform beginning distal to the TM domain has been observed as a secreted soluble protein (secENPP1) (Belli, van Driel and Goding, 1993; Jansen et al., 2012).
  • K173 residue is in the extracellular somatomedin B-like 2 (SMB2) domain, and therefore should be present in both memENPP1 and secENPP1.
  • SMB2 extracellular somatomedin B-like 2
  • plasma from K173Q donors (which would contain only soluble secENPP1) exhibited similar ENPP1 activity as plasma from WT donors ( Figure 6D, 6L).
  • the striking difference in K173Q impact between the membrane and soluble pool of ENPP1 from donors was puzzling to us, since secENPP1 and memENPP1 are typically correlated in most cell lines (Figure 6Q).
  • K173Q ENPP1 has enhanced ATP hydrolysis activity (Figure 6H). Since K173 is located distal to the catalytic pocket of ENPP1 we would not expect it to differentially affect cGAMP or ATP hydrolysis. [0204] To investigate why K173Q ENPP1 protein has enhanced hydrolysis activity only when membrane-anchored, we tested whether K173Q affects memENPP1 dimerization, given its location in the SMB domain that has been implicated in disulfide bond formation in hENPP1 (Gijsbers, Ceulemans and Bollen, 2003; Bellacchio, 2012; Jansen et al., 2012).
  • EXAMPLE 10 Transmembrane and secreted ENPP1 isoforms dictate primary tumor growth and metastasis respectively [0205] Given that a common ENPP1 variant has enhanced activity specifically in the transmembrane pool, but not in the secreted pool, we next addressed the relative contributions of transmembrane and secreted ENPP1 to the tumor-promoting effects of this enzyme. To experimentally investigate this question, we first sought to look for an ENPP1 variant that selectively produces the transmembrane over the secreted isoform.
  • the secENPP1 isoform is likely to be proteolytically processed by the signal peptide complex (SPC) owing to a predicted motif at A84/K85 ( Figure 7A, 7G) (Teufel et al., 2022). Additionally, there is previous evidence arguing against splice isoforms or ectodomain shedding as mechanisms of isoform origin (Davis et al., 2006; Kato et al., 2018) Therefore, we expressed seven ENPP1 variants with mutations at or near the putative signal peptidase cleavage site in 293T cGAS ENPP1-/- cells, seeking mutants that would manipulate the mem:sec ratio.
  • SPC signal peptide complex
  • ENPP1 drives distinct long-term immune-suppressive programs in primary and metastatic niche [0208] To thoroughly characterize long-term immunological changes in primary and metastatic sites in 4T1 breast tumor model, and to delineate contribution by memENPP1 and secENPP1, we performed single-cell RNA-seq (scRNAseq) on primary tumors and lungs collected from mice in the above experiment bearing ENPP1T238A-OE, ENPP1WT-OE, or ENPP1A84S-OE tumors. After strict quality control and filtration, we collected a total of 43,752 cells. We performed unsupervised graph-based clustering on all cells and identified 11 major clusters based on canonical cell markers (Figure 8A, 8O).
  • scRNAseq single-cell RNA-seq
  • fibroblasts a unique cluster enriched in tumors overexpresses myofibroblast marker Acta2, as well as Tgfb1 and Tgfb2 and are known as myofibroblastic cancer-associated fibroblasts (myCAF) and overtly immunosuppressive (Mhaidly and Mechta-Grigoriou, 2020; Mao et al., 2021) [0209]
  • myCAF myofibroblastic cancer-associated fibroblasts
  • we performed unsupervised clustering on this subcluster (Figure 8B, 8P).
  • Tox has well documented roles in CD8+ T cell exhaustion (Khan et al., 2019; Sekine et al., 2020) and its involvement in CD4+ T cell started to gain understanding (Miggelbrink et al., 2021). Sox4 has been reported to drive immune evasion in breast cancer (Bagati et al., 2021) and its expression is upregulated by Casc1570 kb downstream of Sox4 (Sun et al., 2021).
  • MemENPP1 also reprogramed antigen presenting H2-ab1 expressing macrophages into immunosuppressive Tgfb1 and Arg1 expressing macrophages (Figure 8G), consistent with it antagonizing cGAMP’s role in M1-like macrophage polarization.
  • pDCs are a subtype of DCs that normally specializes in INF-I production (Zhou, Lawrence and Liang, 2021).
  • Tumor infiltrating na ⁇ ve B cells can serve as professional APCs to CD8+ and CD4+ T cells in human breast carcinoma and correlate with positive prognosis in patients (Laumont et al., 2022).
  • our scRNAseq analysis of tumors expressing WT ENPP1 or only membrane-bound ENPP1 provided strong support for our working model that tumor secreted cGAMP is dampened by ENPP1, which leads to a suppressive immune environment.
  • ENPP1 induced immunological changes in the lungs. Unlike primary tumors, we observed little changes in innate immune cells, perhaps because most of the innate immune education occurred before cancer colonization into lungs. We, however, observed differences in adaptive immune cells.
  • Enpp1 undergoes a stepwise up-regulation in immunosuppressive macrophages (Figure 2H, 9A). Enpp1 is also significantly upregulated in myCAFs compared to fibroblasts ( Figure 9B). When looking across conditions, we saw that Enpp1 is significantly higher in myCAFs associated with ENPP1T238A-OE condition with presumably the highest amount of intratumoral cGAMP, suggesting that Enpp1 may be upregulated by pathways downstream of cGAMP signaling ( Figure 3F, 9C).
  • cGAMP is elevated in cases of ultraviolet (UV) exposure from sunlight (Skopelja-Gardner et al., 2020) and autoimmune conditions (Haag et al., 2018), and we investigated if ENPP1 RNA is upregulated in these contexts outside of cancer.
  • ENPP1 increased from non-exposed skin to sun exposed skin to melanoma ( Figure 9I).
  • ENPP1 also increased from endothelial/epithelial cells in skin biopsies from health individuals to lupus patients, which tracks with increase in ISGs and the putative transcriptional/epigenetic regulators we discovered (Figure 9J).
  • ENPP1 is also an ISG.
  • serum ENPP1 activity from a breast cancer survivor and healthy donors from multiple blood draws across dates.
  • the breast cancer survivor has significantly higher cGAMP degradation activity in the serum compared to health donors (Figure 9K), and this activity difference can be attributed to ENPP1 as treating serum with ENPP1 inhibitor STF- 1623 (Carozza, Brown, et al., 2020) collapsed the differences.
  • ENPP1 is likely upregulated transcriptionally, and this difference can be readily measured in the serum.
  • EXAMPLE 13 Low ENPP1 expression correlates with immune infiltration and pathological complete response (pCR) to Pembrolizumab (anti-PD1) and Veliparib + Carboplatin (PARP inhibitor) in breast cancer patients [0219] After delineating the molecular and cellular mechanisms governing the deterministic role of ENPP1 in metastasis of murine breast cancers, we asked whether these mechanistic insights and disease outcomes are conserved in humans.
  • ENPP1 inhibition could potentially synergize with existing therapies that are upstream of cGAMP production, such as PARP inhibitor (PARPi) that induces DNA damages (Ding et al., 2018), or downstream of STING mediated immune infiltration, such as anti-PD1/PDL1 (Tumeh et al., 2014).
  • PARP inhibitor PARPi
  • STING mediated immune infiltration such as anti-PD1/PDL1
  • patients may have achieved complete response to Pembrolizumab at least partially due to enhanced cGAMP-STING mediated immune infiltration permitted by an ENPP1-low setting, which confers long term advantage in prognosis.
  • ENPP1-low setting confers long term advantage in prognosis.
  • ENPP1 blockade is therefore a promising immuno-oncology strategy that could synergize with existing anti-PD-1/PD-L1 therapies in the clinics.
  • ENPP1 As a previous study postulated that the ENPP1/cGAMP/adenosine axis may contribute more to metastasis than to primary tumors using overexpression experiments (Li, Duran, Dhanota, Chatila, Bettigole, Kwon, Sriram, Humphries, Salto-Tellez, James, Hanna, Melms, Vallabhaneni, Litchfield, Usaite, Biswas, Bareja, Li, Martin, Dorsaint, Cavallo, Li, Pauli, Gottesdiener, DiPardo, et al., 2021).
  • ENPP1 A84S SNP leads to increased membrane ENPP1 activity, but abolishes its secretion.
  • ENPP1 expression increases with cGAMP-STING signaling.
  • ENPP1 expression correlates with levels of four transcriptional regulators ETS1, AFF3, BACH2, and SATB1, and they are likely induced by STING signaling. The exact mechanism warrants future exploration.
  • host tissue ENPP1 plays an important role in cancers that originate from tissues with high ENPP1 activity.
  • the ENPP1 status of the tissue in which the cancer develops could dictate the extent of ENPP1’s role in tumor development and the efficacy of potential ENPP1 blockade therapy.
  • Elevated ENPP1 activity is even readily observable in serum from a breast cancer patient. Combining all pieces of evidence, we could tantalize the contribution of cGAMP signaling to Enpp1 upregulation along the oncogenic trajectory and in chronic inflammation or autoimmune conditions. Enpp1 upregulation is likely wired into a negative feedback loop to enable immune tolerance during autoimmunity but highjacked by cancers for immune evasion. [0225] Our data highlighted the notion that host-derived ENPP1 is not a passive bystander, but rather actively involved in shaping the tumor immune microenvironment.
  • ENPP1 status of the tissue in which cancer develops either as the primary site or the site of metastasis, and ENPP1 allele or expression level variations could dictate the extent of ENPP1’s role in tumor development.
  • ENPP1 While we previously identified ENPP1 as a cGAMP hydrolase, there has been significant debate as to whether its ability to deplete cGAMP and thereby dampen STING signaling is central to its pro-tumorigenic effects, as ENPP1 has other enzymatic activities toward ATP and other nucleotide triphosphates but also generates eADO – a cancer-associated metabolite – as a byproduct of its cGAMP hydrolase activity.
  • Using an unbiased scRNA-seq approach we systematically characterized the immunological impacts and signaling events upon overexpression of ENPP1’s catalytic activity in orthotopically implanted 4T1 cancer cells.
  • ENPP1-high cancer cells promote breast tumor growth by shunting the immunostimulatory cGAMP-STING pathway to the immunosuppressive eADO pathway, while fostering an angiogenic TME for tumor survival (Figure 3P).
  • cGAMP is the relevant substrate in in vivo cancer models in a manner dependent on downstream STING signaling.
  • ENPP1 contribute to different stages of tumor development including initiation, progression, and metastasis was not well understood. Importantly, our work provides the first evidence that ENPP1 promotes breast cancer initiation (Figure 5B). Comparing between primary tumors and metastases, we noticed a stronger contribution of cGAMP-STING inhibition to the pro-metastatic phenotype of ENPP1 in our scRNA-seq analyses. We posit that this could be due to elevated cGAMP production along the oncogenic trajectory, as we showed that CIN-high pro- metastatic Kif2c+ cancer cells (Bakhoum et al., 2018b) expressed higher levels of Cgas (Figure 3A).
  • ENPP1 on the surface of cGAMP-producing and cGAMP-sensing cells would be ideally poised to snatch a freshly exported or soon-to-be imported cGAMP molecule in close proximity to cGAMP transporters (Ritchie et al., 2019; Lahey et al., 2020; Cordova et al., 2021), thereby circumventing paracrine activation of the STING pathway within the TME.
  • ENPP1 is broadly expressed by previously known immunosuppressive immune and stromal cells such as the macrophages of the “M2” like phenotype myCAFs.
  • Enpp1 expression in myCAFs in both primary and metastatic niches of ENPP1 T238A-OE 4T1 compared to ENPP1 WT-OE 4T1, suggesting ENPP1 expression is inducible either directly or indirectly by extracellular cGAMP and or ATP.
  • ENPP1 should be targeted in tumors with high myeloid content and fibrosis. Future examination of ENPP1 in other mouse cancer models and patient cohorts are warranted to test our hypotheses.
  • ENPP1-low breast cancer patients are significantly more likely benefit from anti-PD- 1 and PARPi therapies than their ENPP1-high counterparts.
  • ENPP1-high breast cancer patients will greatly benefit from a combination of ENPP1 inhibition with anti-PD-1/anti-PD-L1 or PARPi treatments.
  • ENPP1 is a promising target for cancer immunotherapy that may bolster our arsenal of ICB therapeutics as a druggable innate immune checkpoint.
  • a method for treating breast cancer in a subject comprising: a) obtaining a sample from the subject; b) measuring ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in the sample from the subject, c) classifying the sample, wherein the sample has a high classification when its ENPP1 levels are increased; and d) if the sample has the high classification, then administering to the subject: i) an ENPP1 inhibitor and a PD1 inhibitor, when the breast cancer is responsive to a PD1 inhibitor; ii) an ENPP1 inhibitor and a PDL1 inhibitor, when the breast cancer is responsive to a PDL1 inhibitor, iii) an ENPP1 inhibitor and a PARP inhibitor, when the breast cancer is responsive to a PARP inhibitor, thereby treating the breast cancer in the subject.
  • ENPP1 ectonucleotide pyrophosphatase/phosphodiesterase 1
  • the method of a preceding clause wherein the breast cancer is triple negative, microsatellite instability-high (MSI-H), or mismatch repair deficient (dMMR) when the breast cancer is responsive to a PD1 inhibitor; or the breast cancer has a BRCA1 mutation, a BRCA2 mutation, is HER2 negative, is locally advanced, or is metastatic when the breast cancer is responsive to a PARP inhibitor.
  • the step d) further comprises iii) administering chemotherapy.
  • the chemotherapy is paclitaxel.
  • the sample is a breast cancer tissue sample.
  • the measuring is selected from the group consisting of ENPP1 mRNA expression, ENPP1 immunohistochemical levels, ENPP1 gene amplification levels, and ENPP1 K173Q homozygosity.
  • the measuring is ENPP1 mRNA expression, and the high classification occurs when the breast cancer tissue sample ENPP1 mRNA expression has, relative to a reference, a value selected from the group consisting of 6.2 or above, 6.39 or above, 6.4 or above, 7.0 or above, 7.4 or above, 7.8 or above, 8.0 or above, 8.9 or above, 9.2 or above, 9.4 or above, 9.6 or above, 9.8 or above, 10.0 or above.
  • the method of clause 8, wherein the measuring is ENPP1 immunohistochemical levels, and the high classification occurs when a percentage of cells positive for ENPP1 expression in the breast cancer tissue sample is 0.5 or above, 0.75 or above, 0.9 or above, 1 or above, 10 or above, or 50 or above. [0247] 13. The method of clause 8, wherein the measuring is ENPP1 gene amplification levels, and the high classification occurs when ENPP1/CEP6 probe ratio > 2, when ENPP1 copy number > 6, or when ENPP1 copies are > 6 if a single probe is used. [0248] 14. The method of clause 8, wherein the measuring is ENPP1 K173Q homozygosity, and the high classification occurs when the breast cancer tissue sample is homozygous for the K173Q mutation.
  • the PD1 inhibitor when the breast cancer is responsive to a PD1 inhibitor, then the PD1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, and toripalimab. [0253] 19. The method of clause 18, wherein the PD1 inhibitor is pembrolizumab. [0254] 20. The method of a preceding clause, wherein when the breast cancer is responsive to a PDL1 inhibitor, the PDL1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab. [0255] 21.
  • the PARP inhibitor when the breast cancer is responsive to a PARP inhibitor, is selected from the group consisting of veliparib, olaparib, rucaparib, niraparib, and talazoparib. [0256] 22. The method of clause 21, wherein the PARP inhibitor is veliparib. [0257] 23.
  • the step d) further comprises administering a platinum complex selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin.
  • a platinum complex selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin.
  • a method for treating a subject for a solid tumor comprising: a) assessing mRNA expression in a solid tumor sample from the subject to obtain an mRNA expression result; and b) administering to the subject a therapy for the solid tumor based on the determined mRNA expression result; to treat the subject for the solid tumor.
  • step a) is assessing ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) mRNA expression in the solid tumor sample from the subject to obtain a subject ENPP1 mRNA expression result, wherein an increased subject ENPP1 mRNA expression result compared to reference value ranges for ENPP1 mRNA expression in a control solid tumor (“reference ENPP1 mRNA expression result”) indicates that the subject has high ENPP1 mRNA expression in the solid tumor sample, wherein a decreased subject ENPP1 mRNA expression result compared to the reference ENPP1 mRNA expression result indicates that the subject has low ENPP1 mRNA expression in the solid tumor sample.
  • ENPP1 mRNA expression result an increased subject ENPP1 mRNA expression result compared to reference value ranges for ENPP1 mRNA expression in a control solid tumor
  • a decreased subject ENPP1 mRNA expression result compared to the reference ENPP1 mRNA expression result indicates that the subject has low ENPP1 mRNA expression in
  • step b) is administering to the subject: i) an anti- ENPP1 therapy; and ii) a member selected from the group consisting of an anti-PD1 therapy, an anti-PDL1 therapy, and a PARP inhibitor, or a combination thereof, or if the subject has the low ENPP1 mRNA expression in the solid tumor sample, then step b) is administering to the subject a member selected from the group consisting of an anti-PD1 therapy, an anti-PDL1 therapy, and an anti-PARP therapy, or a combination thereof.
  • step b) is administering to the subject: i) an anti- ENPP1 therapy; and ii) a member selected from the group consisting of an anti-PD1 therapy, an anti-PDL1 therapy, and a PARP inhibitor, or a combination thereof, or if the subject has the low ENPP1 mRNA expression in the solid tumor sample, then step b) is administering to the subject a member selected from the group consisting of an anti-PD1 therapy, an anti
  • the solid tumor is breast cancer.
  • the breast cancer is: estrogen receptor positive (ER + ); and/or human epidermal growth factor receptor 2 positive (HER2 + ).
  • the anti-PD1 therapy is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, and toripalimab.
  • the anti-PD1 therapy is pembrolizumab.
  • the anti-PDL1 therapy is selected from the group consisting of atezolizumab, avelumab, and durvalumab.
  • the anti-PARP therapy is selected from the group consisting of veliparib, olaparib, rucaparib, niraparib, and talazoparib.
  • the anti-PARP therapy further comprises a platinum complex selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin.
  • a platinum complex selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin.
  • a method for diagnosing a solid tumor in a subject comprising: a) assessing mRNA expression in a solid tumor sample from the subject to obtain a subject mRNA expression result, thereby diagnosing the solid tumor in a subject. [0276] 42.
  • the diagnosing is for high ENPP1 mRNA expression in the solid tumor
  • the step a) is assessing ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) mRNA expression in the solid tumor sample from the subject to obtain a subject ENPP1 mRNA expression result, wherein an increased subject ENPP1 mRNA expression result compared to reference value ranges for ENPP1 mRNA expression in a control solid tumor (“reference ENPP1 mRNA expression result”) indicates that the subject has high ENPP1 mRNA expression in the solid tumor sample, thereby diagnosing the solid tumor in a subject for high ENPP1 mRNA expression.
  • the step a) is assessing ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) mRNA expression in the solid tumor sample from the subject to obtain a subject ENPP1 mRNA expression result, wherein an increased subject ENPP1 mRNA expression result compared to reference value ranges for ENPP1
  • a method for treating a subject with a solid tumor with a K173Q mutation in ENPP1 comprising: a) obtaining a solid tumor sample from the subject; b) identifying the subject as having the K173Q mutation in ENPP1, c) if the subject has the K173Q mutation in ENPP1, administering an anti-ENPP1 therapy to the subject, thereby treating the subject with a solid tumor with a K173Q mutation in ENPP1.
  • a method for diagnosing a subject as having a solid tumor with a K173Q mutation in ENPP1, comprising: a) obtaining a solid tumor sample from the subject; b) identifying the subject as having the K173Q mutation in ENPP1, thereby diagnosing the subject with a solid tumor with a K173Q mutation in ENPP1.
  • 46. A method for treating or preventing metastasis in a subject in need thereof, the method comprising administering to the subject an anti-ENPP1 therapy.
  • the anti-ENPP1 therapy is administered to the subject individually as a single agent prophylaxis or therapy (monotherapy) or as a first therapy in combination with at least one additional therapy.
  • a method of predicting distant metastasis-free survival for a subject with breast cancer comprising: a) obtaining a breast cancer tissue sample from the subject; b) measuring ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) mRNA expression in the breast cancer tissue sample from the subject; c) classifying the breast cancer tissue sample, wherein the breast cancer tissue sample has a high classification when its ENPP1 mRNA expression has, relative to a reference, a value which is 8.3 or above; or the breast cancer tissue sample has a low classification when its ENPP1 mRNA expression has, relative to a reference, a value which is 8.3 or below, and wherein the reference is the ENPP1 mRNA expression in a population of breast cancer tissue samples; and d) predicting a 100% chance of distant metastasis-free survival for up to 7 years with the low classification, and an 88% chance of distant metastasis-free survival at 4 years with the high classification, thereby predicting distant metastas
  • each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc.
  • all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above.
  • a range includes each individual member.
  • a group having 1-3 articles refers to groups having 1, 2, or 3 articles.
  • a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth.

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Abstract

The present disclosure relates generally to methods of inhibiting and preventing metastasis as well as methods of diagnosing a subject as being at risk of developing a cancer.

Description

METHODS OF INHIBITING TUMOR PROGRESSION AND METASTASIS BY INHIBITION OF ENPP1 GOVERNMENT SUPPORT [0001] This invention was made with Government support under grant nos. DP2CA228044, 5R01CA258427-02, U01 CA225427, R01 CA132870, P01 CA210961, P01 CA2109610, and DP2 CA239597 awarded by the National Institutes of Health. This invention was made with Government support under grant no. W81XWH-2210121 awarded by the National Science Foundation. The Government has certain rights in the invention. CROSS-REFERENCE TO RELATED APPLICATIONS [0002] Pursuant to 35 U.S.C. § 119(e), this application claims priority to the filing dates of United States Provisional Patent Application Serial Nos. 63/496,281 filed April 14, 2023, as well as 63/505,520 filed June 1, 2023, the disclosure of each of which is herein incorporated by reference in their entirety. BACKGROUND [0003] The strategy of blocking adaptive immune checkpoints (PD-1, PD-L1, and/or CTLA-4) offers curative immunotherapy for some patients with otherwise terminal cancer diagnoses – however, only a minority of patients respond to immune checkpoint blockade (ICB) therapy and many cancer types remain inaccessible by this treatment. (Haslam and Prasad, PMID: 31050774). ICB resistance can occur through a variety of mechanisms, one of which is insufficient lymphocyte infiltration into the tumor, which is determined by our innate immune system’s ability to detect and communicate the presence of cancer. Cancer cells have a variety of strategies for suppressing innate immunity; therefore, a deep understanding of innate immune checkpoints holds the potential to unlock the full power of immunotherapy against immunologically “cold” tumors. [0004] The cytosolic double-stranded DNA (dsDNA)-sensing STING pathway is a key innate immune pathway that detects and responds to chromosomal instability (CIN) and extrachromosomal DNA present in cancer cells (Harding et al., 2017; MacKenzie et al., 2017; Turner et al., 2017; Bakhoum et al., 2018). Cytosolic dsDNA is detected by the enzyme cyclic- GMP-AMP synthase (cGAS), which synthesizes the cyclic dinucleotide cGAMP. Once produced by cancer cells, cGAMP is secreted into the extracellular matrix and taken up by surrounding host cells, where it binds and activates the STING pathway, leading to production of type I interferons (IFNs) and downstream immune cell infiltration in a paracrine fashion (Marcus et al., 2018; Luteijn et al., 2019; Ritchie et al., 2019; Jacqueline A. Carozza, Böhnert, et al., 2020; Lahey et al., 2020; Cordova et al., 2021; Maltbaek, Snyder and Stetson, 2021). Previous work has demonstrated that STING activation is required for the efficacy of ICB therapies (PMID: 25517615). Moreover, extracellular cGAMP is important for the curative effect of ionizing radiation (IR) in the E0771 mouse breast cancer model (Carozza, et al., 2020), highlighting the importance of the extracellular cGAMP-STING axis in cancer therapy. [0005] However, extracellular cGAMP is rapidly degraded by the extracellular enzyme ENPP1 (Li 2014, Carozza 2020). There is mounting evidence that ENPP1 promotes cancer. ENPP1 is upregulated in about 50% of human solid tumors (Tang et al., 2017). In breast cancer patients, ENPP1 overexpression correlates with reduced survival and tamoxifen resistance (Li et al., 2021; Umar et al., 2009). In our previous work, ENPP1 inhibitors have shown efficacy in murine models of breast and pancreatic cancers as a single agent and in combination with IR (Jacqueline A. Carozza, Böhnert, et al., 2020; Jacqueline A. Carozza, Brown, et al., 2020). Despite the evidence linking ENPP1 to tumor promotion phenotypically, its dominant mechanisms of action remain unclear, particularly because ENPP1 may impact multiple aspects of cancer signaling and metabolism. ENPP1 expression has been linked to cancer stem cell populations in breast cancer, lung cancer, and glioblastoma (Takahashi et al., 2015; Hu et al., 2019; Bageritz et al., 2014). In addition to interfering with the extracellular cGAMP-STING axis for immune recognition of tumors, ENPP1 has also been shown to produce immunosuppressive adenosine as a downstream metabolite of cGAMP hydrolysis, which has been linked to metastasis of murine CIN-high breast cancers (Li 2021) and self-seeding of circulating breast tumor cells (Cordoba 2021). Therefore, numerous questions remain surrounding the mechanisms of ENPP1’s tumorigenic activity, ENPP1’s contribution to different stages of tumor development including initiation, progression and metastasis, how ENPP1 shape the local and distant tumor immune microenvironment (TIME), and how ENPP1 genetic variants present across the human population may contribute to cancer risk. [0006] The disclosure provided here provides solutions to the problems existing with previous attempts to understand the role of ENPP1 is cancer and potentially offers improved methods for prevention and treatment of metastasis as well as improved cancer diagnostics. SUMMARY [0007] The present disclosure relates generally to methods of treating and preventing metastatic cancer. In another aspect, the present disclosure relates to methods of diagnosing a subject as having cancer or being at risk for cancer. [0008] ENPP1 inhibition can synergize with existing cancer therapies that are upstream of cGAMP production, such as PARP inhibitors, or with existing therapies that are downstream of cGAMP production, such as anti-PD1 or anti-PDL1 therapies. Herein, it is shown that breast cancer subjects who were responsive to PARP inhibitors, anti-PD1, or anti-PDL1 therapies had low levels of ENPP1 expression. Accordingly, the invention also provides methods for treating cancer, including breast cancer, by administering to subjects having high levels of ENPP1 expression (or simply positive for ENPP1 protein expression when examining a tumor biopsy with immunohistochemistry) with both an ENPP1 inhibitor and either a PARP inhibitor, an anti- PD1 therapy, and/or an anti-PDL1 therapy. In some cases, such methods for treating cancer are for subjects who are positive for ENPP1 expression and whose tumors were not responsive to PARP inhibitor, anti-PD1, or anti-PDL1 therapy. [0009] In another aspect, the present disclosure relates to ENPP1 expression as a prognostic biomarker for anti-PD1 and PARP inhibitors. [0010] The foregoing summary is illustrative only and is not intended to be in any way limiting. In addition to the illustrative embodiments and features described herein, further aspects, embodiments, objects and features of the disclosure will become fully apparent from the drawings and the detailed description and the claims. BRIEF DESCRIPTION OF THE DRAWINGS [0011] The features of the present disclosure are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the disclosure are utilized, and the accompanying drawings. [0012] Figures 1A-1F show ENPP1 contributions to tumor progression and metastasis in humans and mice. (A) shows disease free survival of breast cancer patients in the ENPP1-high group (n = 59) and ENPP1-low group (n = 1926). The number of patients in each group were stratified by stages. Threshold for high versus low expression was set at which the P value was the smallest. (B) WT or Enpp1-/- 4T1-luc cells (5 x 104) were orthotopically injected into WT or Enpp1-/- BALB/chi mice. Tumors underwent local irradiation (12 Gy) when they reached 100 20 mm3. Tumor volumes for each genotype combination were plotted as mean SEM. n = 7, 17, 9, 16 mice for KO x KO, KO x WT, WT x KO, and WT x WT cancer x mice genotype combinations. P values of the last tumor measurement were determined by multiple unpaired t test with Welch correction. **P 0.01, ***P 0.001, ****P 0.0001; not significant (ns). (C) Ex vivo relative cGAMP hydrolysis activity calculated from kinetic analysis by tumor lysates (left) and sera (right) from randomly selected mice in (B) reaching experimental end point (n = 2-6 biological replicates). P values were determined by unpaired t test with Welch correction. (D) Schematic of metastasis by tail vein injection experiment. WT or Enpp1 KO 4T1-luc cells (2.5 x 104) were intravenously injected into WT or Enpp1 KO BALB/cJ mice. After 32 days, lungs were harvested, dissociated, and cultured in media containing 6-TG (60 M) for 7 days. Metastatic colonies were fixed and visualized with methylene blue dye. (E) Representative plates containing metastatic colonies from (D). (F) Quantification of the percentage of mice with metastasis (left) and the percentage of plates covered by metastatic colonies (right) from (E). n = 4, 5, 3, 3 mice for KO x KO, KO x WT, WT x KO, and WT x WT cancer x mice genotype combinations. P values were determined by unpaired t test. [0013] Figure 1G shows ENPP1 degradation activity in 4T1 and its derived cell lines as assessed by TLC (Thin Layer Chromatography). [0014] Figure 1H shows ENPP1-GFP expression (top) and ENPP1 degradation activity (bottom) of ENPP1WT-OE 4T1 and ENPP1T238A-OE 4T1 clones as assessed by western blotting and TLC. Bolded clones were pooled for experiments. [0015] Figure 1I shows ENPP1-GFP degradation activity (left) and ENPP1 expression (right) of ENPP1WT-OE 4T1 and ENPP1T238A-OE 4T1 pooled clones as assessed by TLC and western blotting. [0016] Figure 1J shows proliferation of ENPP1WT-OE and ENPP1T238A-OE 4T1 pooled clonal cell lines compared with WT 4T1 cells over time (n = 3 biological replicates). [0017] Figure 1K shows ENPP1 expression in patients with stage 1-4 breast cancer. Shown as box plots of median and interquartile levels. P value was determined by the nonparametric Mann-Whitney U test. [0018] Figure 1L shows primary tumor volumes and quantification of lung metastases of WT BALB/cJ mice bearing ENPP1T238A-OE and ENPP1WT-OE 4T1 tumors (n = 5 and 9 mice). Tumor growth curves were plotted as mean ± SEM. Metastasis data were plotted as mean. P values were determined by unpaired t test with Welch correction. [0019] Figure 1M shows an experimental schematic of ex vivo culture of 4T1 metastasis by orthotopic injection. [0020] Figure 1N shows the number of live 4T1 cells (left) or mouse lung fibroblasts (right) 6 and 9 days after 0, 30, or 60 ^M 6-thioguanine (6-TG) treatment. Data were plotted as mean ± SD. P values were determined by unpaired t test. TLC stands for thin layer chromatography. [0021] Figure 1O shows UMAP plots of the annotated clusters of ENPP1T238A-OE and ENPP1WT- OE 4T1 primary tumors and metastasis colonized lungs. [0022] Figure 1P shows barplots comparing immune cell compositions (containing C08-C18) between ENPP1T238A-OE and ENPP1WT-OE 4T1 primary tumors and metastasis colonized lungs. *P ≤ 0.05.; P value is shown if it is between 0.05 - 0.15. [0023] Figure 1Q shows scRNA-seq analysis of 4T1 murine primary tumors and lung metastases. The bubble heatmap shows expression of selected marker genes for cluster annotation. Dot size indicates fraction of expressing cells, colored based on average expression levels. [0024] Figure 1R shows a UMAP plot of the annotated clusters of ENPP1T238A-OE and ENPP1WT-OE 4T1 primary tumors and metastasis colonized lungs. [0025] Figure 1S shows barplots comparing non-immune cell compositions (containing C01- C05) between ENPP1T238A-OE and ENPP1WT-OE 4T1 primary tumors and metastasis colonized lungs. [0026] Figures 2A-2C show that ENPP1 catalytic activity promotes immune suppression in primary tumors and lung metastases. Violin plots of indicated transcripts in indicated cell types comparing between ENPP1T238A-OE and ENPP1WT-OE 4T1 tumors or metastases. (A) Arg1 in macrophages in primary tumors and lung metastases. (B) Itgae and H2-Ab1 in cDC1s in primary tumors. (C) Cd69, Ilr2a, Pdcd1, and Tox in T cells in primary tumors and lung metastases. P values were determined by the nonparametric Mann-Whitney U test. cDC1 stands for conventional dendritic cell type 1. [0027] Figures 2D-2E show Violin plots of indicated transcripts of indicated cell types comparing between ENPP1T238A-OE and ENPP1WT-OE 4T1 tumors or metastases. (D) Arg1 in monocytes in primary tumors and lung metastases. (E) Itgae and H2-Ab1 in cDC1s in lung metastases (E). P values were determined by nonparametric Mann-Whitney U test. cDC1 stands for conventional dendritic cell type 1. [0028] Figure 2F shows the UMAP plot of the annotated subclusters of macrophages. [0029] Figure 2G shows a bubble heatmap showing expression of marker genes for macrophage subcluster annotation and functional markers indicating M1-like versus M2-like macrophage cell phenotypes. Dot size indicates fraction of expressing cells, colored based on average expression levels. [0030] Figure 2H shows barplots comparing macrophage subcluster compositions (containing Ma1-Ma4) between ENPP1T238A-OE and ENPP1WT-OE 4T1 primary tumors and metastasis colonized lungs. [0031] Figure 2I shows the UMAP plot of the annotated subclusters of T cells. [0032] Figure 2J shows a bubble heatmap showing expression of marker genes for T cell subcluster annotation. Dot size indicates fraction of expressing cells, colored based on average expression levels. [0033] Figure 2K shows barplots comparing T cell subcluster compositions (containing T01- T11) between ENPP1T238A-OE and ENPP1WT-OE 4T1 primary tumors and metastasis colonized lungs. P values were determined by unpaired t test. *P ≤ 0.05.; P value is shown if it is between 0.05 - 0.15. [0034] Figures 3A-3L shows ENPP1 expressed on cancer and responder cells blocks paracrine cGAMP-STING activation. P values were determined by nonparametric Mann-Whitney U test unless otherwise noted. [0035] Figure 3A shows bar graphs of Cgas and Sting1 expression across the annotated clusters. [0036] Figure 3B shows Violin plots of Lrrc8c, Lrrc8a, Lrrc8e and Ifitm2 across the annotated clusters. Schematic of cGAMP responder cells and their putative transporters: LRRC8A:C in endothelial cells, TAM and cDC; LRRC8A:E in fibroblasts. [0037] Figure 3C shows bar graphs of indicated transcripts in indicated cell types comparing between ENPP1T238A-OE and ENPP1WT-OE 4T1 tumors or metastases. Ifitms and Vegfc in endothelial cells in primary tumors. [0038] Figure 3D shows Ifitms in macrophages in primary tumors and metastases. [0039] Figure 3E shows Ifitms in myCAFs in primary tumors and metastases. [0040] Figure 3F shows Enpp1 in myCAFs in primary tumors and metastases. [0041] Figure 3G shows bar graphs of Enpp1 expression across the annotated clusters. [0042] Figure 3H shows bar graphs of Enpp1 and Ifitms across the annotated macrophage subclusters. P values were determined by ordinary one-way ANOVA test. [0043] Figure 3I shows differentially expressed genes in Enpp1-high vs. Enpp1-low groups. Bars represent mean ± SEM. TAM stands for tumor-associated macrophages. cDC stands for conventional dendritic cell, combining both cDC1 and cDC2. myCAF stands for myofibroblastic cancer-associated fibroblast. [0044] Figure 3J shows expression of additional genes in the STING pathway. Violin plots of Irf3, Tbk1, Infra1, Ifitm1, and Ifitm3 across the annotated clusters. [0045] Figure 3K shows bar graphs of indicated transcripts in indicated cell types comparing between ENPP1T238A-OE and ENPP1WT-OE 4T1 tumors or metastases. Ifitms and Vegfc in endothelial cells (C04) in lung metastases; Shown as mean ± SEM. P values were determined by nonparametric Mann-Whitney U test. [0046] Figure 3L shows bar graphs of indicated transcripts in indicated cell types comparing between ENPP1T238A-OE and ENPP1WT-OE 4T1 tumors or metastases. Lrrc8c in macrophages (C10) in primary tumors and lung metastases. Shown as mean ± SEM. P values were determined by nonparametric Mann-Whitney U test. [0047] Figure 3M shows the contribution of the eADO pathway and HP secretion in ENPP1 overexpression. Violin plots are shown of Entpd1, Nt5e, Adora2a and Adora2b across the annotated clusters. [0048] Figure 3N shows bar graphs of indicated transcripts in indicated cell types comparing between ENPP1T238A-OE and ENPP1WT-OE 4T1 tumors or metastases. Adora2b, Tgfb1, Il10rb, Hp in macrophages in primary tumors and lung metastases. [0049] Figure 3O shows Hp in cancer cells, Kif2c+ cancer cells, neutrophils, and monocytes in primary tumors and lung metastases. [0050] Figure 3P shows a proposed model of mechanism of action in ENPP1 overexpression. Bars represent mean ± SEM. P values were determined by nonparametric Mann-Whitney U test. HP stands for Haptoglobin. TAM stands for tumor-associated macrophages. cDC stands for conventional dendritic cell. [0051] Figure 3Q shows bar graphs of ENPP1 expression in normal breast tissue (n = 179), tumor adjacent tissue (n = 113), and primary tumor (n = 1092) in breast invasive carcinoma. Shown as box plots of median and interquartile levels. RNA sequencing data for normal tissue are from GTEx database, and for tumor adjacent tissue and primary tumor are from TCGA database, all queried using UCSC Xena portal. [0052] Figure 4A shows tissue- and cancer-derived ENPP1 contributions to tumor progression. Primary tumor volumes of WT or Enpp1-/- 4T1 BALB/cJ mice orthotopically injected with WT or Enpp1-/- 4T1 (n = 7, 17, 9, 16 mice for Enpp1 KO x KO, KO x WT, WT x KO, and WT x WT cancer x tissue genotype combinations). Data were plotted as mean ± SEM. P values of the last tumor measurement were determined by multiple unpaired t test with Welch correction. [0053] Figure 4B shows tissue- and cancer-derived ENPP1 contributions to tumor metastasis. Quantifications of lung metastatic colonies of WT or Enpp1-/- 4T1 BALB/cJ mice intravenously injected with WT or Enpp1-/- 4T1 (n = 4, 5, 3, 3 mice for Enpp1 KO x KO, KO x WT, WT x KO, and WT x WT cancer x tissue genotype combinations). Data were plotted as mean ± SD. P values were determined by unpaired t test. [0054] Figures 4C-4J show that the antitumoral and immunostimulatory effect of ENPP1 deficiency associates with extracellular cGAMP preservation. (C) schematic of experimental strategies of comparing between wildtype, Enpp1 knockout, and extracellular cGAMP depletion through genetic manipulation and cGAMP neutralization. Structures of dimer ENPP1 (PWD: 4B56), monomer ENPP1 (PWD: 6XKD), mSTING (PWD: 4KCO) and mSTING bound with DMXAA (PWD: 4LOL). [0055] Figure 4D shows primary tumor volumes of Enpp1 WT mice receiving Enpp1 WT 4T1 and R237A non-binding STING injection (WT + NB) (n = 3 biological replicates), Enpp1 KO mice receiving Enpp1 KO 4T1 and NB STING injection (KO + NB) (n = 4 biological replicates), and Enpp1 KO mice receiving Enpp1 KO 4T1 and WT neutralizing STING injection (KO + Neu) (n = 3 biological replicates). [0056] Figure 4E shows the percentage of Ly6G-Ly6Clow cells out of MHC-II+CD11b+CD11c- macrophages. Data were plotted as mean ± SD. P values were determined by unpaired t test with Welch correction. *P ≤ 0.05., **P ≤ 0.01; P value is shown if between 0.05 and 0.2; not significant (ns). [0057] Figure 4F shows the geometric mean of pIRF3 of F40/80+CD206- M1-like macrophages. [0058] Figure 4G shows the percentage of MHC-IIhiCD103+ cells out of CD11b-CD11c+ cells. [0059] Figure 4H shows the geometric mean of CD69 (H) of CD3+CD4-CD8+ cytotoxic T lymphocytes (CTLs). [0060] Figure 4I shows the geometric mean of CD25 (I) of CD3+CD4-CD8+ cytotoxic T lymphocytes (CTLs). [0061] Figure 4J shows the geometric mean of Foxp3 of CD45+CD3hiCD4+CD8- regulatory T cells (Tregs). [0062] Figure 4K shows relative cGAMP hydrolysis activity calculated from kinetic analysis by tumor lysates and sera from randomly selected mice reaching experimental endpoint (n = 2-6 biological replicates). Data represent mean ± SD. P values were determined by unpaired t test with Welch correction. [0063] Figure 4L shows images of metastatic colonies of WT or Enpp1-/- 4T1 BALB/cJ orthotopically injected with WT or Enpp1-/- 4T1 respectively. [0064] Figure 4M shows representative images of lung metastatic colonies and the percentage of mice with lung metastasis from Figure 4B. WT or Enpp1-/- 4T1 BALB/cJ mice were intravenously injected with WT or Enpp1-/- 4T1 (n = 4, 5, 3, 3 mice for Enpp1 KO x KO, KO x WT, WT x KO, and WT x WT cancer x tissue genotype combinations). [0065] Figures 5A-5G show selective disruption of ENPP1’s cGAMP hydrolysis activity delays breast cancer onset, growth, and metastasis. Data are shown as mean. P values comparing mice with lung metastasis % were determined by chi-squared test. P values comparing number of colonies were determined by nonparametric Mann-Whitney U test. P value for Kaplan-Meier curves were determined by log-rank Mantel-Cox test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. not significant (ns). (A) shows survival of WT, Enpp1H362A, Sting-/-, and Enpp1H362A x Sting1-/- C57BL/6J mice (n = 27, 32, 10, 15 mice) bearing orthotopic E0771 breast tumors. Survival was measured by time taken for orthotopic E0771 breast tumors to reach 1000m3. (B) shows Tumor- free survival of MMTV and Enpp1H362A x MMTV mice (n = 15 and 18 mice) that developed spontaneous breast tumors. Tumor-free survival was measured by onset of the first spontaneous breast tumors. (C) shows a schematic of the experiment. (D) shows representative images of WT, Enpp1H362A, Enpp1-/-. Sting1-/- and Enpp1H362A x Sting1-/- C57BL/6J mice (n = 10, 8, 9, 9, 9 mice) intravenously injected with E0771.lmb.PuroR cells. (E) shows quantification of lung metastatic colonies of WT, Enpp1H362A, Enpp1-/-. Sting1-/- and Enpp1H362A x Sting1-/- C57BL/6J mice (n = 10, 8, 9, 9, 9 mice) intravenously injected with E0771.lmb.PuroR cells. (F) shows individual tumor growth curves in WT and Enpp1H362A mice in (A). (G) shows individual tumor growth curves in Sting-/-, and Enpp1H362A x Sting1-/- in (A). [0066] Figures 6A-6Q shows that a common human ENPP1 variant, K173Q, exhibits increased cGAMP hydrolysis activity at the cell membrane. For A – Q. Bars represent mean SD. P values were determined by unpaired t test. *P 0.05; not significant (ns). PBMC stands for peripheral blood mononuclear cells. hENPP1 stands for human ENPP1. TM stands for transmembrane. SMB stands for somatomedin B-like. (A) shows domain structure of hENPP1 and allele frequency of the K173Q variant reported by gnomAD (website: gnomad.broadinstitute.org/). (B and C) show the relative cGAMP hydrolysis activity calculated from kinetic analysis of cGAMP degradation by purified PBMCs (2.5 x 106) from donors with reference A/A sequence (Lys), heterozygous variant sequence A/C, and homozygous variant sequence C/C (Gln) at 517 nucleotide (n = 5 donors per genotype, averaged across 3 technical replicates). (D) shows the relative cGAMP hydrolysis activity calculated from kinetic analysis of cGAMP degradation by plasma from donors with A/A, A/C, and C/C sequence at 517 nucleotide (n = 4-5 per genotype, averaged across 3 technical replicates). (E) shows the relative cGAMP hydrolysis activity normalized by ENPP1 protein expression calculated from kinetic analysis of cGAMP degradation by lysates of 293T cGAS ENPP1-/- cells overexpressing WT or K173Q hENPP1 proteins (n = 8-9 biological replicates). (F) shows the relative cGAMP hydrolysis activity normalized by ENPP1 protein expression calculated from kinetic analysis of cGAMP degradation by supernatants of 293T cGAS ENPP1-/- cells overexpressing WT or K173Q hENPP1 proteins (n = 4 biological replicates). (G) shows the percentage of dimer hENPP1 in lysates of 293T cGAS ENPP1-/- cells overexpressing WT or K173Q hENPP1 proteins assessed by non-reducing western blot. (H) shows the relative ATP hydrolysis activity normalized by ENPP1 protein expression calculated from kinetic analysis of ATP degradation by lysates of 293T cGAS ENPP1-/- cells overexpressing WT or K173Q hENPP1 proteins (n = 3 biological replicates). (I) shows the proposed mechanism of how the K173Q could affect ENPP1’s cGAMP and ATP hydrolysis activity. Structure of hENPP1 dimers (PDB:6weu). The K173 residue (red) is in the SMB domains (yellow) proximal to cell membrane and away from the catalytic domain (light purple). The catalytic pocket (dark purple) is determined by side chains of residues within 5 Å of Zn2+ (violet). K173 could interact with other transmembrane proteins and/or the cell membrane to impact activity. (J) shows the representative images of DNA sequences of donors with the A/A reference sequence (Lys) or the variant C/C sequence (Gln) at residue 173. (K) shows the ENPP1 RNA expression in PBMCs from donors with A/A, A/C, and C/C at 517 nucleotide assessed by qRT-PCR (n = 4-5 donors per genotype, averaged across 3 technical replicates.) (L) shows the relative cGAMP hydrolysis activity calculated from kinetic analysis of cGAMP degradation by purified PBMCs (2.5 x 106) from individual donors (n = 3 technical replicates). (M) shows the schematic of transfection of WT or mutant ENPP1 expressing plamids into 293T cGAS ENPP1-/-, followed by isolation of memENPP1 in lysate and secENPP1 in supernatant. (N) shows the representative images of hENPP1-FLAG expression assessed by western blot and cGAMP degradation assessed by TLC (left) and quantification of the relative ENPP1 to Tubulin protein expression (right) of lysates of 293T cGAS ENPP1-/- cells overexpressing WT or K173Q hENPP1 proteins (n = 8-9 biological replicates). (O) shows the representative blots of hENPP1-FLAG expression in supernatant or lysate (left) and quantification of secENPP1 to memENPP1 protein expression (right) of 293T cGAS ENPP1-/- cells overexpressing WT or K173Q hENPP1 proteins (n = 7 biological replicates). (P) shows the dimer and monomer hENPP1-FLAG expression assessed by non-reducing western blot of lysates of 293T cGAS ENPP1-/- cells overexpressing WT or K173Q hENPP1 proteins (n = 3 biological replicates). (Q) shows the amount of cGAMP degraded in 3 hours by memENPP1 versus secENPP1 of cell lines assessed by TLC. Red dots represent cancer cell lines. Data are fit with simple linear regression with R2 shown. [0067] Figures 7A-7M shows that transmembrane and secreted ENPP1 isoforms contribute to primary tumor growth and metastasis. (A) shows the domain structure of memENPP1 and secENPP1. (B) shows the schematic of levels of transmembrane versus secreted fraction of WT, A84S, and T238A ENPP1. Structure of mouse ENPP1 monomer (PDB:6XKD) and dimer (PDB: 4B56). (C) shows the Ex vivo relative cGAMP hydrolysis activity calculated from kinetic analysis by tumor lysates (left) and sera (right) at physiological condition (pH 7.4, 37oC). ENPP1WT-OE (n = 5 mice), ENPP1A84S-OE (n = 10 mice), or ENPP1T238A-OE (n = 4 mice) 4T1 cells (2.5 x 104) were orthotopically injected into WT BALB/cJ mice, and tumors and sera were collected in mice reaching experimental endpoint (n = 3-8 biological replicates). Data are represented as mean +/- SD. P values were determined by unpaired t test with Welch correction. (D) shows tumor volumes in experiment C plotted as mean +/- SEM with some error bars too small to visualize. P values were determined by multiple unpaired t test with Welch correction. (E) shows quantification of lung metastasis in experiment C. Tumor growth curves were plotted as mean ± SEM. Metastasis data were plotted as mean. P values were determined by unpaired t test with Welch correction. (F) shows proposed model of transmembrane and secreted ENPP1 action. (G) shows the secretory signal peptide prediction of ENPP1 using signalP 6.0. (H) shows the representative blots (bottom) and quantification (top) of mem- and secENPP1-FLAG expression in 293T cGAS ENPP1-/- cells overexpressing WT and A84S residue mutant ENPP1 proteins (n = 2 biological replicates). (I) shows the representative blots (bottom) and quantification (top) of mem- and secENPP1-FLAG expression in 293T cGAS ENPP1-/- cells overexpressing WT and membrane proximal domain (MPD) mutant ENPP1 proteins (n = 2 biological replicates). (J and K) show the representative images (J) and quantification (K) of cGAMP degradation in 4 hours by lysate and supernatant of ENPP1WT-OE, ENPP1A84S-OE, and ENPP1T238A-OE cells. (L) shows a repeat experiment of (C). (M) shows the primary tumor sizes at the time of experimental endpoints in experiment (C). [0068] Figures 8A-8V shows ENPP1 drives distinct long-term immuno-suppressive programs in primary and metastatic niche. [0069] Figures 9A-9K shows that ENPP1 expression is inducible in stromal cells. [0070] Figure 10A shows ENPP1 expression in Immune+ vs. immune- patients across 10 treatment arms in the I-SPY2 breast cancer clinical trial. P value for Kaplan-Meier curves were determined by log-rank Mantel-Cox test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. not significant (ns). Immune+ stands for immune-positive; immune- stands for immune-negative; pCR stands for pathological complete response. [0071] Figure 10B shows ENPP1 expression in pCR vs. no pCR patients across 10 treatment arms in the I-SPY2 breast cancer clinical trial. [0072] Figure 10C shows that all of ENPP1-low patients (ENPP1 < 8.34) remained free of distant metastasis nearly seven years after Paclitaxel + Pembrolizumab neoadjuvant therapy followed by surgical resection, whereas 12% ENPP1-hig patients (ENPP1 > 8.34) developed distant metastasis within 5 years after Paclitaxel + Pembrolizumab neoadjuvant therapy followed by surgical resection. [0073] Figure 10D shows a proposed model of mechanism of action in ENPP1 inhibition. [0074] Figure 10E shows pre-treatment ENPP1 mRNA expression in pCR vs. no pCR patients in Paclitaxel + Pembrolizumab treatment arm in the I-SPY2 breast cancer clinical trial. The ENPP1 mRNA threshold of 6.39 categorizes patients who likely have pCR (ENPP1 < 6.39) versus who have a chance of not having pCR (ENPP1 > 6.39) following Paclitaxel + Pembrolizumab neoadjuvant therapy. [0075] Figure 10F shows pre-treatment ENPP1 mRNA expression in pCR vs. no pCR patients in Paclitaxel + Veliparib + Carboplatin treatment arm in the I-SPY2 breast cancer clinical trial. The ENPP1 mRNA threshold of 6.78 categorizes patients who likely have pCR (ENPP1 < 6.78) versus who have a chance of not having pCR (ENPP1 > 6.78) following Paclitaxel + Veliparib + Carboplatin neoadjuvant therapy. DETAILED DESCRIPTION OF THE DISCLOSURE [0076] The present disclosure relates generally to detection and regulation of ENPP1 for the purposes of the treatment and prevention of metastatic cancer as well as the diagnosis of cancer in a subject. Without being bound by theory, provided herein is evidence that ENPP1 promotes breast cancer progression and metastasis in a STING-dependent manner by dampening the innate-adaptive immune crosstalk in the tumor microenvironment (TME). Both tumor- and tissue-derived ENPP1 anchored on the cell surface can be important for its cancer-promoting activity, while soluble ENPP1 released into the serum can play a minor role. In some embodiments, tumors can induce expression of ENPP1 in the TME. A potential solid tumor biopsy diagnostic is to assay for increased expression of membrane bound ENPP1 on tumor cells and/or in the surrounding TME (as compared to a reference, normal subject), such as with immunohistocytochemistry. Furthermore, although soluble ENPP1 might play a minor functional role, increased levels of soluble ENPP1 in the serum (over basal levels of soluble ENPP1 from the serum of normal subjects) can be used as a diagnostic or prognostic for tumor onset and growth, primary progression, and/or metastasis. The increased levels of soluble ENPP1 in the serum can be stratified and correlated with tumor state and prognosis. As described herein, results show that the cGAMP hydrolysis activity of ENPP1 is required for its tumorigenic effect, and a common human genetic variant of ENPP1 (K173Q) with increased catalytic activity is identified. Accordingly, in some aspects, ENPP1 is an innate immune checkpoint restraining STING pathway activation in the TME, and that therapeutically targeting ENPP1 catalytic activity could boost anti-tumor immunity in ways that could synergize with adaptive ICB therapy. [0077] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. [0078] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment. DEFINITIONS [0079] Unless otherwise defined, all terms of art, notations, and other scientific terms or terminology used herein are intended to have the meanings commonly understood by those of skill in the art to which this application pertains. In some cases, terms with commonly understood meanings are defined herein for clarity and/or for ready reference, and the inclusion of such definitions herein should not necessarily be construed to represent a substantial difference over what is generally understood in the art. Many of the techniques and procedures described or referenced herein are well understood and commonly employed using conventional methodology by those skilled in the art. [0080] The singular form “a”, “an”, and “the” include plural references unless the context clearly dictates otherwise. For example, the term “a cell” includes one or more cells, comprising mixtures thereof. “A and/or B” is used herein to include all of the following alternatives: “A”, “B”, “A or B”, and “A and B”. [0081] The terms “administration” and “administering”, as used herein, refer to the delivery of a bioactive composition or formulation by an administration route comprising, but not limited to, intranasal, transdermal, intravenous, intra-arterial, intramuscular, intranodal, intraperitoneal, subcutaneous, intramuscular, oral, intravaginal, and topical administration, or combinations thereof. The term includes, but is not limited to, administering by a medical professional and self-administering. [0082] The term “effective amount”, “therapeutically effective amount”, or “pharmaceutically effective amount” of a composition of the disclosure, e.g., nucleic acid constructs, srRNAs, recombinant cells, and/or pharmaceutical compositions, generally refers to an amount sufficient for the composition to accomplish a stated purpose relative to the absence of the composition (e.g., achieve the effect for which it is administered, stimulate an immune response, prevent or treat a disease, or reduce one or more symptoms of a disease, disorder, infection, or health condition). An example of an “effective amount” is an amount sufficient to contribute to the treatment, prevention, or reduction of a symptom or symptoms of a disease, which could also be referred to as a “therapeutically effective amount.” A “reduction” of a symptom means decreasing of the severity or frequency of the symptom(s), or elimination of the symptom(s). The exact amount of a composition including a “therapeutically effective amount” will depend on the purpose of the treatment, and will be ascertainable by one skilled in the art using known techniques (see, e.g., Lieberman, Pharmaceutical Dosage Forms (vols.1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); Pickar, Dosage Calculations (1999); and Remington: The Science and Practice of Pharmacy, 20th Edition, 2003, Gennaro, Ed., Lippincott, Williams & Wilkins). [0083] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the disclosure. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure. [0084] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number. If the degree of approximation is not otherwise clear from the context, “about” means either within plus or minus 10% of the provided value, or rounded to the nearest significant figure, in all cases inclusive of the provided value. In some embodiments, the term “about” indicates the designated value ± up to 10%, up to ± 5%, or up to ± 1%. [0085] The term “pharmaceutically acceptable excipient” as used herein refers to any suitable substance that provides a pharmaceutically acceptable carrier, additive, or diluent for administration of a compound(s) of interest to a subject. As such, “pharmaceutically acceptable excipient” can encompass substances referred to as pharmaceutically acceptable diluents, pharmaceutically acceptable additives, and pharmaceutically acceptable carriers. As used herein, the term “pharmaceutically acceptable carrier” includes, but is not limited to, saline, solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds (e.g., antibiotics and additional therapeutic agents) can also be incorporated into the compositions. [0086] As used herein, a “subject” or an “individual” includes animals, such as human (e.g., human individuals) and non-human animals. In some embodiments, a “subject” or “individual” is a patient under the care of a physician. Thus, the subject can be a human patient or an individual who has, is at risk of having, or is suspected of having a health condition of interest (e.g., rabies infection) and/or one or more symptoms of the health condition. The subject can also be an individual who is diagnosed with a risk of the health condition of interest at the time of diagnosis or later. The term “non-human animals” includes all vertebrates, e.g., mammals, e.g., rodents, e.g., mice, non-human primates, and other mammals, such as e.g., sheep, dogs, cows, chickens, and non-mammals, such as amphibians, reptiles, etc. [0087] A solid tumor sample can be obtained by any means available to those skilled in the art. In certain embodiments, the solid tumor sample is obtained from a biopsy, such as a needle biopsy, for example, a core needle biopsy (CNB). In certain embodiments, the solid tumor sample is obtained from a percutaneous core needle biopsy. In other embodiments, the solid tumor sample is obtained from a surgical biopsy. Core biopsy can be done guided by imaging or palpation (freehand). Routinely, stereotactic biopsy (needle biopsy guided by mammography done in 2 planes and analyzed by computer to produce a 3-dimensional image) or ultrasound- guided biopsy can be used to improve accuracy. Clips are placed at the biopsy site to identify it. If core biopsy is not possible (eg, the lesion is too posterior), surgical biopsy can be done; a guidewire is inserted, using imaging for guidance, to help identify the biopsy site. In other embodiments, the solid tumor sample is obtained through surgery, such as surgical resection or surgical removal of the solid tumor sample. The solid tumor can be classified or predicted to be invasive recurrent or indolent based on analysis of the features identified herein. The determination of the aggressiveness phenotype of the solid tumor sample can be used to develop a treatment plan for the subject with the solid tumor and to treat the patient accordingly. [0088] As used herein, “pathologic complete response” or pCR, in certain embodiments is defined as a reduction of at least 80%, or of at least 85%, or of at least 90%, or of at least 95%, or of at least 98%, or of at least 99%, or of at least 99.5% of invasive cancer in the breast after completion of a therapeutic regimen. In certain embodiments, pCR is defined as the absence of residual invasive cancer on hematoxylin and eosin evaluation of the complete resected breast specimen and all sampled regional lymph nodes following completion of neoadjuvant systemic therapy (i.e. ypT0/Tis ypN0 in the current AJCC staging system). In other embodiments, pCR is defined as the absence of residual invasive cancer and in situ cancer on hematoxylin and eosin evaluation of the complete resected breast specimen and all sampled regional lymph nodes following completion of neoadjuvant systemic therapy (i.e. ypT0 ypN0 in the current AJCC staging system.) [0089] It is understood that aspects and embodiments of the disclosure described herein include "comprising", "consisting", and "consisting essentially of" aspects and embodiments. As used herein, "comprising" is synonymous with "including", "containing", or "characterized by", and is inclusive or open-ended and does not exclude additional, unrecited elements or method steps. As used herein, "consisting of" excludes any elements, steps, or ingredients not specified in the claimed composition or method. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claimed composition or method. Any recitation herein of the term "comprising", particularly in a description of components of a composition or in a description of steps of a method, is understood to encompass those compositions and methods consisting essentially of and consisting of the recited components or steps. [0090] All genes, gene names, and gene products disclosed herein are intended to correspond to homologs from any species for which the compositions and methods disclosed herein are applicable. Thus, the terms include, but are not limited to genes and gene products from humans and mice. It is understood that when a gene or gene product from a particular species is disclosed, this disclosure is intended to be exemplary only, and is not to be interpreted as a limitation unless the context in which it appears clearly indicates. Thus, for example, for the genes or gene products disclosed herein, which in some embodiments relate to mammalian nucleic acid and amino acid sequences, are intended to encompass homologous and/or orthologous genes and gene products from other animals including, but not limited to other mammals, fish, amphibians, reptiles, and birds. In some embodiments, the genes, nucleic acid sequences, amino acid sequences, peptides, polypeptides and proteins are human. The term “gene” is also intended to include variants thereof. [0091] It is appreciated that certain features of the disclosure, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the disclosure, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub- combination. All combinations of the embodiments pertaining to the disclosure are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations of the various embodiments and elements thereof are also specifically embraced by the present disclosure and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein. METHODS OF TREATING METASTASIS [0092] Aspects of the disclosure include methods that involve administering to a subject with cancer a therapeutically effective amount of an ENPP1 inhibitor to prevent or treat the subject for metastasis. In some embodiments, the subject is one who is diagnosed with or suspected of having cancer. In some embodiments, the subject is one who is diagnosed with metastatic cancer. In some embodiments, the methods of treatment are not directed to non-metastatic cancer, for example the cancers described in WO2019051269. [0093] As used herein, metastasis includes the process by which a cancer spreads or transfers from the site of origin to other regions of the body with the development of a similar cancerous lesion at the new location. A “metastatic” or “metastasizing” cell is one that loses adhesive contacts with neighboring cells and migrates via the bloodstream or lymph from the primary site of disease to invade neighboring body structures. [0094] Any convenient ENPP1 inhibitors can be used in the subject methods of treating cancer. Exemplary ENPP1 inhibitors include, without limitation, those described in WO2019051269. In some embodiments, the ENPP1 inhibitor is compound 1 from WO2019051269. In some embodiments, the ENPP1 inhibitor is STF-1623 (Carozza, Brown, et al., 2020). [0095] In certain embodiments, the ENPP1 inhibitor is a member selected from the group consisting of:
Figure imgf000020_0001
Figure imgf000021_0001
Figure imgf000022_0001
[0096] In certain embodiments, the ENPP1 inhibitor is a member selected from the following table: Table A
Figure imgf000022_0002
Figure imgf000023_0001
Figure imgf000024_0001
Figure imgf000025_0001
Figure imgf000026_0001
[0097] In certain embodiments, the ENPP1 inhibitor is a member selected from the following table B:
Figure imgf000026_0002
Figure imgf000027_0001
Figure imgf000027_0002
[0098] In certain embodiments, the ENPP1 inhibitor is described in WO2020/160333. In certain embodiments, the ENPP1 inhibitor is described in Table 1 in WO2020/160333. In certain embodiments, the ENPP1 inhibitor is described in Table 2 in WO2020/160333. In certain embodiments, the ENPP1 inhibitor is described in Table 3 in WO2020/160333. In certain embodiments, the ENPP1 inhibitor is described in Table 3a in WO2020/160333. [0099] In certain embodiments, the ENPP1 inhibitor is described in WO2022/125614, WO2022/125613, WO2022/119928, WO2020/190912, WO2020/028724, WO2021/158829, or WO2019/023635. In certain embodiments, the ENPP1 inhibitor is described in WO2022/212488, WO2022/197734, WO2020/210649, or WO2020/140001. [0100] In some embodiments, the subject is mammalian. In some embodiments, the subject is human. Other subjects can include domestic pets (e.g., dogs and cats), livestock (e.g., cows, pigs, goats, horses, and the like), rodents (e.g., mice, guinea pigs, and rats, e.g., as in animal models of disease), as well as non-human primates (e.g., chimpanzees, and monkeys). The subject may be in need of treatment for cancer. In some instances, the subject methods include diagnosing cancer, including any one of the cancers described herein. In some embodiments, the compound is administered as a pharmaceutical preparation. [0101] The ENPP1 inhibitors are administered to a subject, e.g., a subject having, suspected of having, or at risk of developing cancer selected from, but not limited to, a pancreatic cancer, an endometrial cancer, a non-small cell lung cancer (NSCLC), a renal cell carcinoma ((RCC), e.g. clear cell RCC, non-clear cell RCC), a urothelial cancer, a head and neck cancer (e.g. head and neck squamous cell cancer), a melanoma (e.g., advanced melanoma such as Stage III-IV high- risk melanoma, unresectable or metastatic melanoma), a bladder cancer, a hepatocellular carcinoma, a breast cancer (e.g., triple negative breast cancer, ER+/HER2− breast cancer), an ovarian cancer, a gastric cancer (e.g. metastatic gastric cancer or gastroesophageal junction adenocarcinoma), a colorectal cancer, a glioblastoma, a biliary tract cancer, a glioma (e.g., recurrent malignant glioma with a hypermutator phenotype), Merkel cell carcinoma (e.g., advanced or metastatic Merkel cell cancer), Hodgkin lymphoma, non-Hodgkin lymphoma (e.g. primary mediastinal B-cell lymphoma (PMBCL)), a cervical cancer, an advanced or refractory solid tumor, a small cell lung cancer (e.g., stage IV non-small cell lung cancer), a non-squamous non-small cell lung cancer, desmoplastic melanoma, a pediatric advanced solid tumor or lymphoma, a mesothelin-positive pleural mesothelioma, an esophageal cancer, an anal cancer, a salivary cancer, a prostate cancer, a carcinoid tumor, a primitive neuroectodermal tumor (pNET), and a thyroid cancer. [0102] In some embodiments, the ENPP1 inhibitor is administered to the tumor tissue microenvironment (TME). As described in the Examples herein, tissue ENPP1 expression plays a dominant role in promoting primary breast tumor growth and metastasis. [0103] In some embodiments, a "therapeutically effective amount" is an amount of a subject inhibitor that, when administered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to decrease metastatic tumor burden in the subject by about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%, compared to metastatic tumor burden in the individual in the absence of treatment with the inhibitor, or alternatively, compared to the metastatic tumor burden in the subject before or after treatment with the inhibitor. As used herein the term “metastatic tumor burden" refers to the total mass of metastatic tumor tissue carried by a subject with cancer. [0104] In some embodiments, a "therapeutically effective amount" is an amount of a subject inhibitor that, when administered to an individual in one or more doses, in monotherapy or in combination therapy, is effective to prevent metastasis of a primary tumor in a subject. [0105] For the treatment of cancer, the ENPP1 inhibitor can be administered in combination with a chemotherapeutic agent selected from the group consisting of alkylating agents, nitrosoureas, antimetabolites, antitumor antibiotics, plant (vinca) alkaloids, steroid hormones, taxanes, nucleoside analogs, steroids, anthracyclines, thyroid hormone replacement drugs, thymidylate- targeted drugs, Chimeric Antigen Receptor T cell therapies, Chimeric Antigen Receptor/NK cell therapies, apoptosis regulator inhibitors (e.g., B cell CLL/lymphoma 2 (BCL-2) BCL-2-like 1 (BCL- XL) inhibitors), CARP-1/CCAR1 (Cell division cycle and apoptosis regulator 1) inhibitors, colony- stimulating factor-1 receptor (CSF1R) inhibitors, CD47 inhibitors, cancer vaccine (e.g., a Thl7- inducing dendritic cell vaccine, or a genetically modified tyrosinase such as Oncept®) and other cell therapies. [0106] Specific chemotherapeutic agents of interest include, but are not limited to, Gemcitabine, Docetaxel, Bleomycin, Erlotinib, Gefitinib, Lapatinib, Imatinib, Dasatinib, Nilotinib, Bosutinib, Crizotinib, Ceritinib, Trametinib, Bevacizumab, Sunitinib, Sorafenib, Trastuzumab, Ado- trastuzumab emtansine, Rituximab, Ipilimumab, Rapamycin, Temsirolimus, Everolimus, Methotrexate, Doxorubicin, Abraxane, Folfirinox, Cisplatin, Carboplatin, 5-fluoro uracil, Teysumo, Paclitaxel, Prednisone, Levothyroxine, Pemetrexed, navitoclax, and ABT-199. Peptidic compounds can also be used. Cancer chemotherapeutic agents of interest include, but are not limited to, dolastatin and active analogs and derivatives thereof; and auristatin and active analogs and derivatives thereof (e.g., Monomethyl auristatin D (MMAD), monomethyl auristatin E (MMAE), monomethyl auristatin F (MMAF), and the like). See, e.g., WO 96/33212, WO 96/14856, and U.S.6,323,315. Suitable cancer chemotherapeutic agents also include maytansinoids and active analogs and derivatives thereof (see, e.g., EP 1391213; and Liu et al (1996) Proc. Natl. Acad. Sci. USA 93:8618-8623); duocarmycins and active analogs and derivatives thereof (e.g., including the synthetic analogues, KW-2189 and CB 1- TM1); and benzodiazepines and active analogs and derivatives thereof (e.g., pyrrolobenzodiazepine (PBD). [0107] The ENPP1 inhibitors can be administered to a subject alone or in combination with an additional, i.e., second, active agent. Combination therapeutic methods where the ENPP1 inhibitors may be used in combination with a second active agent or an additional therapy, e.g., radiation therapy. The terms "agent," "compound," and "drug" are used interchangeably herein. For example, ENPP1 inhibitors can be administered alone or in conjunction with one or more other drugs, such as drugs employed in the treatment of diseases of interest, including but not limited to, immunomodulatory diseases and conditions and cancer. In some embodiments, the subject method further includes coadministering concomitantly or in sequence a second agent, e.g., a small molecule, a chemotherapeutic, an antibody, an antibody fragment, an antibody-drug conjugate, an aptamer, a protein, or a checkpoint inhibitor. In some embodiments, the method further includes performing radiation therapy on the subject. [0108] The terms "co-administration" and "in combination with" include the administration of two or more therapeutic agents either simultaneously, concurrently or sequentially within no specific time limits. In one embodiment, the agents are present in the cell or in the subject's body at the same time or exert their biological or therapeutic effect at the same time. In one embodiment, the therapeutic agents are in the same composition or unit dosage form. In other embodiments, the therapeutic agents are in separate compositions or unit dosage forms. In certain embodiments, a first agent can be administered prior to (e.g., minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapeutic agent. [0109] "Concomitant administration" of a known therapeutic drug or additional therapy with a pharmaceutical composition of the present disclosure means administration of the compound and second agent or additional therapy at such time that both the known drug and the composition of the present invention will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug with respect to the administration of a subject compound. Routes of administration of the two agents may vary, where representative routes of administration are described in greater detail below. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs or therapies and compounds of the present disclosure. [0110] In some embodiments, the compounds (e.g., a subject compound and the at least one additional compound or therapy) are administered to the subject within twenty-four hours of each other, such as within 12 hours of each other, within 6 hours of each other, within 3 hours of each other, or within 1 hour of each other. In certain embodiments, the compounds are administered within 1 hour of each other. In certain embodiments, the compounds are administered substantially simultaneously. By administered substantially simultaneously is meant that the compounds are administered to the subject within about 10 minutes or less of each other, such as 5 minutes or less, or 1 minute or less of each other. [0111] In some embodiments, the ENPP1 inhibitor is combined with PD1 inhibitor and PARP inhibitor regimens. As shown in the Examples, low ENPP1 expression correlates with higher tumor immune infiltration and response to PD1 inhibitor and PARP inhibitor regimens, but not other treatments. Accordingly, for those subjects with positive ENPP1 status, addition of anti- ENPP1 to existing PD1 inhibitor and PARP inhibitor regimens can increase individuals’ chance of completely responding to the therapies. [0112] In an exemplary embodiment, the PD1 inhibitor regimen involves administering 200 mg of a PD1 inhibitor described herein (such as pembrolizumab) every 3 weeks, or 400 mg every 6 weeks as an intravenous infusion over 30 minutes. In an exemplary embodiment, the anti-PD1 regimen involves administering 500 mg every 3 weeks for 4 doses followed by 1,000 mg every 6 weeks as an intravenous infusion over 30 minutes. [0113] In an exemplary embodiment, the PARP inhibitor regimen involves 1 mg of a PARP inhibitor described herein (such as talazoparib) taken as a single oral daily dose, with or without food. In an exemplary embodiment, the PARP inhibitor regimen involves 300 mg of a PARP inhibitor described herein (such as olaparib) taken orally twice daily with or without food [0114] The ENPP1 inhibitor and second chemotherapeutic agent, as well as any additional therapeutic agents for combination therapies, can be administered orally, subcutaneously, intramuscularly, parenterally, or other route. The ENPP1 inhibitor and second chemotherapeutic agent may be administered by the same route of administration or by different routes of administration. The therapeutic agents can be administered by any suitable means including, but not limited to, for example, oral, rectal, nasal, topical (including transdermal, aerosol, buccal and sublingual), vaginal, parenteral (including subcutaneous, intramuscular, intravenous and intradermal), intravesical or injection into an affected organ. [0115] The ENPP1 inhibitor may be administered in a unit dosage form and may be prepared by any methods well known in the art. Such methods include combining the subject compound with a pharmaceutically acceptable carrier or diluent which constitutes one or more accessory ingredients. A pharmaceutically acceptable carrier is selected on the basis of the chosen route of administration and standard pharmaceutical practice. Each carrier must be "pharmaceutically acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the subject. This carrier can be a solid or liquid and the type is generally chosen based on the type of administration being used. [0116] Examples of suitable solid carriers include lactose, sucrose, gelatin, agar and bulk powders. Examples of suitable liquid carriers include water, pharmaceutically acceptable fats and oils, alcohols or other organic solvents, including esters, emulsions, syrups or elixirs, suspensions, solutions and/or suspensions, and solution and or suspensions reconstituted from non-effervescent granules and effervescent preparations reconstituted from effervescent granules. Such liquid carriers may contain, for example, suitable solvents, preservatives, emulsifying agents, suspending agents, diluents, sweeteners, thickeners, and melting agents. Preferred carriers are edible oils, for example, corn or canola oils. Polyethylene glycols, e.g. PEG, are also good carriers. [0117] Any drug delivery device or system that provides for the dosing regimen of the instant disclosure can be used. A wide variety of delivery devices and systems are known to those skilled in the art. METHODS OF DIAGNOSING CANCER [0118] As described in the Examples provided herein, subjects with heterozygous or homozygous lysine to glutamine mutation at position 173 (K173Q) exhibit elevated ENPP1 activity in PBMC lysates, with homozygous individuals displaying higher levels than heterozygous individuals. Accordingly, provided herein are methods of diagnosing a subject with a K173Q mutation in ENPP1 as having cancer or being at risk of developing cancer. [0119] In some embodiments, the method includes obtaining a biopsy from a subject. A biopsy can include a biological sample obtained from a subject. A biopsy may be used for analysis (e.g., diagnosis) to determine the presence or status of disease (e.g., type of disease, severity of disease, or cause of disease). A biopsy may be used to direct disease treatment or provide a prognosis. [0120] In some embodiments, a liquid biopsy is obtained from a subject. A liquid biopsy is a simple and non-invasive alternative to surgical biopsies which enables doctors to discover a range of information about a tissue (e.g., a tumor) through a simple blood sample. Liquid biopsy is a type of technique for sampling and analyzing of non-solid biological tissues, mainly used in disease diagnosis. In certain embodiments, liquid biopsies provide an opportunity for detecting, analyzing and monitoring cancer in various body effluents such as blood or urine instead of a fragment of cancer tissue. It is composed of different biological matrices such as circulating tumor cells (CTCs), cell free nucleic acids (e.g., cfDNA), exosomes or tumors. In addition to representing a non- or minimally invasive procedure, liquid biopsy may represent a better view of tumor heterogeneity and allows for real-time monitoring of cancer evolution and treatment. Several liquid biopsy approaches are known in the art, such as those described in the following: Yadav et al., “Detection of circulating tumour cells in colorectal cancer: Emerging techniques and clinical implications,” World J Clin Oncol. 2021 Dec 24; 12(12): 1169- 118; Bunduc et al., “Exosomes as prognostic biomarkers in pancreatic ductal adenocarcinoma a systematic review and meta-analysis,” Transl Res. 2022 Jan 20:S1931- 5244; Takami H, et al., “Advances in Molecular Profiling and Developing Clinical Trials of CNS Germ Cell Tumors: Present and Future Directions,” Curr Oncol Rep.2022 Jan 20; and Li et al., “Liquid biopsy in lung cancer: significance in diagnostics, prediction, and treatment monitoring,” Mol Cancer.2022 Jan 20;21(l):25; Underwood et al., “Liquid biopsy for cancer: review and implications for the radiologist,” Radiology, Nov 19, 2019. [0121] As described herein, a liquid biopsy obtained from a subject with cancer (e.g., breast cancer) may identify the subject as having heterozygous or homozygous lysine to glutamine mutation at position 173 (K173Q) of ENPP1. [0122] In some embodiments, the biopsy sample is assayed using methods including, but not limited to, sequencing (e.g., next-generation sequencing methods), real-time PCR, digital droplet PCR, and other known PCR and sequencing methods of analyzing DNA. [0123] In some embodiments, the next generation sequencing method comprises a method selected from the group consisting of Ion Torrent, Illumina, SOLiD, 454; Massively Parallel Signature Sequencing, solid phase reversible dye terminator sequencing; and DNA nanoball sequencing. As used herein, “next generation sequencing” refers to the speeds that were not possible with conventional sequencing methods (e.g., Sanger sequencing) by reading thousands of millions of sequencing reactions simultaneously. Next generation sequencing techniques and sequencing primer designs are well known in the art (e.g., Shendure, et al., “Next-generation DNA sequencing,” Nature, 2008, vol.26, No.10, 1135-1145; Mardis, “The impact of next- generation sequencing technology on genetics, “Trends in Genetics, 2007, vol.24, No.3, pp 133-141; Su, et al., “Next generation sequencing and its applications in molecular diagnostics,” Expert Rev Mol Diagn, 2011, 11 (3): 333-43; Zhang et al., “The impact of next- generation sequencing on genomics,” J Genet Genomics, 2011, 38 (3): 95-109; (Nyren, P. et al. Anal Biochem 208: 17175 (1993): Bentley, DR Curr Opin Genet Dev 16: 545-52 (2006); Strausberg, RL, et al. Drug Disc Today 13: 569-77 (2008); U.S. Patent No.7,282,337; U.S. Patent No. 7,279,563; U.S. Patent No.7,226,720; No.7,220,549; No. U.S. Patent No. 7,169,560; see and No.20070070349); U.S. Pat. No. 6,818,395; U.S. Pat. No. 6,911,345; U.S. Patent Application Publication No.2006/0252077; No. 2007/0070349. The entire contents of these general references on next-generation sequencing are incorporated herein by reference. [0124] In some embodiments, sequencing the sample further includes identifying the presence of mutations in ENPP1 which are indicative of cancer or the risk of developing cancer. In some embodiments, the subject is identified as having a cancer or being at risk for developing cancer when sequencing results in the detection of a K173Q mutation. [0125] In some embodiments, a tissue biopsy is obtained from a subject. The term "tissue biopsy" as used herein refers to a tissue sample obtained from a subject (e.g., breast tissue). The presence of K173Q mutation in ENPP1 which is indicative of cancer or the risk of developing cancer can be identified with the use of one or more antibodies specific for the K173Q mutation in a tissue sample. Detection methodologies suitable for use include, but are not limited to, immunohistochemistry of cells in a tumor sample, enzyme linked immunosorbent assays (ELISAs) including antibody sandwich assays of cells in a tumor sample, mass spectroscopy, immuno-PCR, FACS, and protein microarrays. METHODS OF PROGNOSING CANCER [0126] As described herein, in breast cancer patients, low ENPP1 expression correlates with higher tumor immune infiltration and response to anti-PD1 and PARP inhibitors, but not other treatments. Thus, in some embodiments, an ENPP1-low status can be used as a prognostic biomarker for anti-PD1 and PARP inhibitors. In some embodiments, ENPP1 expression levels can be added to biomarker panels for deciding which patients should receive anti-PD-1 and PARP inhibitors. Giving these therapies to ENPP1-negative patients can increase the precision in selecting complete responders. METHODS OF TREATING BREAST CANCER [0127] In a certain aspect, the invention provides a method for treating breast cancer in a subject, comprising: a) obtaining a sample from the subject; b) measuring ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in the sample from the subject; c) classifying the sample, wherein the sample has a high classification when its ENPP1 levels are increased; and d) if the sample has the high classification, then administering to the subject: i) an ENPP1 inhibitor; and ii) a PD1 inhibitor, wherein the breast cancer is responsive to a PD1 inhibitor, thereby treating the breast cancer in the subject. In an exemplary embodiment, the method further comprises assessing that the breast cancer is responsive to a PD1 inhibitor. In an exemplary embodiment, the treating is achieving a pathological complete response (pCR). In an exemplary embodiment, the breast cancer is triple negative, microsatellite instability-high (MSI- H), or mismatch repair deficient (dMMR). In an exemplary embodiment, the step d) further comprises iii) administering chemotherapy. In an exemplary embodiment, the chemotherapy is paclitaxel. In an exemplary embodiment, the sample is a breast cancer tissue sample. In an exemplary embodiment, the measuring is selected from the group consisting of ENPP1 mRNA expression, ENPP1 immunohistochemical levels, ENPP1 gene amplification levels, and ENPP1 K173Q homozygosity. In an exemplary embodiment, the measuring is ENPP1 mRNA expression, and the high classification occurs when the breast cancer tissue sample ENPP1 mRNA expression has, relative to a reference, a value selected from the group consisting of 6.2 or above, 6.39 or above, 6.4 or above, 7.0 or above, 7.4 or above, 7.8 or above, 8.0 or above, 8.9 or above, 9.2 or above, 9.4 or above, 9.6 or above, 9.8 or above, 10.0 or above. In an exemplary embodiment, the reference is the ENPP1 mRNA expression in a population of breast cancer tissue samples. In an exemplary embodiment, the measuring is ENPP1 immunohistochemical levels, and the high classification occurs when a percentage of cells positive for ENPP1 expression in the breast cancer tissue sample is 0.1 or above, 0.5 or above, 0.75 or above, 0.9 or above, 1 or above, 10 or above, or 50 or above. In an exemplary embodiment, the measuring is ENPP1 immunohistochemical levels, and the high classification occurs when a percentage of cells positive for ENPP1 expression in the breast cancer tissue sample is 1 or above. In an exemplary embodiment, the measuring is ENPP1 gene amplification levels, and the high classification occurs when ENPP1/CEP6 probe ratio > 2, when ENPP1 copy number > 6, or when ENPP1 copies are > 6 if a single probe is used. In an exemplary embodiment, the measuring is ENPP1 K173Q homozygosity, and the high classification occurs when the breast cancer tissue sample is homozygous for the K173Q mutation. In an exemplary embodiment, the sample is a blood sample. In an exemplary embodiment, the measuring is of ENPP1 activity through plasma ENPP1 cGAMP degradation half-life, and the high classification is when the cGAMP degradation half-life is less than 60 minutes, or less than 50 minutes or less than 70 minutes. In an exemplary embodiment, the measuring is of ENPP1 plasma protein levels, and the high classification is when the ENPP1 plasma protein level is > 4.4 ^g/L, > 3.4 ^g/L, or > 5.4 ^g/L. In an exemplary embodiment, the PD1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, and toripalimab. In an exemplary embodiment, the PD1 inhibitor is pembrolizumab. In an exemplary embodiment, the ENPP1 inhibitor is described herein. [0128] In another aspect, the invention provides a method for treating breast cancer in a subject, comprising: a) obtaining a sample from the subject; b) measuring ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in the sample from the subject; c) classifying the sample, wherein the sample has a high classification when its ENPP1 levels are increased; and d) if the sample has the high classification, then administering to the subject: i) an ENPP1 inhibitor; and ii) a PDL1 inhibitor, wherein the breast cancer is responsive to a PDL1 inhibitor, thereby treating the breast cancer in the subject. In an exemplary embodiment, the method further comprises assessing that the breast cancer is responsive to a PDL1 inhibitor. In an exemplary embodiment, the treating is achieving a pathological complete response (pCR). In an exemplary embodiment, the breast cancer is triple negative, microsatellite instability-high (MSI- H), or mismatch repair deficient (dMMR). In an exemplary embodiment, the step d) further comprises iii) administering chemotherapy. In an exemplary embodiment, the chemotherapy is paclitaxel. In an exemplary embodiment, the sample is a breast cancer tissue sample. In an exemplary embodiment, the measuring is selected from the group consisting of ENPP1 mRNA expression, ENPP1 immunohistochemical levels, ENPP1 gene amplification levels, and ENPP1 K173Q homozygosity. In an exemplary embodiment, the measuring is ENPP1 mRNA expression, and the high classification occurs when the breast cancer tissue sample ENPP1 mRNA expression has, relative to a reference, a value selected from the group consisting of 6.2 or above, 6.39 or above, 6.4 or above, 7.0 or above, 7.4 or above, 7.8 or above, 8.0 or above, 8.9 or above, 9.2 or above, 9.4 or above, 9.6 or above, 9.8 or above, 10.0 or above. In an exemplary embodiment, the reference is the ENPP1 mRNA expression in a population of breast cancer tissue samples. In an exemplary embodiment, the measuring is ENPP1 immunohistochemical levels, and the high classification occurs when a percentage of cells positive for ENPP1 expression in the breast cancer tissue sample is 0.1 or above, 0.5 or above, 0.75 or above, 0.9 or above, 1 or above, 10 or above, or 50 or above. In an exemplary embodiment, the measuring is ENPP1 immunohistochemical levels, and the high classification occurs when a percentage of cells positive for ENPP1 expression in the breast cancer tissue sample is 1 or above. In an exemplary embodiment, the measuring is ENPP1 gene amplification levels, and the high classification occurs when ENPP1/CEP6 probe ratio > 2, when ENPP1 copy number > 6, or when ENPP1 copies are > 6 if a single probe is used. In an exemplary embodiment, the measuring is ENPP1 K173Q homozygosity, and the high classification occurs when the breast cancer tissue sample is homozygous for the K173Q mutation. In an exemplary embodiment, the sample is a blood sample. In an exemplary embodiment, the measuring is of ENPP1 activity through plasma ENPP1 cGAMP degradation half-life, and the high classification is when the cGAMP degradation half-life is less than 60 minutes, or less than 50 minutes or less than 70 minutes. In an exemplary embodiment, the measuring is of ENPP1 plasma protein levels, and the high classification is when the ENPP1 plasma protein level is > 4.4 ^g/L, > 3.4 ^g/L, or > 5.4 ^g/L. In an exemplary embodiment, the PDL1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab. In an exemplary embodiment, the ENPP1 inhibitor is described herein. [0129] In another aspect, the invention provides a method for treating breast cancer in a subject, comprising: a) obtaining a sample from the subject; b) measuring ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in the sample from the subject; c) classifying the sample, wherein the sample has a high classification when its ENPP1 levels are increased; and d) if the sample has the high classification, then administering to the subject: i) an ENPP1 inhibitor; and ii) a PARP inhibitor, wherein the breast cancer is responsive to a PARP inhibitor, thereby treating the breast cancer in the subject. In an exemplary embodiment, the method further comprises assessing that the breast cancer is responsive to a PARP inhibitor. In an exemplary embodiment, the treating is achieving a pathological complete response (pCR). In an exemplary embodiment, the breast cancer has a BRCA1 mutation, a BRCA2 mutation, is HER2 negative, is locally advanced, or is metastatic. In an exemplary embodiment, the step d) further comprises administering chemotherapy. In an exemplary embodiment, the chemotherapy is paclitaxel. In an exemplary embodiment, the sample is a breast cancer tissue sample. In an exemplary embodiment, the measuring is selected from the group consisting of ENPP1 mRNA expression, ENPP1 immunohistochemical levels, ENPP1 gene amplification levels, and ENPP1 K173Q homozygosity. In an exemplary embodiment, the measuring is ENPP1 mRNA expression, and the high classification occurs when the breast cancer tissue sample ENPP1 mRNA expression has, relative to a reference, a value selected from the group consisting of 6.4 or above, 6.78 or above, 7.0 or above, 7.4 or above, 7.8 or above, 8.0 or above, 8.9 or above, 9.2 or above, 9.4 or above, 9.6 or above, 9.8 or above, 10.0 or above, 10.2 or above, 10.4 or above, and 10.5 or above. In an exemplary embodiment, the reference is the ENPP1 mRNA expression in a population of breast cancer tissue samples. In an exemplary embodiment, the measuring is ENPP1 immunohistochemical levels, and the high classification occurs when a percentage of cells positive for ENPP1 expression in the breast cancer tissue sample is 0.1 or above, 0.5 or above, 0.75 or above, 0.9 or above, 1 or above, 10 or above, or 50 or above. In an exemplary embodiment, the measuring is ENPP1 immunohistochemical levels, and the high classification occurs when a percentage of cells positive for ENPP1 expression in the breast cancer tissue sample is 1 or above. In an exemplary embodiment, the measuring is ENPP1 gene amplification levels, and the high classification occurs when ENPP1/CEP6 probe ratio > 2, when ENPP1 copy number > 6, or when ENPP1 copies are > 6 if a single probe is used. In an exemplary embodiment, the measuring is ENPP1 K173Q homozygosity, and the high classification occurs when the breast cancer tissue sample is homozygous for the K173Q mutation. In an exemplary embodiment, the sample is a blood sample. In an exemplary embodiment, the measuring is of ENPP1 activity through plasma ENPP1 cGAMP degradation half-life, and the high classification is when the cGAMP degradation half-life is less than 60 minutes, or less than 50 minutes or less than 70 minutes. In an exemplary embodiment, the measuring is of ENPP1 plasma protein levels, and the high classification is when the ENPP1 plasma protein level is > 4.4 ^g/L, > 3.4 ^g/L, or > 5.4 ^g/L. In an exemplary embodiment, the PARP inhibitor is selected from the group consisting of veliparib, olaparib, rucaparib, niraparib, and talazoparib. In an exemplary embodiment, the PARP inhibitor is veliparib. In an exemplary embodiment, the step d) further comprises administering a platinum complex selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin. In an exemplary embodiment, the platinum complex is carboplatin. In an exemplary embodiment, the ENPP1 inhibitor is described herein. [0130] All publications and patent applications mentioned in this disclosure are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. [0131] No admission is made that any reference cited herein constitutes prior art. The discussion of the references states what their authors assert, and the Applicant reserves the right to challenge the accuracy and pertinence of the cited documents. It will be clearly understood that, although a number of information sources, including scientific journal articles, patent documents, and textbooks, are referred to herein; this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art. [0132] The discussion of the general methods given herein is intended for illustrative purposes only. Other alternative methods and alternatives will be apparent to those of skill in the art upon review of this disclosure, and are to be included within the spirit and purview of this application. [0133] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims. EXAMPLES [0134] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as Sambrook, J., & Russell, D. W. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory and Sambrook, J., & Russel, D. W. (2001). Molecular Cloning: A Laboratory Manual (3rd ed.). Cold Spring Harbor, NY: Cold Spring Harbor Laboratory (jointly referred to herein as “Sambrook”); Ausubel, F. M. (1987). Current Protocols in Molecular Biology. New York, NY: Wiley (including supplements through 2014); Bollag, D. M. et al. (1996). Protein Methods. New York, NY: Wiley-Liss; Huang, L. et al. (2005). Nonviral Vectors for Gene Therapy. San Diego: Academic Press; Kaplitt, M. G. et al. (1995). Viral Vectors: Gene Therapy and Neuroscience Applications. San Diego, CA: Academic Press; Lefkovits, I. (1997). The Immunology Methods Manual: The Comprehensive Sourcebook of Techniques. San Diego, CA: Academic Press; Doyle, A. et al. (1998). Cell and Tissue Culture: Laboratory Procedures in Biotechnology. New York, NY: Wiley; Mullis, K. B., Ferré, F. & Gibbs, R. (1994). PCR: The Polymerase Chain Reaction. Boston: Birkhauser Publisher; Greenfield, E. A. (2014). Antibodies: A Laboratory Manual (2nd ed.). New York, NY: Cold Spring Harbor Laboratory Press; Beaucage, S. L. et al. (2000). Current Protocols in Nucleic Acid Chemistry. New York, NY: Wiley, (including supplements through 2014); and Makrides, S. C. (2003). Gene Transfer and Expression in Mammalian Cells. Amsterdam, NL: Elsevier Sciences B.V., the disclosures of which are incorporated herein by reference. [0135] Additional embodiments are disclosed in further detail in the following examples, which are provided by way of illustration and are not in any way intended to limit the scope of this disclosure or the claims. EXAMPLE 1 Materials and Methods [0136] Mouse strains. C57BL/6J (strain #000664), BALB/cJ (strain #000651), BALB/cJ- Enpp1asj-2J/GrsrJ (referred to as Enpp1-/-, strain #019107), C57BL/6J-Stinggt/J (referred to as Sting-/-, strain #017537), C57BL/6J-Enpp1asj/GrsrJ (strain #012810), and FVB/N-Tg(MMTV- PyVT)634Mul/J (referred to as MMTV-PyMT, strain #002374) mice were purchased from the Jackson Laboratory. C57BL/6J-Enpp1H362A and C57BL/6J-Enpp1H362A x Sting1-/- mice were and characterized in house (Carozza et al., 2021). FVB/N-Tg(MMTV-PyVT) 634Mul/J mice were bred with C57BL/6J-WT or C57BL/6J-Enpp1H362A to generate B6;FVB-MMTV and B6;FVB- Enpp1H362A x MMTV respectively. For MMTV spontaneous tumor model, female mice from the second generation of both B6;FVB-MMTV and B6;FVB-Enpp1H362A x MMTV genotypes were used for experiment. For all other breast tumor experiments, female mice between 6-15 weeks old were used for tumor experiments. Mice were maintained at Stanford University in compliance with the Stanford University Institutional Animal Care and Use Committee (IACUC) regulations. All procedures were approved by the Stanford University Administrative Panel on Laboratory Animal Care (APLAC). [0137] Mammalian cell lines and primary cells.4T1, E0771.lmb, MDA-MB-231, NMuMG, MCF-7, Neuro-2a, mouse embryonic fibroblast (MEF), U937, L-929, and EMT6 cells were procured from ATCC. E0771 cells were procured from CH3 BioSystems. Panc02 were procured from the DTP/DCTD/NCI Tumor Repository. Human umbilical vein endothelial cells (HUVEC) pooled from six different donors were procured from Lonza. 4T1-luciferase (4T1-luc) cells were a gift from C. Contag, Stanford University, Stanford, CA, USA (Vilalta et al., 2014).4T1-luc Enpp1-/- pooled clonal cell line and 293T cGAS ENPP1-/- single clonal cell line were generated in a previous study (Jacqueline A. Carozza, Böhnert, et al., 2020). Primary human PBMCs were isolated by subjecting enriched buffy coat from whole blood (Stanford Blood Center) to a Percoll density gradient. Primary mouse lung fibroblasts were isolated by plating dissociated mouse lungs and propagating attached cells for 2 weeks. Primary mouse lung fibroblasts were isolated by incubating the minced lungs with 1 mg/mL collagenase from Clostridium histolyticum (Sigma-Aldrich) and 20 μg/mL DNase I (Sigma-Aldrich) for 1 hour, then passed through 100 μM cell strainer, spun down, treated with RBC lysis buffer for 5 minutes and plated in 10cm dish until all fibroblasts attached. To isolate metastasis from the blood or distant organs in animal studies, blood, draining inguinal lymph nodes (dLNs), lungs, livers, and brains were collected at experimental end point. Blood was dispensed into a 15-ml conical tube containing 10 mL 1x HBSS (Gibco), centrifuged at 1500 rpm at room temperature for 5 minutes before plated. dLNs were teased apart by forcing through a 100 μM strainer before plated. All other organs were minced with scissors and forces and digested in collagenase from Clostridium histolyticum (Sigma-Aldrich) at various conditions: 4°C for 75 minutes for lungs, 37°C for 30 minutes for livers, and 37°C for 120 minutes for brains. Digested organs were passed through a 100 μM cell strainer, washed twice with 1x HBSS (Gibco) before plating. 4T1, E0771, E0771.LMB, EMT6, and their derived cell lines were maintained in RPMI (Corning Cellgro) supplemented with 10% FBS (R&D Systems), 10 mM HEPES (Gibco), and 1% penicillin-streptomycin (ThermoFisher). 293T cGAS ENPP1-/-, MDA-MB-231, L929, Neuro-2a, MEF, and mouse lung fibroblast cells were maintained in DMEM (Corning Cellgro) supplemented with 10% FBS (R&D Systems) and 1% penicillin-streptomycin (Thermo Fisher). MCF-7 and NMuMG cells were maintained in DMEM (Corning Cellgro) supplemented with 10% FBS (R&D Systems), 10 ^g/mL bovine insulin (Sigma-Aldrich) and 1% penicillin-streptomycin (Thermo Fisher). U937 and PBMC cells were maintained in RPMI (Corning Cellgro) supplemented with 10% heat-inactivated FBS (R&D Systems) and 1% penicillin-streptomycin (Thermo Fisher). HUVEC cells were maintained in EMG-2 supplemented growth media (Lonza). 4T1 and E0771.lmb.PuroR metastasis were cultured for 7-10 days with minimal disturbance in IMDM (Gibco) supplemented with 10% FBS, 1% penicillin-streptomycin, and 60 μM 6-thioguanine (Sigma- Aldrich) or 1.5 μg/mL puromycin (Sigma-Aldrich) respectively. All cells were maintained in a humidified incubator at 37°C and 5% CO2. All cell lines tested negative for mycoplasma contamination. [0138] Reagents and antibodies.2’3’-cyclic-GMP-AMP (cGAMP) and [32P] cGAMP was synthesized in house as described below. The following antibodies were used for flow cytometry: Alexa Fluor 594 anti-CD8a (1:200), Alexa Fluor 700 anti-CD45 (1:800), APC anti- F4/80 (1:200), Brilliant Violet 421 anti-CD11b (1:400), Brilliant Violet 510 anti-F4/80 (1:400), Brilliant Violet 570 anti-Ly-6C (1:200), Brilliant Violet 605 anti-CD335 (1:800), Brilliant Violet 650 anti-CD45 (1:100), Brilliant Violet 650 anti-CD206 (1:100), Brilliant Violet 785 anti-CD8a (1:200), Brilliant Violet 785 anti-CD11c (1:400), Brilliant Violet 785 anti-CD62L (1:200), FITC anti-I-A/I-E (1:800), PE anti-CD11c (1:200), PerCP-Cy5.5 anti-I-A/I-E (1:100), and TruStain FcX (1:100) were purchased from BioLegend. APC anti-FoxP3 (1:100) was purchased from Tonbo Biosciences. BUV 395 anti-CD103 (1:400), BUV 563 anti-CD115 (1:200), BUV 563 anti-Ly-6G (1:200), and BUV 805 anti-CD4 (1:200) were purchased from BD Biosciences. eFluor 450 anti-CD25 (1:200), PE anti-EOMES (1:100), and PerCP-eFluor 710 anti-CD3e (1:200) are purchased from eBioscience Invitrogen. VioGreen anti-Ly-6C (1:400) was purchased from Miltenyi Biotec. Rabbit anti-phospho-STING (1:200) was purchased from Cell Signaling Technology. The following antibodies were used for western blotting: mouse anti-tubulin (1:2000), rabbit anti-DYKDDDDK (FLAG, 1:1000), and rabbit anti-GFP (1:1000) were purchased from Cell Signaling. IRDye 800CW goat anti-rabbit (1:15,000) and IRDye 680RD goat anti-mouse (1:15,000) were purchased from LI-COR Biosciences. [0139] Synthesis and purification of cGAMP and [32P] cGAMP. To enzymatically synthesize cGAMP (Ritchie et al., 2019), 1 μM purified sscGAS was incubated testis DNA (Sigma) for 24 h. The reaction was then heated at 95°C for 3 min and filtered through a 3-kDa filter. cGAMP was purified from the reaction mixture using a PLRP-S polymeric reversed phase preparatory column (100 Å, 8 μm, 300 x 25 mm; Agilent Technologies) on a preparatory HPLC (1260 Infinity LC system; Agilent Technologies) connected to UV-vis detector (ProStar; Agilent Technologies) and fraction collector (440-LC; Agilent Technologies). The flow rate was set to 25 mL/min. The mobile phase consisted of 10 mM triethylammonium acetate in water and acetonitrile. The mobile phase started as 2% acetonitrile for the first 5 min. Acetonitrile was then ramped up to 30% from 5-20 min, then to 90% from 20-22 min, maintained at 90% from 22-25 min, and then ramped down to 2% from 25-28 min. Fractions containing cGAMP were lyophilized and resuspended in water. The concentration was determined by measuring absorbance at 280 nm. To enzymatically synthesize [32P] cGAMP, 1 μM purified sscGAS was incubated with 20 mM Tris-HCl pH 7.4, 250 μCi (3000 Ci/mmol) [α-32P] ATP (Perkin Elmer), 1 mM GTP, 20 mM MgCl2, and 100 μg/mL herring testis DNA (Sigma) in a reaction volume of 100 μL for 24 h. The reaction was purified by preparatory TLC on a HP-TLC silica gel plate (Millipore), eluted in water, and filtered through a 3-kDa filter to remove silica gel. [0140] STING expression and purification. Neutralizing STING (WT) or non-binding STING (R237A) were expressed and purified using previously published methods (Jacqueline A. Carozza, Böhnert, et al., 2020). In brief, pTB146 His-SUMO-mSTING (residues 139-378) was expressed in Rosetta (DE3) pLysS competent cells (Sigma-Aldrich). Cells were grown in 2xYT medium with 100 μg/mL ampicillin until they reached an OD600 of 1. They were then induced with 0.75 mM IPTG at 16°C overnight. Cells were pelleted and resuspended in 50 mM Tris pH 7.5, 400 mM NaCl, 10 mM imidazole, 2 mM DTT, and protease inhibitors (cOmplete, EDTA- free protease inhibitor cocktail, Sigma-Aldrich). The cells were then flash frozen and thawed twice before sonication to lyse the cells. The supernatant was incubated with HisPur cobalt resin (Thermo Scientific) for 30 min at 4°C. The resin-bound protein was washed with 50 column volumes of 50 mM Tris pH 7.5, 150 mM NaCl, 2% triton X-114; 50 column volumes of 50 mM Tris pH 7.5, 1 M NaCl; and 20 column volumes of 50 mM Tris pH 7.5, 150 mM NaCl. Protein was eluted from resin with 600 mM imidazole in 50 mM Tris pH 7.5, 159 mM NaCl. Fractions containing His-SUMO-STING were pooled, concentrated, and dialyzed against 50 mM Tris pH 7.5, 150 mM NaCl while incubating with SUMOlase His-ULP1 to remove the His-SUMO tag overnight. The solution was incubated with the HisPur cobalt resin again to remove the His- SUMO tag, and STING was collected from the flowthrough. Protein was dialyzed against 20 mM Tris pH 7.5, loaded onto a HitrapQ anion exchange column (GE Healthcare) using Äkta FPLC (GE Healthcare), and eluted with a NaCl gradient. Fractions containing sting were pooled, buffer exchanged into PBS, and stored at -80°C until use. [0141] Recombinant DNA. To clone guide RNA plasmid targeting Enpp1, Enpp1 guide sequences listed in Table S1 were cloned into the BbsI site of PX458-GFP or P458-mCherry backbones (synthesized by Addgene) following the protocol from Ryuji Morizane Lab at Harvard in order. To clone pLenti-CMV-mENPP1-WT-GFP-Puro plasmid, mENPP1-WT sequence was amplified from pcDNA3-mENPP1-FLAG (synthesized by Genscript) using pLenti_mENPP1_fwd and pLenti_mENPP1_rev primers in Table S1 and inserted into the XbaI- BamHI sites of pLenti-CMV-GFP-Puro (Addgene). To clone pLenti-CMV-mENPP1-T238A- GFP-Puro plasmid, T238A point mutants were first introduced into pcDNA3-mENPP1-FLAG using QuikChange mutagenesis. mENPP1-T238A sequence was then introduced into the XbaI- BamHI sites of pLenti-CMV-GFP-Puro. pLenti-TetONE-FLAG-Puro were synthesized by Addgene and used to generate E0771.lmb.PuroR cell line. The pcDNA3- mouseENPP1(mENPP1)-FLAG plasmids were synthesized by Genscript. The A84X and membrane proximal domain (MPD) point mutants were introduced into pcDNA-mENPP1- FLAG using QuikChange mutagenesis (Agilent) with primers in Table S1 and sequenced by verifying the region of the mutation. For creation of pLenti-CMV-mENPP1-WT-GFP-Puro plasmid, mENPP1-WT sequence was amplified from pcDNA3-mENPP1-FLAG using pLenti_mENPP1_fwd and pLenti_mENPP1_rev primers in Table S1 and inserted into the XbaI- BamHI sites of pLenti-CMV-GFP-Puro (Addgene). For creation of pLenti-CMV-mENPP1- T238A-GFP-Puro and pLenti-CMV-mENPP1-A84S-GFP-Puro plasmids, T238A and A84S point mutants were first introduced into pcDNA3-mENPP1-FLAG using QuikChange mutagenesis (Agilent). mENPP1-T238A and mENPP1-A84S sequences were then introduced into the XbaI-BamHI sites of pLenti-CMV-GFP-Puro (Addgene). The pcDNA3- humanENPP1(hENPP1)-FLAG plasmid was synthesized by Genscript. For creation of pcDNA3- hENPP1-K173Q-Flag, two sequences flanking the point mutation site was amplified from pcDNA3-hENPP1-Flag using hENPP1_K173Q_F1_fwd and hENPP1_K173Q_F1_rev, hENPP1_K173Q_F2_fwd and hENPP1_K173Q_F2_rev, and re-introduced into BamHI-EcoRI site of pcDNA3-hENPP1-FLAG using via Gibson assembly. PX458-GFP and PX458-mCherry plasmids were synthesized by Addgene. The guide sequences listed in Table S1 were cloned into the BbsI site of PX458 backbone following the protocol from Ryuji Morizane Lab at Harvard. pLenti-TetONE-FLAG-Puro were synthesized by Addgene. All oligonucleotide sequences are in Table S1. Table S1. Oligonucleotides used in this study
Figure imgf000045_0001
Figure imgf000046_0001
Supplemental Table 1. Oligonucleotide Sequences
Figure imgf000046_0002
Figure imgf000047_0001
[0142] Generation of transient CRISPR edited cell line. 4T1 Enpp1-/- cells were created the same way as 4T1-luc Enpp1-/- cells previously described (Carozza, Böhnert, et al., 2020). Briefly, 4T1 underwent transient transfection with Lipofectamine 3000 of the following pairs of sgRNAs targeting mouse Enpp1: PX458-mENPP1_sgRNA1-GFP and PX458- mENPP1_sgRNA2-mCherry; PX458- mENPP1_sgRNA3-GFP and PX458- mENPP1_sgRNA1- mCherry. Double GFP and mCherry positive cells were sorted with FACS (SONY) and underwent single-cell cloning. Sequence knockout was confirmed with PCR using mENPP1_sgRNA12_seq_fwd and mENPP1_sgRNA12_seq_rev, or mENPP1_sgRNA34_seq_fwd and mENPP1_sgRNA34_seq_rev primer pairs. Functional knockout was confirmed with activity assay (commercial antibodies are not sensitive enough for verification of protein expression). Multiple clean knockout clones were pooled to generate the 4T1 Enpp1-/- cell line. [0143] Generation of stable expression cell lines. 4T1 Enpp1-/- cells were virally transfected to stably express WT, T238A, or A84S mouse ENPP1, giving rise to ENPP1WT-OE, ENPP1T238A-OE, ENPP1A84S-OE cell lines. Briefly, lentiviral packaging plasmids (pHDM-G, pHDM-Hgmp2, pHDM-tat1b, and pRC.CMV-rev1b) were purchased from Harvard Medical School.500 ng of pLenti-CMV-mENPP1-GFP-Puro, pLenti-CMV-mENPP1-T238A-GFP-Puro, or pLenti-CMV-mENPP1-A84S-GFP-Puro plasmid, and 500 ng of each of the packaging plasmids were transfected into 293T cells using FuGENE 6 transfection reagent (Promega). The viral media was exchanged after 24 h, harvested after 48 h and passed through a 0.45 μm filter, and used to transduce 4T1 Enpp1-/- cells (4T1 cells are used as 4T1-luc cells already carry puromycin resistance which will interfere with subsequent drug selection process).48 h post- transduction cells were selected with 1–2 μg/ml puromycin and single-cell cloned, and multiple clones were pooled after verification by activity assay. E0771.LMB cells were virally transfected with empty pLenti-TetONE-FLAG-Puro vector to stably carry puromycin resistance following the previously described lentiviral transfection protocol, giving rise to the E0771.LMB.Puro cell line. [0144] Lysate and supernatant collection from cell lines. Cells are plated in 6 well plate.24 h before collection and when cells reach around 60-70% confluency, media is replaced with serum-free media consisting of DMEM or PRMI, 1%Insulin-Transferrin-Selenium (ThermoFisher), and 1% penicillin-streptomycin (Thermo Fisher). For 293T expression experiments, WT or mutant mouse or human pcDNA-ENPP1-FLAG plasmids were transiently transfected into 293T cGAS ENPP1-/- cells with polyethylenimine (PEI) at ratio of 1 μg plasmid to 3 μg PEI per well of a 6 well plate. After 24 h, replace media with 1 mL of serum-free media consisting of DMEM, 1% Insulin-Transferrin-Selenium (ThermoFisher), and 1% penicillin- streptomycin (Thermo Fisher).24 h after culturing in serum-free condition, media supernatant is collected, centrifuged at 1000 x g for 3 minutes followed by 12000 x g for 1 minute to get rid of dead cells and debris. Supernatant is concentrated using 10KDa Amicon Ultra-0.5 Centrifugal Filter Unit (EMD Millipore). Cells were washed off the plate in 1 mL PBS, centrifuged at 100 x g for 3 minutes, and lysed in 40-100 μL10 mM Tris pH 7.5, 150 mM NaCl, and 1% NP-40 for western blotting and activity assays. Lysates were stored at -20°C. If only lysates are collected, cells are left in regular media until 80-90% confluent before collection. [0145] Mouse serum, tumor, and tissue collections. Mouse blood was collected through terminal cardiac puncture and spun at 2,000 x g for 5 min. The resulting serum layer was collected and stored at -80°C until use. Cell lysate preparation for cGAMP degradation activity assay is as following: cells from a confluent well in a 6 well plate was collected in 1 mL of PBS, centrifuged at 1000 x g for 3 minutes, lysed in 50-100 μL of lysis buffer (10 mM Tris pH 9, 150 mM NaCl, 10 μM ZnCl2, 1% NP-40), and stored in -20°C until use. For western blotting, cells were lysed on the plate in 100-250 μL of Laemmli sample buffer, boiled at 95°C for 5 minutes and sonicated. Mouse tumor lysate (100 mg/mL) for cGAMP degradation activity assay were generated by lysing tissues in 10 mM Tris pH 7.5, 150 mM NaCl, 10 μM ZnCl2, 1.5% NP-40, and freshly added protease inhibitors (cOmplete, EDTA-free protease inhibitor cocktail, Sigma- Aldrich). Organ lysates were then homogenized with a bead homogenizer (Omni International) and stored at -20°C until use. [0146] Human PBMC and plasma isolation. Buffy coat (obtained de-identified from the Stanford Blood Center) was diluted 1:3 with PBS. Diluted buffy coat was layered on top of 50% Percoll (GE Healthcare) containing 140 mM NaCl and centrifuged at 600 x g for 30 min. The separated plasma and PBMC layer were collected. PBMCs were washed once with PBS and once with RPMI. Human PBMCs were then lysed in 10 mM Tris pH 7.5, 150 mM NaCl, and 1% NP-40 to generate 2.5-5x106 cells/^L lysates. Lysates were stored at -80°C. Genomic DNA (gDNA) of human PBMCs were isolated using DNeasy Blood & Tissue Kit (Qiagen). hENPP1 exon 4 region was amplified from donor gDNA templates and sequenced with hENPP1_K173Q_seq_fwd and hENPP1_K173Q_seq_rev primers (Table S1). To generate complementary DNA (cDNA), cells were lysed with 0.5-0.75 mL TRIzol (Invitrogen) and total RNA from cells were extracted using Direct-zol RNA Kit (Zymo). RNA was reverse transcribed in 20 ^L reactions containing 500 ng total RNA, 50 pmol Random Hexamer (Thermo Scientific) and 50 pmol Oligo(dT) Primers (Thermo Scientific), 0.5 mM dNTPs (NEB), 40 U RNaseOUT (Invitrogen), 1x Maxima RT Buffer (Thermo Scientific), and 200 U Maxmia Reverse Transcriptase (Thermo Scientific). RT reactions were incubated for 10 min at 25°C, then for 30 min at 50°C, and terminated by incubating for 5 min at 85°C. The entire hENPP1 exon region was amplified from donor cDNA templates and sequenced with hENPP1_seq_1-10 primers (Table S1). Human sample collection is approved by Stanford Institutional Review Board (Protocol #43809). [0147] ENPP1 activity assay by [32P] cGAMP degradation thin-layer chromatography assays. cGAMP activity assays (20-30 μl total) were composed of the following: cell lysate, organ lysate, mouse serum, or human plasma (50-75%), cGAMP (1 to 5 μM, with trace [32P] cGAMP spiked in), and buffer (standard assay buffer unless otherwise noted was 50 mM Tris pH 9 or pH 7.5, 250 mM NaCl, 0.5 mM CaCl2, 1 μM ZnCl2). At indicated times, 1 μl aliquots of the reaction were quenched by spotting on HP-TLC silica gel plates (Millipore). The TLC plates were run in mobile phase (85% ethanol, 5 mM NH4HCO3) and exposed to a phosphor screen (GE BAS-IP MS). Screens were imaged on a Typhoon 9400 scanner and the 32P signal was quantified using ImageJ. For cell lysates and supernatants, assays were run in standard buffer listed above at pH 9.0 at room temperature with 1 μM cGAMP spkied with [32P] cGAMP. For lysates and serum or plasma from mice, assays were run in PBS or physiological buffer (50 mM Tris, 150 mM NaCl, 1.5 mM CaCl2, 10 μM ZnCl2) at pH 7.4 or pH 9.0 at 37C with 5 μM cGAMP. For lysates and plasma collected from blood bank buffy coat, assays were ran in high metal buffer (50 mM Tris, 250 mM NaCl, 3 mM CaCl2, 250 μM ZnCl2) to overcome trace EDTA during blood collection process at pH 9.0 and 37C. For human plasma collected by ourselves, assays were ran in PBS at 37C with or without STF-1623. [0148] For experiments reported in Figure 1G, 1H, 1I, cGAMP degradation activity assay (20 μl) for cells containing 50% cell lysate, cGAMP (1 μM, with trace [32P] cGAMP spiked in), and standard ENPP1 activity buffer (50 mM Tris pH 9, 250 mM NaCl, 0.5 mM CaCl2, 1 μM ZnCl2) took place in room temperature. [0149] For experiments reported in Figure 4K, cGAMP degradation activity assay for moues organs (30 μl) containing 75% organ lysate (100 mg/mL), 5 μM cGAMP, and PBS took place in 37°C. Lastly, cGAMP degradation activity assay (20 μl) for mouse serum containing 50% serum, 5 μM cGAMP and physiological ENPP1 activity buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1.5 mM CaCl2, 10 μM ZnCl2) took place in 37°C. At indicated times, 1 μl aliquots of the reaction were quenched by spotting on HP-TLC silica gel plates (Millipore). The TLC plates were run in mobile phase (85% ethanol, 5 mM NH4HCO3) and exposed to a phosphor screen (GE BAS-IP MS). Screens were imaged on a Typhoon 9400 scanner and the 32P signal was quantified using ImageJ. The sample size and statistical tests of computation are indicated in respective figure legend. [0150] Western blotting. For western blotting, 5x Lameli sample buffer (LSB) or non-reducing SDS buffer (LSB lacking BME) were added to cell lysates and boiled at 95°C for 10 minutes. Cell lysates were separated on an SDS-polyacrylamide gel (Genscript) and transferred to a nitrocellulose membrane using a wet transfer system (BioRad). Primary antibody (Anti-FLAG, GFP, Tubulin, Cell Signaling) was added overnight at 4°C, followed by three washes in TBS-T (1x TBS-0.1% tween). Secondary antibody IRDye 800CW goat anti-rabbit (1:15,000) and IRDye 680RD goat anti-mouse (1:15,000) were purchased from Li-COR Biosciences and added for 1 h at room temperature, followed by three additional washes in TBS-T. Blots were imaged in IR using a LI-COR Odyssey Blot Imager. Bands were quantified using ImageJ. [0151] Mouse primary tumor models (4T1, 4T1-luc, E0771). For the 4T1 model, six- to eleven-week-old female WT or Enpp1asj (Enpp1 KO) BALB/cJ mice were inoculated with 2.5– 5 x 104 ENPP1WT-OE or ENPP1T238A-OE 4T1 cells suspended in 50 μL of PBS into the fifth mammary fat pad. For the 4T1-luc model, seven- to twelve-week-old female WT or Enpp1 KO BALB/cJ mice were inoculated with 5 x 104 WT or Enpp1 KO 4T1-luc cells suspended in 50 μL of PBS into the fifth mammary fat pad. When 4T1-luc tumor volume (determined by length2 x width / 2) reached 100 ± 20 mm3, tumors were either irradiated or not irradiated with 12 Gy using a 225 kVp cabinet X-ray irradiator filtered with 0.5 mm Cu (IC-250, Kimtron Inc., CT). Mice were anesthetized with a mixture of 80 mg/kg ketamine (VetaKet) and 5 mg/kg xylazine (AnaSed) prior to irradiation. Anaesthetized animals were shielded with a 3.2 mm lead shield with a 15 x 20 mm aperture where the tumor was placed. For the STING neutralization experiment with 4T1 model, 5 x 104 WT or Enpp1 KO 4T1-luc cells were injected into WT or Enpp1 KO BALB/cJ mice. The next day, mice were intratumorally injected with 100 μL of 100 μM neutralizing or non-binding STING and every other day following that up ting day 15. Mice were euthanized on day 15 and tumors were extracted for FACS analysis. For the E0771 model, six- to fifteen-week-old female WT or Enpp1H362A C57BL/6J mice were inoculated with 2.5 x 104 E0771 cells suspended in 50 μL of PBS into the fifth mammary fat pad. For all three models, tumor volumes were recorded and analyzed by averaging tumor volumes in a group using unpaired t test with Welch correction to test for significance until the first death event. Animal death was plotted in a Kaplan-Meier curve using Graphpad Prism and statistical significance was assessed using the log-rank Mantel-Cox test. [0152] Mouse tumor metastasis models (4T1 and E0771.LMB). For 4T1 metastasis from orthotopic tumors, we implanted 2.5 x 104 WT or Enpp1 KO 4T1 into WT or Enpp1 KO BALB/cJ respectively following protocols above. At day 33, we sacrificed the animals and collected blood, draining inguinal lymph nodes, primary tumors, lungs, livers, and brains. Blood is dispensed into a 15-ml conical tube containing 10 mL 1x HBSS, centrifuged at 1500 rpm at room temperature for 5 minutes and plated into IMDM supplemented with 60 μM 6-MP, 10% FBS and 1% penicillin-streptomycin. Organs are cut into small pieces with scissors and forces, except for lymph nodes which were teased apart by forcing through a 100 μM strainer. Organs are digested in 1 mg/mL collagenase IV (Sigma, C5138): 37C for 30 minutes for primary tumor and liver, 4C for 75 minutes for lung, and 37C for 30 minutes for liver. Dissociated organs are washed twice with 1x HBSS buffer before plated into 60 μM 6-MP containing IMDM media. Cells are cultured for 6-12 days without disturbance. Then clonogenic metastases are fixed and visualized with 0.03% (w/v) methylene blue). Metastatic colonies were quantified with Fuji Image J. For 4T1 metastasis from tail vein injections, we injected 2.5 x 104 WT or Enpp1 KO 4T1 into WT or Enpp1 KO BALB/cJ directly into the tail vein and monitored the mice weights until any mouse’s weight starts to fall for more than 10%, around day 20-30. We collected lungs and quantified metastatic burden following process described above. ENPP1T238A-OE, ENPP1WT-OE, ENPP1A84S-OE metastasis from orthotopic tumors followed the same protocol as above, except having initial cell injection number of 2.5 x 106. For E0771.LMB models, E0771.LMB cells were transfected with an empty vector containing the puromycin resistant gene (denoted as E0771.LMB.PuroR), so that we could selectively culture cancer cells from lung explants ex vivo. We intravenously injected E0771.LMB.PuroR through the tail vein into WT, Enpp1H362A, Sting-/-, and Enpp1H362A x Sting-/- C57BL/6J mice.30 days later, we cultured dissociated lungs using 1 mg/mL type IV collagenase for 30 minutes from these mice in 1μg/μL puromycin for 9 days. Metastatic colonies were visualized with methylene blue dye and quantified with image J. [0153] 4T1 murine breast tumor models. For experiment reported in Figure 1 and Figure 7, BALB/cJ female mice were orthotopically injected with 2.5x106 ENPP1WT-OE or ENPP1T238A-OE 4T1 suspended in 100 μL of PBS cells in the 4th mammary fat pad (MFP). When tumors reached 1000 mm3, we sacrificed the animals and collected primary tumors and lungs. Tissues were processed into single cell suspension following steps described above. Half of the lung suspension were plated into 60 μM 6-thioguanine (Sigma-Aldrich) and 10% heat-inactivated FBS (R&D Systems) containing IMDM (ThermoFisher) media and cultured for 6-12 days without disturbance. At the end of the experiment, colonies of metastases were fixed in methanol and visualized with 0.03% (w/v) methylene blue (Sigma-Aldrich). Metastatic colonies were quantified with Fuji Image J. The rest half of the lung suspension and the tumor suspension were cryopreserved and later thawed for scRNA-seq. For primary tumor experiment reported in Figure 4A and Figure 4K, we orthotopically injected 5 x 104 WT or Enpp1-/- 4T1-luc cells into WT or Enpp1-/- BALB/cJ. When tumors were palpable with an average tumor volume of 100 ± 20 mm3 (determined by length2 x width / 2), 10-12 days after cell inoculation, tumors were irradiated with 12 Gy using a 225 kVp cabinet X-ray irradiator filtered with 0.5 mm Cu (IC-250, Kimtron Inc., CT) following previously described procedures (Carozza, Böhnert, et al., 2020). We sacrificed mice when their tumors reached 1000 mm3 and collected their primary tumors and sera for cGAMP degradation activity following steps described above. For tumor metastasis experiment reported in Figure 4B, 4M, we intravenously injected 5 x 104 WT or Enpp1-/- 4T1 cells into the tail veins of WT or Enpp1-/- BALB/cJ mice. We collected their lungs around day 30 and quantified metastatic burden following process describe above. For tumor metastasis experiment reported in Figure 4L, we orthotopically injected 2.5 x 104 WT or Enpp1-/- 4T1 cells into the tail veins of WT or Enpp1-/- BALB/cJ mice, respectively. At day 33, we sacrificed the animals and collected blood, draining inguinal lymph nodes, primary tumors, lungs, livers, and brains for metastasis assay. The sample size and statistical tests of computation are indicated in respective figure legend. [0154] E0771 murine breast tumor models. For primary tumor experiment in Figure 5A, we orthotopically injected 2.5 x 104 E0771 cells into the 4th MFP of WT, Enpp1H362A, Sting1-/-, or Enpp1H362A x Sting1-/- C57BL/6J mice. We measured animal survival by the time it took for the tumors to reach 1000 mm3. For tumor metastasis experiment in Figure 5C-5E, we intravenously injected E0771.lmb.PuroR cells into the tail veins of WT, Enpp1H362A, Enpp1-/-, Sting1-/-, and Enpp1H362A x Sting1-/- C57BL/6J mice.30 days later, we cultured dissociated lungs in 1μg/μL puromycin for 9 days before methylene blue visualization. The sample size and statistical tests of computation are indicated in respective figure legend. [0155] Spontaneous breast tumor model (MMTV-PyMT). MMTV-PyMT mice (JAX, 002374) on an FVB/NJ background were obtained from The Jackson Laboratories. Homozygous C57BL/6J-Enpp1H362A mice were generated as described (Carozza et al., 2022). FVB/N- Tg(MMTV-PyVT) 634Mul/J mice were bred with C57BL/6J-WT or C57BL/6J-Enpp1H362A to generate B6;FVB-MMTV and B6;FVB-Enpp1H362A x MMTV respectively. For MMTV spontaneous tumor model, female mice from the second generation of both B6;FVB-MMTV and B6;FVB-Enpp1H362A x MMTV genotypes were used for the experiment. Mice were fed a standard rodent chow diet and were housed in a special pathogen-free facility. Mice were maintained at Stanford University in compliance with the Stanford University Institutional Animal Care and Use Committee regulations. All procedures were approved by the Stanford University Administrative Panel on Laboratory Animal Care. Mice were euthanized by CO2 inhalation. Mice were monitored and assessed for tumor onset. Tumors were measured in two dimensions used digital calipers and tumor volume was calculated by the modified ellipsoidal formula , where length is the longest diameter and width is the shortest
Figure imgf000053_0001
diameter. Tumor onset was defined by the date on which a palpable tumor (tumor volume, V > 0.5 mm3) was observed. Tumor-free survival was plotted in a Kaplan-Meier curve with GraphPad Prism 9.5, and statistical significance was assessed by log-rank Mantel-Cox test. [0156] Flow cytometry analysis of tumors. In certain experiments, seven- to twelve-week-old female WT or Enpp1 KO BALB/cJ mice were inoculated with 5 x 104 WT or Enpp1 KO 4T1-luc cells, respectively, suspended in 50 μL of PBS into the fifth mammary fat pad. Upon reaching 100 ± 20 mm3, tumors were irradiated with 12 Gy and excised four days later. Alternatively, starting on day one after inoculation of 5 x 104 WT 4T1-luc cells into WT BALB/cJ mice, 100 μL of 100 μM neutralizing STING (WT) or non-binding STING (R237A) were injected intratumorally into the right fifth mammary fat pad every other day until the end of the experiment on day 15. Alternatively, 5 x 104 WT or Enpp1-/- 4T1-luc cells were orthotopically injected into WT or Enpp1-/- BALB/cJ mice respectively. Starting the next day, mice were intratumorally injected with 100 μL of 100 μM neutralizing (WT) or non-binding (R237A) STING every other day up to day 13. [0157] Subsequently, tumors were extracted and incubated in RPMI + 10% FBS with 20 μg/ml DNase I type IV (Sigma-Aldrich) and 1 mg/ml Collagenase from Clostridium histolyticum (Sigma-Aldrich) at 37 °C for 30 min. Tumors were passed through a 100 μm cell strainer (Fisher Scientific) and red blood cells were lysed using red blood cell lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA) for 5 min at room temperature. Cells were stained with Live/Dead fixable nearIR or blue dead cell staining kit (ThermoFisher). Samples were then fixed and permeabilized with eBioscience Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), Fc-blocked for 10 min using TruStain fcX (BioLegend) and subsequently antibody- stained with antibodies. Cells were analyzed using a Symphony (BD Biosciences), or an Aurora analyzer (Cytek). Data was analyzed using FlowJo V10 software (BD) and Prism 9.1.0 software (Graphpad) for statistical analysis and statistical significance was assessed using the unpaired t test with Welch’s correction. [0158] Single-cell RNA-seq of mouse primary tumors and lungs. In certain experiments, cryopreserved single cell suspensions of dissociated tumors and lungs from the ENPP1T238A- OE, ENPP1WT-OE, ENPP1A84S-OE metastasis experiments were thawed into warm RPMI media with 10% HI-FBS. Cells were handled gently with wide-bore tips from this point up until 10x GEM generation. Cells were washed with PBS and 0.04% BSA, passed through 40 μm Flowmi cell strainer (Bel-Art, 974-25244) before measuring concentration and viability with an automated cell counter. Viability for tumor samples were between 72-87% and for lung samples were between 91-96%. Cells were resuspended in PBS + 0.04% BSA to 1000 cells/μL. Single- cell suspensions were processed using the 10x Gemonics Single Cell 3’v3 RNA-seq kit. Gene expression libraries were prepared according to the manufacturer’s protocol with targeted cell recovery of 6000. Final 12 libraries were pooled and sequenced on the NextSeq 2000 platform (Illumina). Raw sequencing reads from the gene expression libraries were processed using CellRanger v3.0.2, aligning reads to the mm10 build of the mouse genome. Main processing, quality control, and analyses were performed with Cellenics platform (website: scp.biomage.net/data-management). [0159] Alternatively, Mammary fat pad primary tumors and lungs containing metastases were harvested from two ENPP1WT-OE or ENPP1T238A-OE tumor bearing mice when primary tumors reached 1000 mm3 (Figure 1). Cryopreserved single cell suspensions were thawed into warm RPMI media with 10% HI-FBS. Cells were washed with PBS and 0.04% w/v BSA, passed through 40 μm Flowmi cell strainer (Bel-Art, 974-25244), and assessed for concentration and viability with an automated cell counter. Cells were resuspended in PBS and 0.04% w/v BSA to 1000 cells/μL. Single-cell suspensions were processed with a Chromium Controller microfluidic device (10X Genomics), using the Chromium Next GEM Single Cell 3’ HT Reagent Kits v3.1 (Dual Index). Library preparation was performed according to the manufacturer's instructions (single cell 3’ HT reagent kits v3.1 protocol, Rev D, 10x Genomics). Briefly, cells were incorporated into gel bead-in-emulsions (GEM) and reverse-transcribed. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the appropriate molecular weight fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Illumina, R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. Pooled libraries were sequenced on NextSeq 2000 (Illumina). FASTQs were demultiplexed, mapped to the murine reference genome (Ensembl release 93 GRCm38), and gene counts were quantified using Cell Ranger (version 3.1.0). In total, we obtained 653,549,810 reads across 38,637 cells, and detected on average (median) 1,445 genes per cell. [0160] ScRNA-seq clustering and cell type annotation. All subsequent processing, quality control, and analyses were performed with Cellenics (website: scp.biomage.net/data- management). We filtered out cells with high mitochondrial content and high doublet count. We then applied Harmony data integration with HVGs = 2000 and performed dimensionality reduction using 27 principal components (90.18% variation). We performed Louvain clustering with resolution set to be 0.8. We manually annotated 18 clusters based on known cell markers (Figure 1Q). Specifically, we identified cancer cells based on markers enriched in 4T1 compared to BALB/cJ mammary fat pad (Schrörs et al., 2020). A separate cancer cluster was annotated for overexpressing Kif2c, which has been reported to drive CIN and metastasis (Bakhoum et al., 2018a) and additional genes related to metastasis (Schrörs et al., 2020). Among fibroblasts, a unique cluster enriched in tumors overexpresses myofibroblast marker Acta2, as well as Tgfb1 and Tgfb2 and are known as myofibroblastic cancer-associated fibroblasts (myCAF) and overtly immunosuppressive (Mhaidly and Mechta-Grigoriou, 2020; Mao et al., 2021). We also performed detailed subcluster analysis on macrophages (Figure 6J-6K) and T cells (Figure 6L-6M). Cluster frequency analysis, gene expression analysis, and differential gene expression are performed in Cellenics. [0161] ISPY-2 trial and patient dataset analysis. The ISPY-2 is an ongoing multicenter, Phase II neoadjuvant (therapies administered before surgery) platform trial for high-risk, early- stage breast cancer designed to rapidly identify new treatments and treatment combinations with increased efficacy compared to standard-of-care (sequential weekly paclitaxel followed by doxorubicin/cyclophosphamide [T-AC] chemotherapy (Wolf et al., 2022). Multiple investigational treatment regimens are simultaneously and adaptively randomized against the shared control arm. The primary efficacy end point is pathologic complete response (pCR). pCR is a known prognostic biomarker for long-term outcomes; achieving pCR after neoadjuvant therapy implies approximately 80% reduction in recurrence rate (Yee et al., 2020). Gene expression arrays were one of the two primary biomarker platforms assayed pre-treatment to identify biomarkers predictive of pCR and long-term outcomes of experimental neoadjuvant therapies and combinations. Details on conducting the gene expression array is listed in the section herein entitled “Molecular profiling of ENPP1 level using tissue mRNA expression”. The transcriptomic data of the study is available in NCBI’s Gene Expression Omnibus (GEO), SubSeries GSE 194040 (mRNA) (website: www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc= GSE194040). Normalized, batch corrected microarray data and sample metadata were retrieved from GSE194040. Individual two-tailed t-tests were performed comparing ENPP1 expression between patients who achieved a pCR and those who did not, within each ISPY-2 clinical arm. Similarly, ENPP1 expression was compared between tumors that were likely to respond to immunotherapy (immune+) and those that were not using two-tailed t-tests. Immune+ status was estimated based on the average dendritic cell and STAT1 signatures (Wolf et al., 2022). Additionally, distant-metastasis free survival (DMFS) of ENPP1-high versus ENPP1-low group was compared. The threshold for ENPP1-high versus ENPP1-low was set so that the P-value was the smallest. Specifically, “surv_cutpoint” function from the survminer package (www.sthda.com/english/wiki/survminer-0-2-4#determine-the-optimal-cutpoint-for-continuous- variables) in RStudio was used. [0162] Quantification and Statistical Analysis. In cGAMP degradation assays, half-life was obtained by one phase exponential decay fitting with Prism software with intercept (Y0) set up to be the initial cGAMP concentration, and plateau set to be 0. In proliferation assays, proliferation rate was obtained by exponential fitting with Prism software. In human PBMC and plasma cGAMP degradation activity assays, their relationship was obtained by linear regression fitting with Prism software. All statistical tests were performed using GraphPad Prism software and are noted in the figure legends. Data are presented as the mean ± standard deviation unless otherwise stated. [0163] Key resources table
Figure imgf000057_0001
Figure imgf000058_0001
Figure imgf000059_0001
Figure imgf000060_0001
[0164] Breast biopsy processing. Core needle biopsy (CNB) processing followed published protocols (Wolf et al., 2022; Joe and Esserman, 2023). Briefly, a small skin incision was made through which the core biopsy needle (typically 9 to 14 gauge [approximately 2.1 mm outer diameter]) was introduced. The shortest path to the lesion was typically chosen. Patient safety (eg, staying parallel to the chest wall to avoid pneumothorax) was an important factor in the approach. CNB was typically performed under local anesthesia. CNB of 16-gauge were taken from the primary breast tumor before treatment. Collected tissue samples were immediately frozen in O.C.T. embedding media and then stored in -80℃ until further processing. An 8^M section was stained with hematoxylin and eosin (H&E) and pathologic evaluation performed to confirm the tissue contained at least 30% tumor. A tissue sample meeting the 30% tumor requirement was further cryosectioned at 30^M for downstream analysis. [0165] Molecular profiling of ENPP1 level using tissue mRNA expression. Gene expression arrays of breast sections described in the section herein entitled “Breast biopsy processing” were performed following published protocol (Wolf et al., 2022). Briefly, twenty to thirty sections were collected and emulsified in 0.5ml Qiazol solution and the tubes underwent RNA extraction and gene expression profiling on Agilent 44K (Agilent_human_DiscoverPrint_15746 with annotation GPL30493 (update of GPL16233); n=333) or 32K (Agendia32627_DPv1.14_SCFGplus with annotation GPL20078; n=654) expression arrays. For each array, the green channel mean signal was log2-tranformed and centered within array to its 75th quantile as per the manufacturer’s data processing recommendations. All values indicated for non-conformity are NA’d out; and a fixed value of 9.5 was added to avoid negative values. [0166] ENPP1 mRNA level was used to predict pCR of the following neoadjuvant therapies in the I-SPY2 trial. More details are described in Example 13. 1. Paclitaxel + Pembrolizumab: i. ENPP1 mRNA ≤ 6.0, will have pCR ii. ENPP1 mRNA > 6.0, have a chance of not having pCR 1. ENPP1 mRNA > 8.9, have a heightened chance of not being pCR 2. Paclitaxel + ABT888 + Carboplatin: i. ENPP1 mRNA ≤ 6.5, will be pCR ii. ENPP1 mRNA > 6.5, have a chance of not being pCR 1. ENPP1 mRNA > 8.7, have a heightened chance of not being pCR 2. ENPP1 mRNA > 10.4, will not be pCR [0167] ENPP1 mRNA level was used to predict DMFS of Paclitaxel + Pembrolizumab neoadjuvant therapy in the I-SPY2 trial. More details are described in Example 13. 1. Paclitaxel + Pembrolizumab: i. ENPP1 mRNA < 8.3, 0% chance of developing distant metastasis up to 7 years. ii. ENPP1 mRNA ≥ 8.3, have a 12% chance of developing distant metastasis within 4 years. [0168] Molecular profiling of ENPP1 status using immunohistochemistry (IHC). IHC of ENPP1 can be performed following previously published protocol (Li et al., 2021). Briefly, one to five sections obtained as described in the section herein entitled “Breast biopsy processing” can undergo processing. The antigen is retrieved using sodium citrate pH6 buffer for 30 min. Following blockage with Background Buster (Innovex), the slides are incubated with 2.5 μg/ml anti-ENPP1 antibody (Abcam ab4003 at 1:200) for 4 hr, and then incubated with the biotinylated secondary antibody for 30 minutes. The Streptavidin-HRP D (DABMap kit, Ventana Medical Systems) and the DAB detection kit (Ventana Medical Systems) are used to detect the signal according to the manufacturer instructions. Then the slides are counterstained with hematoxylin and are mounted with Permount mounting medium. Tumor necrosis is assessed semi quantitatively by a certified pathologist based on the cross-sectional area containing necrosis. [0169] ENPP1 IHC level can be used to predict ENPP1 status: Any staining above 1% cells positive for ENPP1 expression per slide is considered positive for ENPP1. [0170] Molecular profiling of ENPP1 gene amplification testing using fluorescent in situ hybridization (FISH). Tissue biopsy obtained as described in the section herein entitled “Breast biopsy processing” can undergo FISH preparation following published protocol and updated guideline (Sauter et al., 2009; Wolff et al., 2023). Briefly, formalin-fixed paraffin-embedded (FFPE) specimens can be sampled at thickness of 3-4 mm and fixed for 18-24 hours in 10% neutral-buffered formalin. The tissues can be dehydrated in a series of alcohols and cleared through xylene, followed by impregnation with molten paraffin wax, heled at no more than 60 ℃. Tissue specimens can be sectioned between 4-6 ^M. Tissue sections mounted on charged slides can be held for up to 12 months at 2–8 °C before staining. Following sectioning, it is recommended that slides are incubated at 60 °C for one hour. Stained sections should be stored at -20 °C to preserve fluorescent signal and prevent fading. Each test section will be stained with the ENPP1 probes designed by Integrated DNA Technologies (Table below) and centromeric chromosome 6 control probe (CEP6, Empire Genomics). Slides will be visualized following the fluorescence microscope manufacturer protocol. A total of 20 cells of invasive carcinoma with optimal nuclear signals were randomly selected in 2–4 separate fields for evaluation. Signals of ENPP1 and CEP6 can be counted manually according to the specification of the kit. ENPP1 gene amplification status is defined below. In cases with equivocal results, a repeat counting of additional 20 cells in another 2–4 separate fields will be performed. Table. Top 5 ENPP1 FISH Probes (Integrated DNA Technologies).
Figure imgf000062_0001
[0171] ENPP1 FISH result can be used to predict ENPP1 status: Dual probe (ENPP1 and chromosome 6 control probes) 1. Positive: ENPP1/control ratio ≥ 2, OR ENPP1 copy number ≥ 6 regardless of ratio by ISH 2. Equivocal: ENPP1/control ratio < 2, AND ENPP1 copy number ≥ 4 but <6 3. Negative: ENPP1/control ratio < 2, AND ENPP1 copy number < 4 Single probe (ENPP1) 1. Positive: ≥ 6 ENPP1 copies 2. Equivocal: ≥ 4 but <6 ENPP1 copies 3. Negative: < 4 ENPP1 copies [0172] ENPP1 K173Q mutation sequencing: tissue. One tissue section collected as described in the section herein entitled “Breast biopsy processing” will be used. Genomic DNA (gDNA) of human PBMCs were isolated using DNeasy Blood & Tissue Kit (Qiagen). hENPP1 exon 4 region was amplified from donor gDNA templates and sequenced with hENPP1_K173Q_seq_fwd and hENPP1_K173Q_seq_rev primers (Table S1). To generate complementary DNA (cDNA), cells were lysed with 0.5-0.75 mL TRIzol (Invitrogen) and total RNA from cells were extracted using Direct-zol RNA Kit (Zymo). RNA was reverse transcribed in 20 ^L reactions containing 500 ng total RNA, 50 pmol Random Hexamer (Thermo Scientific) and 50 pmol Oligo(dT) Primers (Thermo Scientific), 0.5 mM dNTPs (NEB), 40 U RNaseOUT (Invitrogen), 1x Maxima RT Buffer (Thermo Scientific), and 200 U Maxmia Reverse Transcriptase (Thermo Scientific). RT reactions were incubated for 10 min at 25°C, then for 30 min at 50°C, and terminated by incubating for 5 min at 85°C. The entire hENPP1 exon region was amplified from donor cDNA templates and sequenced with hENPP1_seq_1-10 primers (Table S1). Patient can be A/A (WT), A/C (heterozygous), or C/C (homozygous) allele for K173Q single nucleotide polymorphism. [0173] ENPP1 K173Q mutation sequencing: blood. Collect 10 mL whole blood. Dilute whole blood with sterile PBS at a 1:1 v/v ratio. Prepare a tube with Ficoll density gradient medium (DGM) as per the manufacturer's instructions e.g., a 50 mL tube with 15 ml DGM. Gently, overlay the diluted blood onto the DGM taking care not to mix the layers. Centrifuge at 1000 x g for 20 mins at room temperature and with the brake off to generate distinct plasma, peripheral blood mononuclear cells (PBMCs), DGM, and red blood cell layers. Insert a pipette directly through the plasma layer and carefully harvest the PBMC layer via gentle aspiration and transfer it to a fresh 50 mL tube. Add wash buffer (PBS+0.5% BSA) to the harvested PBMC to give a final volume of 45 mL and mix well. Centrifuge at 400 × g for 10 mins at room temperature with the brake on. Generate cDNA following the same method described in the section entitled “ENPP1 K173Q mutation sequencing: tissue”. [0174] Molecular profiling of circulating ENPP1 level. Collect at least 200uL blood sample into commercially available anticoagulant-treated tubes, e.g., citrate-treated (light blue tops) or heparin-treated (green tops) (ThermoFisher). Avoid using ethylenediaminetetraacetic acid (EDTA) coated tubes for blood collection as it interferes with ENPP1 activity assay. Centrifugation for 15 minutes at 2,000 x g depletes platelets in the plasma sample. Immediately transfer the liquid component (plasma) into a clean polypropylene tube. The samples should be maintained at 2–8°C while handling. If the plasma is not analyzed immediately, the plasma should be apportioned into 0.1 ml aliquots, stored, and transported at –20°C or lower. It is important to avoid freeze-thaw cycles. Plasma ENPP1 level can be analyzed using either activity-based or protein-based assays. Details of an ENPP1 activity-based assay are described in the section herein entitled “ENPP1 activity assay by [32P] cGAMP degradation thin-layer chromatography assays”. Briefly, 22 ^L fresh serum or plasma was reacted with 5 ^M 32P- cGAMP spiked cGAMP at 37 ℃ and pH 7.4. cGAMP degradation is measured at 0, 10, 30, 60, 90, 120 minutes. Half-time of cGAMP degradation is calculated. For protein-based assay, ENPP1 protein levels will be validated using commercially available ENPP1 ELISA kits (MyBioSource.com #MBS451862; American Research Products Inc. #ARP-E1282L; Aviva Systems Biology #OKCD08756; Biomatik #EKF59219). [0175] Circulating ENPP1 levels in a breast cancer patient compared with normal populations can help infer if the patient’s breast cancer overexpresses ENPP1. The threshold is set as following: 1. ENPP1 activity assay: plasma ENPP1 cGAMP degradation half-life less than 60 minutes (the average degradation half-time in healthy donors, Figure 9K) in a breast cancer patient indicates the tumor overexpresses ENPP1 and sheds into circulation and is thus considered positive for ENPP1. 2. ENPP1 protein levels: Plasma ENPP1 levels is > 4.4 ^M/L (the average plasma ENPP1 concentration in healthy population, ProteinAtlas) in a breast cancer patient indicates the tumor overexpresses ENPP1 and sheds into circulation and is thus considered positive for ENPP1. EXAMPLE 2 Tissue- and cancer-derived ENPP1 contribute to tumor progression and metastasis [0176] ENPP1 expression level has been shown to correlate with poor prognosis in several cancer types, and we confirmed the same relationship in the context of breast cancer (Figure 1A). Patients in the METABRIC database with breast tumors expressing high levels of ENPP1 mRNA have significantly worse disease-free survival rate, despite exhibiting a similar distribution across disease stages as the ENPP1-low group (Figure 1A). This observation hints at the potential involvement of ENPP1 in tumorigenesis, and prompted us to ask whether host tissue, tumor, or both sources of ENPP1 expression are important for ENPP1’s tumorigenic role, using breast cancer as a model. To separately examine the contribution of cancer- versus tissue- derived ENPP1, we implanted WT or Enpp1-/- 4T1 breast cancer cells in WT or Enpp1-/- BALB/cJ mice. We treated established tumors with ionizing radiation (IR) to induce cGAMP production (Carozza, Böhnert, et al., 2020) when tumors were palpable around 100 mm3. Depleting either cancer- or tissue-derived ENPP1 had an additive effect on slowing tumor growth, with tissue ENPP1 playing a larger role (Figure 1B, 4A). While cancer and tissue ENPP1 additively contribute to intratumoral cGAMP hydrolysis activity, we found endogenous ENPP1 expression by the tissue is primarily responsible for the level of cGAMP hydrolysis activity in serum (Figure 1C, 4K). Therefore, primary tumor growth is primarily associated with cGAMP degradation activity of ENPP1 in the TME, with contributions from both the tumor and surrounding tissue. [0177] Next, we examined ENPP1’s role in breast cancer metastasis. We orthotopically implanted WT and Enpp1-/- 4T1 cells into WT and Enpp1-/- 4T1 mice respectively (WT x WT vs. KO x KO) and collected primary tumors and various tissues for ex vivo culturing on day 33 (Figure 1M). Since 4T1 cells are resistant to 6-thioguanine (6-TG) (Pulaski and Ostrand‐ Rosenberg, 2000), we used this treatment ex vivo to selectively kill stromal cells but allow growth of 4T1 colonies, allowing us to detect and quantify metastatic 4T1 cells in various destination tissues (Figure 1N). In WT x WT mice, distal metastasis was observed in draining inguinal lymph node (dLN), blood, lung and liver, but not in the brain, whereas in KO x KO mice, no metastasis was detected in any of the examined organs (Figure 4L). To disambiguate metastasis from primary tumor growth rate, we intravenously injected WT or Enpp1-/- 4T1 cells into WT or Enpp1-/- mice (Figure 1D) and again found that loss of ENPP1 in either the injected cells or in the whole animal decreased metastatic tumor burden in the lung (Figure 1E, F, 4B, 4M). Strikingly, total loss of ENPP1 (KO x KO) eliminated lung metastases in two thirds of the animals (Figure 1F, 4M). Therefore, we conclude that ENPP1 directly increases the metastatic potential of 4T1 breast cancer cells. Our results show that cancer- and tissue-derived ENPP1 act in combination to promote primary tumor growth and distal organ metastasis. Importantly, the complete lack of detectable metastasis in most ENPP1-deficient animals harboring ENPP1- deficient tumors indicates a significant therapeutic potential of ENPP1 inhibition to protect against metastasis. ENPP1’s catalytic activity drives breast tumor growth and metastasis by restricting adaptive immune infiltration [0178] ENPP1 expression levels have been shown to correlate with poor prognosis in several cancer types. Patients in the METABRIC database with breast tumors expressing high levels of ENPP1 mRNA have a significantly worse disease-free survival rate, despite exhibiting a similar distribution across disease stages as the ENPP1-low group (Figure 1A). Furthermore, patients with stage IV metastatic disease have significantly higher ENPP1 RNA expression than patients with stage III disease (Figure 1K, 9E). To determine if ENPP1 expression is causally linked to poor prognosis in breast cancer, we sought to perform mechanistic studies in mouse models. We performed orthotopic implantation of Enpp1-knockout 4T1 murine breast cancer cells overexpressing either WT ENPP1 (ENPP1WT-OE) or catalytically dead ENPP1 (ENPP1T238A-OE) (Kato et al., 2018) into WT mice (Figure 1G-I). ENPP1WT-OE 4T1 cells exhibited faster primary tumor growth and more lung metastases (Figure 1L) without affecting cell proliferation (Figure 1J), implicating a non-tumor cell intrinsic mechanism of enhanced tumor growth and metastasis. To thoroughly characterize the impact of ENPP1 overexpression on the tumor microenvironment (TME), we performed single-cell RNA-seq (scRNA-seq) on primary tumors and lungs colonized by metastases collected from this experiment. We observed 32,539 cells that passed quality filters. We performed unsupervised graph-based clustering on all cells and identified 18 major clusters corresponding to reported cell types by manual annotation of lineage markers. (Figure 1O, 1Q-1R). [0179] Although the ENPP1WT-OE condition did not alter the composition of non-immune cells (Figure 1S), it led to a decreased proportion of conventional DC type 1 (cDC1s), T cells, and Gzmb+ cytotoxic NK cells in primary tumors (Figure 1P). Unexpectedly, overexpression of WT ENPP1 resulted in a dramatic decrease in tumor infiltrating naïve B cells in both the primary and metastatic TME compared with the catalytic mutant (Figure 1P). Together, we conclude that ENPP1’s catalytic activity restricts adaptive immune cell infiltration, contributing to its role in promoting breast cancer growth and metastasis. EXAMPLE 3 ENPP1’s catalytic activity promotes immune suppression in primary tumors and lung metastases [0180] Building on our findings that ENPP1 catalytic activity alters the composition of the tumor immune compartment, we next investigated its impact on the functional landscape of tumor- infiltrating immune cells. We first turned our attention to innate immune cells. We found increased expression of Arginase 1 (Arg1) in monocytes and macrophages in mice injected with ENPP1WT-OE 4T1 cells (Figure 2A, 2D), which is associated with pro-tumor myeloid-derived suppressor cells (MDSCs) (Rodriguez et al., 2004) and M2-like macrophages (Yang and Ming, 2014), respectively. We further categorized macrophages into four subtypes based on expression of reported macrophage identity markers (Cheng et al., 2021) and functional markers (Figure 2F, 2G). These four tumor-associated macrophage (TAM) subsets express different levels of anti-tumor M1-like macrophage markers versus pro-tumor M2-like macrophage markers, with Vcan+ TAMs being the most M1-like, and Pparg+ being exclusively immunosuppressive (Figure 2G). We observed a decrease in Vcan+ and C1qc+ TAMs and a concomitant increase in M2-like Spp1+ TAMs in ENPP1WT-OE, suggesting that ENPP1 catalytic activity favors the polarization of M1-like to M2-like TAMs (Figure 2H, 9A). This is consistent with our previously reported effect of extracellular cGAMP depletion on macrophage polarization (Cordova et al., 2021). [0181] Focusing on antigen presenting cells (APCs) that orchestrate innate-adaptive crosstalk, we noticed a decrease in Itgae expressing migratory cDC1 in primary tumors, but not metastases, in the presence of ENPP1 catalytic activity (Figure 2B, 2E), mirroring the effects we previously observed with extracellular cGAMP depletion during ionizing radiation (IR) treatment (Carozza, Böhnert, et al., 2020). Additionally, cDC1s in ENPP1WT-OE primary tumors, but not metastases, express less H2-Ab1, suggesting decreased antigen-presentation capacity (Figure 2B, 2E). We reason that APC recruitment and education at the initial site of cancer encounter is important in mounting a successful adaptive immune response, and this process appears to be diminished when tumors overexpress ENPP1. [0182] Examining changes in adaptive immune cells, we found that the relative abundance of T cell subpopulations was not significantly altered between the WT and catalytic mutant ENPP1 conditions (Figure 2I, J, K). However, compared to ENPP1T238A-OE, WT ENPP1 activity decreased the expression of Cd69 (an early T cell activation marker) and Ilr2a (a late T cell activation marker), and increased the expression of Pdcd1 and Tox (exhausted T cell markers) in the T cells infiltrating both primary tumors and lung metastases (Figure 2C). In summary, our scRNA-seq data suggests that cancer cell-derived ENPP1 catalytic activity attenuated T cell activation while promoting exhaustion in primary tumors and sites of metastasis. Taken together, ENPP1 catalytic activity shapes the immunosuppressive TME both in primary tumors and metastases. EXAMPLE 4 ENPP1 overexpression in cancer cells inhibits STING signaling to suppress anti-tumor immunity [0183] To understand the mechanisms of cancer-derived ENPP1 overexpression in driving immunosuppression, we first analyzed expression of genes in the extracellular cGAMP-STING pathway (Figure 3A-B, 3J). Notably, Cgas expression is the highest in a distinct cancer cell cluster annotated for overexpressing Kif2c, which has been reported to drive CIN and metastasis (Bakhoum et al., 2018a). In contrast, Sting1 is expressed at relatively high levels in endothelial cells, fibroblasts, macrophages, and DCs (Figure 3A). The differential expression of cGAS and STING supports our model that cancers produce and secrete cGAMP, which is then detected by surrounding host cells (Carozza, Böhnert, et al., 2020). We defined potential cGAMP responder cells as those with low expression of Cgas and high expression of Sting1 and interferon- stimulated genes (ISGs) Ifitm1, Ifitm2, and Ifitm3 (Figure 3B, 3J), which matched well with our previous report of cell types that respond to extracellular cGAMP (Cordova et al., 2021). We then examined expression of the only known murine cGAMP transporters, LRRC8A:C and LRRC8A:E complexes (Lahey, Mardjuki, et al., 2020; Zhou et al., 2020), in these responder cells. While endothelial cells, macrophages, and cDCs express genes encoding the LRRC8A:C complex, fibroblasts uniquely express genes encoding the LRRC8A:E complex (Figure 3B). [0184] We next examined how overexpression of WT ENPP1 in cancer cells affected STING activation in cGAMP responder cells as measured by their combined Ifitm1, Ifitm2, and Ifitm3 expression (Ifitms). Looking first at endothelial cells, we found that elevated ENPP1 catalytic activity from ENPP1WT-OE 4T1s suppressed endothelial Ifitms expression, with a more pronounced effect in primary tumors than lung metastases (Figure 3C, 3K). Interferon (IFN) signaling is known to downregulate vascular endothelial growth factors (VEGFs) and inhibit angiogenesis (von Marschall et al., 2003); we observed that endothelial cells with blunted IFN signaling in ENPP1WT-OE primary tumors express more Vegfc (Figure 3C, 3K). Turning our focus to TAMs, we show that in metastasis, ENPP1WT-OE 4T1s with increased cGAMP degradation activity led to decreased expression of Ifitms as expected (Figure 3D). However, we observed the opposite trend in primary tumors where Ifitms expression in TAMs increased in ENPP1WT-OE tumors (Figure 3D). We noticed that Lrrc8c expression in TAMs is also higher in ENPP1WT-OE primary tumors but not metastases (Figure 3L). It is possible that increased cGAMP import activity in ENPP1WT-OE primary tumors contributes to the unexpected increase in their Ifitms expression. [0185] To understand how ENPP1WT-OE 4T1s promote immunosuppressive phenotypes in primary tumor-resident TAMs despite increased ISG expression in these cells, we examined the activation status of the eADO pathway: a potential STING-independent downstream effect of cGAMP hydrolysis (Figure 3M). We observed increased expression of Adora2b (the eADO receptor), Tgfb1 and Il10rb (downstream of eADO signaling), and Hp (a gene that is transcriptionally upregulated by adenosine signaling) in ENPP1WT-OE primary tumor associated macrophages, indicating activation of the eADO pathway in TAMs by ENPP1 catalytic activity specifically in primary tumors but not in metastases (Boison and Yegutkin, 2019; Hatfield et al., 2019; De Córdoba et al., 2022) (Figure 3N). Haptoglobin (HP) secretion by cancer cells upon of eADO signaling has been reported to recruit polymorphonuclear MDSCs (PMN-MDSCs) and promote self-seeding of ENPP1-high circulating tumor cells (CTCs) (De Córdoba et al., 2022). While we did not observe increased Hp expression in ENPP1WT-OE cancer cells, we found that neutrophils and monocytes in the ENPP1WT-OE metastatic niche had the biggest increase and the highest overall expression of Hp, suggesting that they are the potential source of HP production that facilitate metastasis (Figure 3O). Together, our data suggest a model in which ENPP1 overexpression promotes primary tumor growth and metastasis through synergistic stimulation of the eADO pathway and inhibition of the STING pathway (Figure 3P). EXAMPLE 5 ENPP1 expressed on host responder cells suppresses paracrine STING activation [0186] We next turned our attention to a third class of cGAMP responder cells, myofibroblast- like cancer-associated fibroblasts (myCAFs): a subtype of fibroblasts with immunosuppressive functions (Mhaidly and Mechta-Grigoriou, 2020). Like TAMs, myCAFs also exhibited an unexpected, albeit not statistically significant due to small sample sizes, decrease in STING pathway activation as measured by Ifitms expression, with a concomitant increase in Enpp1 expression, in ENPP1T238A-OE primary tumors (Figure 3E, F, 9C). We observed similar increases in ENPP1 expression along the oncogenic trajectory from normal tissue to tumor-adjacent tissue to primary tumor in human breast invasive carcinoma (BRCA) (Figure 3Q, 9F). Previous studies and our analysis so far have focused on ENPP1 expressed by cancer cells. However, these data hint at the importance of the ENPP1 level expressed by a responder cell in regulating its paracrine STING signaling. [0187] To build a more comprehensive picture of Enpp1 expression across host cells in the TME, we examined its expression level and downstream impact on STING activation in stromal and immune cells. Among the responder cells, we found that fibroblasts, macrophages, and DCs express relatively high levels of Enpp1 (Figure 3G). Endothelial cells, on the other hand, do not express measurable levels of Enpp1 (Figure 3G), which could explain the predominant effect of cancer-derived ENPP1 on their STING activation profile (Figure 3C). Additionally, we found that Ifitms expression in subpopulations of TAMs in the metastases anti-correlates with their Enpp1 expression level (Figure 3H). Specifically, there is a step-wise increase in Enpp1 expression and a corresponding decrease in Ifitms expression from the immunostimulatory M1- like macrophages to the immunosuppressive M2-like macrophages (Figure 3H). This result could potentially explain our previous findings that M2-polarized macrophages are less sensitive to cancer-derived extracellular cGAMP than M1-polarized macrophages (Cordova et al., 2021). [0188] Expanding this correlation to other cell types, we performed differential gene expression analysis between Enpp1-high cells (Enpp1 > 1.24, 338 cells) and Enpp1-low cells (0 < Enpp1 < 1.24, 1040 cells) and found that Ifitm2 and Ifitm3 are among the most significantly downregulated genes in Enpp1-high cells (Figure 3I, 9D). Together, our results demonstrate that ENPP1 expressed on cGAMP responder cells potently inhibits these cells’ paracrine STING activation. These findings suggest the need to inhibit both cancer- and host-derived ENPP1 to alleviate its tumorigenic effect and emphasize the impact of host ENPP1 expression level in different cell types on the downstream effects of paracrine STING signaling EXAMPLE 6 ENPP1 alters the tumor immune infiltrate in an extracellular cGAMP-dependent manner [0189] We previously characterized short-term immunological changes induced by extracellular cGAMP in the 4T1 model utilizing cell-impermeable STING protein as a neutralizing agent to deplete extracellular cGAMP as well as R237A mutant STING that does not bind to cGAMP (NB) as a negative control (Carozza, Böhnert, et al., 2020; Cordova et al., 2021). To test if ENPP1 knockout recapitulates extracellular cGAMP mediated immunological changes in breast cancer development, we injected either WT neutralizing STING (Neu) or R237A non-binding STING (NB) into Enpp1 WT x WT or KO x KO mice on day one and every other day following orthotopic 4T1 implantation (Figure 4C). On day 15, we observed a near 75% reduction in tumor sizes upon Enpp1 deletion: an effect that was partially reversed with cGAMP neutralization but not negative control NB STING treatment (Figure 4D), supporting the notion that ENPP1 affects tumor growth at least partially through the modulation of extracellular cGAMP levels. [0190] We also analyzed these primary tumors by flow cytometry to measure changes in STING pathway activation and the immunological compartment in response to Enpp1 knockout with or without extracellular cGAMP depletion. Within the primary TME, we observed a significant increase in the percentage of macrophages in the Enpp1 KO x KO mice, a trend that was reversed upon cGAMP depletion (Figure 4E). We previously reported that antitumoral M1 macrophages directly sense tumor-derived extracellular cGAMP by phosphorylating their IRF3 (Cordova et al., 2021). Here, we observed increased IRF3 phosphorylation in these cells when Enpp1 is knocked out and this trend was again ablated upon cGAMP depletion (Figure 4F). In addition, we observed a cGAMP-dependent increase in antigen presenting CD103+ DCs that are known to be important for antitumoral responses (Figure 4G) (Salmon et al., 2016). As for adaptive immunity, Enpp1 deficiency increases the expression of activation markers CD25 and CD69 in the CD8+ cytotoxic T lymphocytes (CTLs) in an extracellular cGAMP-dependent manner (Figure 4H, 4I). Finally, we observed a decrease of the immunosuppressive regulatory T cell (Treg) marker FoxP3 upon ENPP1 knockout, which is reversed by cGAMP depletion (Figure 4J). Together, our data suggest that endogenous ENPP1 blocks the extracellular cGAMP mediated immune activation to sense and defend against growing primary tumors. EXAMPLE 7 Enpp1 knockout in cancer and tissue cells additively delays tumor growth and abolishes metastasis [0191] Next, we formally tested the relative contribution of cancer- and host-derived ENPP1 on breast tumor growth and metastasis. We implanted WT or Enpp1-/- 4T1 cells into WT or Enpp1-/- BALB/cJ mice and treated established tumors (palpable around 100 mm3) with ionizing radiation (IR) to further induce cGAMP production (Carozza, Böhnert, et al., 2020). Indeed, depleting cancer- or tissue-derived ENPP1 had an additive effect on slowing tumor growth, with tissue ENPP1 playing a larger role (Figure 1B, 4A). Cancer- and host-derived ENPP1 contribute equally to intratumoral cGAMP degradation activity, while tissue-derived ENPP1 is mainly responsible for cGAMP degradation in serum (Figure 1C, 4K). [0192] We also investigated how the ENPP1 source impacts metastasis by orthotopically implanting WT or Enpp1-/- 4T1 cells into WT or Enpp1-/- 4T1 mice, respectively (WT x WT vs. KO x KO) and collected various organs for ex vivo culturing at experimental end point. In WT x WT mice, distal metastasis was observed in the draining inguinal lymph node (dLN), blood, lung and liver, but not in the brain. On the other hand, we observed no metastasis in KO x KO mice (Figure 4L). Since the KO x KO mice also have attenuated primary tumor growth, we reasoned that this could mask direct effects on metastasis, and therefore sought to disambiguate metastasis from primary tumor growth rate by intravenously injecting 4T1 cells. Again, we found that total loss of ENPP1 in cancer cells and host tissue rendered two thirds of the mice metastasis-free (Figure 1E, 1F, 4B, 4M). Our results support a model in which both tissue-derived and tumor- derived ENPP1 act in concert to promote primary tumor growth and distal organ metastasis. Importantly, the complete lack of detectable metastasis in most ENPP1-deficient animals harboring ENPP1-deficient tumors indicates a significant therapeutic potential of ENPP1 inhibition (Carozza, Brown, et al., 2020) to protect against metastasis. The antitumoral and immunostimulatory effect of ENPP1 deficiency associates with extracellular cGAMP preservation [0193] Our scRNA-seq analysis revealed that likely both dampening STING signaling and promoting eADO signaling contribute to an immunosuppressive primary TME in ENPP1 overexpressing tumors. When reversed to consider the ENPP1-deficient condition, we hypothesize that as less eADO will be generated, the extracellular cGAMP-STING signaling will play a dominant role in immune recruitment and activation. To test this hypothesis, we took advantage of previously developed cell-impermeable STING protein as a neutralizing agent to deplete extracellular cGAMP and compared it with R237A mutant STING that does not bind to cGAMP as a negative control (Carozza, Böhnert, et al., 2020; Cordova et al., 2021). We injected either WT neutralizing STING (Neu) or R237A non-binding STING (NB) into WT mice bearing WT 4T1 orthotopic tumors (WT x WT) or Enpp1 KO mice with ENPP1 KO 4T1 orthotopic tumors (KO x KO) (Figure 4C). On day 15, we observed a near 75% reduction in average tumor sizes upon Enpp1 deletion: an effect that was around 50% rescued with extracellular cGAMP neutralization but not with the NB STING treatment (Figure 4D). This trend suggests that Enpp1 knockout slows primary tumor growth at least partially through enhancing extracellular cGAMP levels. [0194] We also analyzed the immunological changes in these primary tumors in response to Enpp1 knockout with or without extracellular cGAMP depletion by flow cytometry. We observed an increase in the percentage of macrophages in KO x KO tumors, a trend that was reversed upon extracellular cGAMP depletion (Figure 4E). In KO x KO tumors, we observed moderate increase in STING activation in M1-like macrophages measured by IRF3 phosphorylation (Figure 4F), number of CD103+ migratory cDCs (Figure 4G), and expression of activation markers CD69 and CD25 in cytotoxic T cells (Figure 4H, I). Additionally, we observed decreased immunosuppressive regulatory T cell (Treg) marker FoxP3, all correlating with a more immunostimulatory TME (Figure 4J). However, these modest effects were abolished upon extracellular cGAMP depletion (Figure 4F-J). Together, our data draw connection between enhanced extracellular cGAMP signaling and immunological control of primary tumor growth upon Enpp1 loss. EXAMPLE 8 Selective silencing of ENPP1’s cGAMP hydrolysis activity delays breast cancer growth and metastasis in a STING-dependent manner [0195] Next, we formally investigated the contribution of suppressing extracellular cGAMP- STING signaling as the mechanism of action for ENPP1’s tumorigenic roles. We took advantage of the previously developed homozygous Enpp1H362A mouse model: a separation-of-function point mutant that does not degrade cGAMP but retains its catalytic activity towards ATP and other nucleotide triphosphate substrates (Carozza et al., 2022). We implanted E0771 syngeneic mouse breast cancer cells into WT or Enpp1H362A C57BL/6J mice and observed a delay in tumor growth (Figure 5F) and significantly improved survival outcomes in Enpp1H362A mice compared to WT mice (Figure 5A). Enpp1H362A mice retarded E0771 tumor growth to a similar degree as Enpp1-/- mice compared to WT mice (Carozza, Böhnert, et al., 2020), suggesting that ENPP1’s tumorigenic phenotype in the E0771 model is mediated mainly through cGAMP hydrolysis. To exclude potential STING-independent roles of cGAMP in this context, we compared orthotopic E0771 breast tumors in Sting-/- and Enpp1H362A x Sting-/- mice. Strikingly, we found that the slowing of primary tumor growth observed in Enpp1H362A mice was abolished in the Sting knockout background, indicating that ENPP1’s tumor-promoting effect is indeed dependent on the cGAMP-STING signaling axis (Figure 5A, 5G). Given that E0771 cells do not express ENPP1 (Carozza, Böhnert, et al., 2020), our data suggest that the endogenous level of ENPP1 in the tissue is sufficient to dampen paracrine STING activation to a level that is conducive to promote primary tumor progression. [0196] Furthermore, we noticed a 69% increase in the tumor-free rate in Enpp1H362A compared with WT mice after E0771 implantation (4/32 [1.25%] vs.2/17 [0.74%]) (Figure 5A). From these results, we hypothesized that Enpp1 depletion also prevents primary tumor onset by increasing cGAMP-mediated immune surveillance. To test this hypothesis, we adopted a spontaneous breast tumor model of hemizygotic mice harboring the mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT, MMTV for short). The median time for female MMTV mice to develop spontaneous breast tumors is 11 months of age (Figure 5B). Remarkably, Enpp1H362A x MMTV mice with impaired extracellular cGAMP degradation activity exhibited delayed median tumor onset by 2 months (Figure 5B). These findings support a role for ENPP1 in promoting breast tumor initiation by dampening cGAMP-STING signaling. [0197] We next asked if the effect of ENPP1 on metastasis is also dependent on the extracellular cGAMP-STING pathway. We intravenously injected E0771.lmb, a derived metastatic cell line from the parental E0771 cells (Johnstone et al., 2015) into WT, Enpp1H362A, Enpp1-/-, Sting-/-, and Enpp1H362A x Sting-/- mice (Figure 5C). While 50% of WT mice had lung metastasis, none of the Enpp1H362A mice or Enpp1-/- had metastasis, suggesting that endogenous ENPP1 promotes metastasis mainly, if not exclusively, through extracellular cGAMP hydrolysis (Figure 5D, 5E). The anti-metastatic effect of blocking ENPP1’s cGAMP hydrolysis in the Enpp1H362A mice is STING-dependent (Figure 5D, 5E), strongly supporting a model in which dampening extracellular cGAMP-STING signaling is the main mechanism of action of ENPP1 in promoting both primary and metastatic breast cancer. Unlike in primary tumor growth where WT mice exhibited better survival than Sting-/- mice (Figure 5A), WT mice had the same metastatic burden as Sting-/- mice (Figure 5D, 5E), indicating that endogenous ENPP1 activity in the tissue is sufficient to keep STING activation below the threshold of meaningful immunological protection specifically against metastasis (Figure 5D, 5E). Selective inhibition of ENPP1’s cGAMP hydrolysis activity abolishes breast cancer metastasis a STING-dependent manner [0198] Apart from its cGAMP hydrolysis activity, ENPP1 is also known to degrade extracellular ATP (Carozza et al., 2022), and generate immunomodulatory adenosine as byproduct (Li et al., 2021). While our experiments above support a model in which extracellular cGAMP signaling is at least partially responsible for the effects of ENPP1 on tumorigenesis, we wanted to formally test the sufficiency of cGAMP hydrolysis to explain ENPP1’s pro-tumorigenic phenotypes. We took advantage of the previously developed homozygous Enpp1H362A mouse model: a separation- of-function point mutant that does not degrade cGAMP but retains its catalytic activity towards ATP and other nucleotide triphosphate substrates (Carozza et al., 2022). Expanding beyond the 4T1 tumor model, we observed delayed primary E0771 tumor growth as measured by improved survival outcomes (time taken for tumors to reach 1000 mm3) in Enpp1H362A mice compared to WT mice (Figure 5A). Enpp1H362A mice retarded E0771 tumor growth to a similar degree as Enpp1-/- mice, as compared to WT mice (Carozza, Böhnert, et al., 2020), and the tumor slowing effects in Enpp1H362A mice were completely abolished in the Sting1 knockout background (Figure 5A). [0199] Furthermore, we noticed a 41% increase in the tumor-free rate in Enpp1H362A compared with WT mice after E0771 implantation (4/32 [12.5%] vs.2/27 [7.4%]) (Figure 5A). Therefore, we hypothesized that Enpp1 depletion also disfavors primary tumor onset. To test this hypothesis, we adopted a spontaneous breast tumor model of hemizygous mice harboring the mouse mammary tumor virus-polyoma middle tumor-antigen (MMTV-PyMT, MMTV for short). The median time for female MMTV mice to develop spontaneous breast tumors is 11 weeks of age (Figure 5B). Remarkably, Enpp1H362A x MMTV mice with impaired extracellular cGAMP degradation activity exhibited delayed median tumor onset by 2 weeks (Figure 5B). These findings support cGAMP-mediated protection from tumor initiation upon ENPP1 blockade. [0200] Lastly, to determine whether blocking ENPP1’s cGAMP hydrolysis activity alone is sufficient for preventing metastasis, we intravenously injected E0771.lmb.PuroR (a derived metastatic cell line from the parental E0771 cells (Johnstone et al., 2015) engineered with puromycin resistance to allow for ex vivo selection) into mice of different genetic backgrounds. 50% of WT mice but none of the Enpp1H362A mice and only 11% of Enpp1-/- mice had lung metastasis. Moreover, the anti-metastatic effect of blocking ENPP1’s cGAMP hydrolysis activity in the Enpp1H362A mice is completely STING-dependent (Figure 5C). While WT had slower primary tumor growth as Sting1-/- mice (Figure 5A), the two genotypes had similar lung metastatic burden (Figure 5C), indicating that endogenous ENPP1 activity in the tissue fully blocks STING mediated immunological protection specifically against metastasis. Therefore, we postulate that deactivating ENPP1’s cGAMP hydrolysis activity to enhance paracrine STING signaling will be a promising therapeutic approach to impede breast cancer metastasis EXAMPLE 9 A common human ENPP1 variant, K173Q, exhibits increased cGAMP hydrolysis activity at the cell membrane [0201] Building on the findings that the cGAMP hydrolysis activity and tissue expression level of ENPP1 make major contributions to breast cancer development, we investigated the human population-level heterogeneity in these parameters. To this end, we collected lysates from peripheral blood mononuclear cells (PBMCs) and plasma from 15 human donors, sequenced their ENPP1 exons, and measured their cGAMP degradation activity. Strikingly, we found that donors with a heterozygous or homozygous lysine to glutamine variant at position 173 (K173Q) (Figure 6A, 6J) exhibit elevated ENPP1 activity in PBMC lysates in a K173Q allele dose- dependent manner, (Figure 6B, 6C), despite all having similar level of ENPP1 RNA expression (Figure 6K). The c.517A>C variant denoting this missense mutation is highly prevalent, with 20.9% allele frequency among the general population and, notably, 78.6% among Africans and African Americans (rs1044498, Genome Aggregation Database (gnomAD) was accessed on April 13, 2023 from website: registry.opendata.aws/broad-gnomad (Figure 6A). The large variation in cGAMP hydrolysis levels that remains after stratifying by K173Q zygosity may be due either to differences in PBMC composition across donors or K173Q-indepenent factors affecting ENPP1 activity, such as post-translational modifications (Figure 6C). [0202] ENPP1 is a type II transmembrane protein with a short N-terminal cytosolic tail, a transmembrane (TM) domain, and an extracellular region containing the extracellular somatomedin B-like (SMB) domains and the catalytic domain (Figure 6A). In addition to its full-length membrane-anchored form (memENPP1), an ENPP1 isoform beginning distal to the TM domain has been observed as a secreted soluble protein (secENPP1) (Belli, van Driel and Goding, 1993; Jansen et al., 2012). The K173 residue is in the extracellular somatomedin B-like 2 (SMB2) domain, and therefore should be present in both memENPP1 and secENPP1. However, plasma from K173Q donors (which would contain only soluble secENPP1) exhibited similar ENPP1 activity as plasma from WT donors (Figure 6D, 6L). [0203] The striking difference in K173Q impact between the membrane and soluble pool of ENPP1 from donors was puzzling to us, since secENPP1 and memENPP1 are typically correlated in most cell lines (Figure 6Q). To confirm that the K173Q substitution increases ENPP1 catalytic activity selectively at the membrane, we overexpressed K173Q or WT human ENPP1 in 293T cGAS ENPP1-/- cells (Figure 6M). The K173Q mutation does not affect memENPP1 protein expression level (Figure 6N), nor the amount of secENPP1 secreted (Figure 6O). While the K173Q lysates containing memENPP1 degraded cGAMP approximately twice as fast as WT lysates (Figure 6E), the K173Q supernatant containing secENPP1 degraded cGAMP at the same rate as WT supernatant (Figure 6F), mirroring the results we obtained from K173Q human donor samples. In addition to its enhanced cGAMP hydrolysis activity, we also found that membrane-tethered K173Q ENPP1 has enhanced ATP hydrolysis activity (Figure 6H). Since K173 is located distal to the catalytic pocket of ENPP1 we would not expect it to differentially affect cGAMP or ATP hydrolysis. [0204] To investigate why K173Q ENPP1 protein has enhanced hydrolysis activity only when membrane-anchored, we tested whether K173Q affects memENPP1 dimerization, given its location in the SMB domain that has been implicated in disulfide bond formation in hENPP1 (Gijsbers, Ceulemans and Bollen, 2003; Bellacchio, 2012; Jansen et al., 2012). Using non- reducing SDS-PAGE followed by western blotting, we could not detect any change in dimerization between the K173Q and WT proteins in 293T cell lysates (Figure 6G, 6P). Because K173Q is in the juxtamembrane region, we hypothesize that it enhances tethered ENPP1 activity by interacting with the cell membrane or other membrane proteins. (Figure 6I) (Costanzo et al., 2001). EXAMPLE 10 Transmembrane and secreted ENPP1 isoforms dictate primary tumor growth and metastasis respectively [0205] Given that a common ENPP1 variant has enhanced activity specifically in the transmembrane pool, but not in the secreted pool, we next addressed the relative contributions of transmembrane and secreted ENPP1 to the tumor-promoting effects of this enzyme. To experimentally investigate this question, we first sought to look for an ENPP1 variant that selectively produces the transmembrane over the secreted isoform. The secENPP1 isoform is likely to be proteolytically processed by the signal peptide complex (SPC) owing to a predicted motif at A84/K85 (Figure 7A, 7G) (Teufel et al., 2022). Additionally, there is previous evidence arguing against splice isoforms or ectodomain shedding as mechanisms of isoform origin (Davis et al., 2006; Kato et al., 2018) Therefore, we expressed seven ENPP1 variants with mutations at or near the putative signal peptidase cleavage site in 293T cGAS ENPP1-/- cells, seeking mutants that would manipulate the mem:sec ratio. From this mutational scan, we identified A84S as a variant that compromised secENPP1 production (Figure 7H, 7I), which correlates to a known human single nucleotide polymorphism (SNP) (A102S, rs1250860929, Genome Aggregation Database (gnomAD) was accessed on April 13, 2023 from website: registry.opendata.aws/broad-gnomad. Other point mutations at position 84 had similar effects (Figure 7H, 7I). [0206] We overexpressed WT, A84S, and catalytic mutant T238A (Kato et al., 2018) versions of ENPP1 in 4T1 Enpp1-/- cells (denoted as ENPP1WT-OE, ENPP1A84S-OE, and ENPP1T238A- OE 4T1s) (Figure 7B). As expected, ENPP1A84S-OE 4T1 cells exhibited an increase in the proportion of memENPP1 without affecting its overall expression level nor the cells’ ability to degrade cGAMP in culture (Figure 7J, 7K). To understand how altering mem:secENPP1 ratio on cancer cells would affect cGAMP activity in the TME and serum respectively, we collected primary tumors and sera from WT BALB/cJ mice with orthotopic 4T1 tumors reaching 1000m3 (Figure 7L) and tested cGAMP degradation activity ex vivo. In both tumor and serum, ENPP1T238A-OE tumors represent the endogenous level of cGAMP hydrolysis activity from tissue ENPP1. First, we observed that ENPP1WT-OE tumors increased cGAMP hydrolysis activity not only in the TME, but also in the serum, suggesting that secENPP1 can be cleared into circulation and that tumors overexpressing ENPP1 can lead to detectable increase in serum cGAMP hydrolysis activity (Figure 7C). Next, we confirmed that compared to ENPP1WT-OE cells, ENPP1A84S-OE cells with increased surface tethering of ENPP1 led to increased intratumoral ENPP1 activity and a corresponding decrease in serum ENPP1 activity similar to the baseline level in ENPP1T238A-OE (Figure 7C). [0207] Primary tumor growth rate correlates with intratumoral cGAMP degradation activity, but not with serum cGAMP degradation activity (Figure 7D), suggesting that memENPP1 exerts local regulation of extracellular cGAMP in the TME and subsequently influences primary tumor progression. In addition to primary tumors, we sacrificed the mice when their primary tumors reached 1000m3 to quantify lung metastatic burden. Strikingly, ENPP1WT-OE with elevated serum ENPP1 activity from secENPP1 had significantly higher amount of lung metastases than ENPP1T238A-OE or ENPP1A84S-OE (Figure 7E). Together, we elucidated distinctive roles by memENPP1 and secENPP1 isoforms in primary cancer growth and metastasis (Figure 7F). EXAMPLE 11 ENPP1 drives distinct long-term immune-suppressive programs in primary and metastatic niche [0208] To thoroughly characterize long-term immunological changes in primary and metastatic sites in 4T1 breast tumor model, and to delineate contribution by memENPP1 and secENPP1, we performed single-cell RNA-seq (scRNAseq) on primary tumors and lungs collected from mice in the above experiment bearing ENPP1T238A-OE, ENPP1WT-OE, or ENPP1A84S-OE tumors. After strict quality control and filtration, we collected a total of 43,752 cells. We performed unsupervised graph-based clustering on all cells and identified 11 major clusters based on canonical cell markers (Figure 8A, 8O). Specifically, we identified cancer cells based on markers enriched in 4T1 compared to BALB/cJ mammary fat pad (Schrörs et al., 2020). A separate cancer cluster was annotated for overexpressing Kif2c, which has been reported to drive CIN and metastasis (Bakhoum et al., 2018) and additional genes related to metastasis (Schrörs et al., 2020). Among fibroblasts, a unique cluster enriched in tumors overexpresses myofibroblast marker Acta2, as well as Tgfb1 and Tgfb2 and are known as myofibroblastic cancer-associated fibroblasts (myCAF) and overtly immunosuppressive (Mhaidly and Mechta-Grigoriou, 2020; Mao et al., 2021) [0209] To better characterize myeloid cells, we performed unsupervised clustering on this subcluster (Figure 8B, 8P). In addition to basophils, plasmacytoid dendritic cells (pDCs), conventional DC class 1 (cDC1), cDC2, and neutrophils, we annotated five subsets of macrophages based on marker genes reported by recent publication, including Vcan, C1qc, Spp1, and Pparg macrophages (Cheng et al., 2021). Notably, Spp1+ and Pparg+ macrophages exist in tumors and lungs specifically, suggesting heterogeneous transcriptomic patterns between macrophages from different tissues. These five macrophage subsets express a spectrum of anti- tumor M1-like markers Cd86, Nos2, Ptgs2, H2-Ab1 and pro-tumor M2-like markers Tgb1, Arg1, Cd200, Mrc1, with Vcan+ macrophages being most M1-like and Pparg+ macrophages being exclusively immunosuppressive (Figure 8Q). We also categorized monocytes into lung monocytes and three subsets of tumor monocytes based on their functional states (Figure 8Q). While monocyte from the lungs showed higher expression of neutrophil-associated gene Csf3r suggestive of its blood-derived origin, tumor-infiltrating monocytes cluster 1-3 progressively expressed more inflammatory cytokines and chemokins, Il1b and Cxcl2, cell growth regulator Egr1, and tissue resident marker Nlrp3, Nfkbia (Cheng et al., 2021). [0210] We also performed detailed subcluster analysis on T and NK cells (Figure 8C, 8R). After annotating naïve (Sell+), effector (Cd44+), and regulatory (Foxp3+) CD4+ T cells, a unique CD4+ cluster (T06) remained. Differential gene analysis between this cluster with the other three CD4+ T cell clusters showed significantly higher expression of Tox, Sox4, lncRNA 2610407P16Rik/Casc15 (Figure 8S). Tox has well documented roles in CD8+ T cell exhaustion (Khan et al., 2019; Sekine et al., 2020) and its involvement in CD4+ T cell started to gain understanding (Miggelbrink et al., 2021). Sox4 has been reported to drive immune evasion in breast cancer (Bagati et al., 2021) and its expression is upregulated by Casc1570 kb downstream of Sox4 (Sun et al., 2021). All three markers point towards an immunosuppressive function of this Tox+ CD4+ T cell cluster. [0211] After thoroughly characterizing cell types in the TME, we then investigated expression genes in the STING pathway (Figure 8D). Enpp1 is expressed by most host cells and is the highest in fibroblasts, explaining the considerable contribution of tissue-derived ENPP1. Interestingly, while Cgas expression is the highest in cancer cells, Sting expression is high in fibroblasts and selective myeloid cells (Figure 8D, 8T), consistent with our model that cancers produce and secret cGAMP, which is then detected by immune cells (Carozza, Böhnert, et al., 2020). Given our model that ENPP1 antagonizes extracellular cGAMP-STING pathway, we turned our attention to the expression of interferon stimulated genes (ISGs) downstream of STING activation (Figure 8D, 8U). Ifitm genes (Ifitm1, Ifitm2, Ifitm3) are the most abundantly and prevalently expressed ISGs and myeloid and cDCs express the highest amount of ISGs. Interestingly, we observed a step-wise decrease in expression of in all three Ifitm genes in more immunosuppressive monocytes (Figure 8E). [0212] We next asked how ENPP1 activity in the TME and serum impact ISG activation in immune cells, immune cell composition and functional states. Our previous work showed that intratumoral monocytes and macrophages directly sense tumor-derived extracellular cGAMP (Cordova et al., 2021). We first looked at these two populations in the primary TME to see if ENPP1 activity antagonizes cGAMP signaling. In the primary TME, memENPP1 expressed by ENPPWT-OE and ENPP1A84S-OE cancer cells promoted immunosuppressive tumor- infiltrating monocytes expressing less Csf3r and more inflammatory cytokines Il1b and Cxcl2 and tissue resident marker Nlrp3 (Figure 8F) (Cheng et al., 2021). MemENPP1 also reprogramed antigen presenting H2-ab1 expressing macrophages into immunosuppressive Tgfb1 and Arg1 expressing macrophages (Figure 8G), consistent with it antagonizing cGAMP’s role in M1-like macrophage polarization. [0213] In addition to these known immune populations, we observed a significant increase in the proportion of inactivated (Cd83-) pDCs out of total cells in ENPP1A84S-OE with high intratumoral ENPP1 (Figure 8H). pDCs are a subtype of DCs that normally specializes in INF-I production (Zhou, Lawrence and Liang, 2021). However, pDCs express low levels of ISGs despite high expression of Sting (Figure 8D), consistent with previous report of impaired INF-I production by pDCs in breast tumors (Sisirak et al., 2012). These pDCs have been reported to favor Treg expansion (Conrad et al., 2012; Sisirak et al., 2012). Indeed, ENPP1A84S-OE with the highest amount of pDCs also have the highest amount of Foxp3+ Tregs (Figure 8I). Additionally, we observed that intratumoral ENPP1 decreased the expression of Ifitm genes in CD8+ T cells and CTLs (Figure 8V), validating our previous finding that T cells can sense extracellular cGAMP (Cordova et al., 2021). As consequences, ENPP1A84S-OE decreased the amount of CD8+ T cells in the TME (Figure 8J). Surprisingly, increased memENPP1 in ENPP1WT-OE and ENPP1A84S-OE dramatically decreased the proportion of naïve B cells (Figure 8K). Tumor infiltrating naïve B cells can serve as professional APCs to CD8+ and CD4+ T cells in human breast carcinoma and correlate with positive prognosis in patients (Laumont et al., 2022). In summary, our scRNAseq analysis of tumors expressing WT ENPP1 or only membrane-bound ENPP1 provided strong support for our working model that tumor secreted cGAMP is dampened by ENPP1, which leads to a suppressive immune environment. [0214] Next, we looked into ENPP1 induced immunological changes in the lungs. Unlike primary tumors, we observed little changes in innate immune cells, perhaps because most of the innate immune education occurred before cancer colonization into lungs. We, however, observed differences in adaptive immune cells. Like primary tumors, metastatic tumors harboring the ENPP1WT-OE and ENPP1A84S-OE genotype have less infiltrating B cells (Figure 8L). ENPP1WT-OE, but not ENPP1A84S-OE led to decreased Ifitm1 expression in CD8+ T cells and CTLs, Ifitm2 in CTLs (Figure 8M), as well as higher exhaustion marker Tox expression in both CD4+ T cells and CTLs, suggesting a role for secENPP1 (Figure 8N). EXAMPLE 12 Enpp1 expression is inducible in stromal cells [0215] To identify transcriptional regulatory mechanisms of ENPP1, we look at changes in Enpp1 expression across clusters and conditions. Enpp1 undergoes a stepwise up-regulation in immunosuppressive macrophages (Figure 2H, 9A). Enpp1 is also significantly upregulated in myCAFs compared to fibroblasts (Figure 9B). When looking across conditions, we saw that Enpp1 is significantly higher in myCAFs associated with ENPP1T238A-OE condition with presumably the highest amount of intratumoral cGAMP, suggesting that Enpp1 may be upregulated by pathways downstream of cGAMP signaling (Figure 3F, 9C). To uncover putative transcriptional or epigenetic regulators, we performed a differential gene expression analysis between cells expression ENPP1-high group (Enpp1 > 1, 592 cells) and ENPP1-low group (0 < Enpp1 < 1, 961 cells) (Figure 3I, 9D). Transcriptional factor Ets1, tissue restricted nuclear transcriptional activator Aff3, transcriptional regulator Bach2, and epigenetic regulator Satb1 were significantly enriched and, therefore, are putative regulators of Enpp1. Notably, Ifitm2 and Ifitm3 are enriched in ENPP1-low group, confirming that ENPP1 on a responder cell dampen its STING activation. [0216] We next sought to examine if inducible ENPP1 expression in stromal cells is generalizable in the context of other cancer studies. Patients with stage IV metastatic disease have significantly higher tumor ENPP1 RNA expression than patients with earlier stage diseases (Figure 1K, 9E). We observed stepwise increases in ENPP1 expression along the oncogenic trajectory from normal tissue to tumor-adjacent tissue to primary tumor in human breast invasive carcinoma, glioblastoma multiforme, and stomach adenocarcinoma (Figure 3Q, 9F), supporting that tumors could induce Enpp1 expression in normal adjacent tissues. Telling of which specific stromal cells have inducible Enpp1 expression, a previous study showed that mouse endothelial cells (ECs) isolated from implanted or spontaneous breast tumors express higher levels of Enpp1 than those from normal breast tissues (Figure 9G) (Jeong et al., 2021). CIN-high cancer cells have been suggested to produce more cGAMP due to increased cytosolic dsDNA (Bakhoum et al., 2018). Additionally, we noted that Cgas is upregulated in Kif2c+ CIN-high cancer cells compared to Kif2c- cancer cells in our experiment (Figure 9H). Together, we identified a positive correlation between extracellular cGAMP buildup and Enpp1 upregulation in the TME. [0217] Furthermore, cGAMP is elevated in cases of ultraviolet (UV) exposure from sunlight (Skopelja-Gardner et al., 2020) and autoimmune conditions (Haag et al., 2018), and we wondered if ENPP1 RNA is upregulated in these contexts outside of cancer. Indeed, ENPP1 increased from non-exposed skin to sun exposed skin to melanoma (Figure 9I). ENPP1 also increased from endothelial/epithelial cells in skin biopsies from health individuals to lupus patients, which tracks with increase in ISGs and the putative transcriptional/epigenetic regulators we discovered (Figure 9J). Given the interferonopathy observed in these patients evident in the elevated ISG production in the same cell types, it is possible that ENPP1 is also an ISG. [0218] Finally, we measured serum ENPP1 activity from a breast cancer survivor and healthy donors from multiple blood draws across dates. The breast cancer survivor has significantly higher cGAMP degradation activity in the serum compared to health donors (Figure 9K), and this activity difference can be attributed to ENPP1 as treating serum with ENPP1 inhibitor STF- 1623 (Carozza, Brown, et al., 2020) collapsed the differences. We did not find mutations in the coding region, excluding alteration in protein activity or secretion. Therefore, ENPP1 is likely upregulated transcriptionally, and this difference can be readily measured in the serum. EXAMPLE 13 Low ENPP1 expression correlates with immune infiltration and pathological complete response (pCR) to Pembrolizumab (anti-PD1) and Veliparib + Carboplatin (PARP inhibitor) in breast cancer patients [0219] After delineating the molecular and cellular mechanisms governing the deterministic role of ENPP1 in metastasis of murine breast cancers, we asked whether these mechanistic insights and disease outcomes are conserved in humans. We hypothesize that, in addition to serving as a monotherapy, ENPP1 inhibition (ENPP1i) could potentially synergize with existing therapies that are upstream of cGAMP production, such as PARP inhibitor (PARPi) that induces DNA damages (Ding et al., 2018), or downstream of STING mediated immune infiltration, such as anti-PD1/PDL1 (Tumeh et al., 2014). In the I-SPY2 clinical trial (Wolf et al., 2022), breast cancer patients who had immune-positive (Immune+) tumors as defined by dendritic cell and STAT1 (downstream of IFN-I signaling) (Ivashkiv and Donlin, 2014) signatures had significantly less ENPP1 universally across all 10 treatment arms (Figure 10A). However, ENPP1-low patients had statistically significantly higher chance of pathological complete response (pCR) in Pembrolizumab (anti-PD-1) and Veliparib (PARP inhibitor) + Carboplatin treatment groups as neoadjuvant therapies, but not in other treatment groups (Figure 10B). These are the only two treatment arms out of the ten arms that have a clear mechanistic connection to cGAMP-STING signaling since anti-PD-1 efficacy requires CD8+ T cell infiltration (Tumeh et al., 2014) and PARP inhibitors (PARPi) induces DNA damage which increases cGAMP production (Ding et al., 2018). Most strikingly, 100% of ENPP1-low patients (n = 32) remained free of distant metastasis to date, near seven years after Pembrolizumab treatment and surgery, while only around 85% of ENPP1-high patients (n = 32) remained free of distant metastasis. (Figure 10C). Together, patients may have achieved complete response to Pembrolizumab at least partially due to enhanced cGAMP-STING mediated immune infiltration permitted by an ENPP1-low setting, which confers long term advantage in prognosis. The strong correlation suggests that lowering ENPP1 activity in humans is also sufficient to elicit cGAMP- STING signaling and provide long term protection to metastasis. ENPP1 blockade is therefore a promising immuno-oncology strategy that could synergize with existing anti-PD-1/PD-L1 therapies in the clinics. Together, we put forward a model that boosting extracellular cGAMP- STING signaling is the major mechanism of action of ENPP1 inhibition in mounting immune protection against breast cancer, a strategy that could synergize with existing PARPi or anti- PD1/PDL1 therapies. (Figure 10D). Discussion [0220] This study is motivated by the mounting correlative evidence of ENPP1’s role in poor prognosis and metastases in breast cancers and a few contradicting mechanisms that have been brought forward. Previous studies linking ENPP1’s tumorgenic effects to its catalytically- and immunologically-independent functions relied on in vitro assays (Bageritz et al., 2014) or immunodeficient mouse models (Takahashi et al., 2015; Hu et al., 2019). Here, using syngeneic tumor models and genetic knock-out mice, we provide definitive evidence that endogenous ENPP1 promotes breast cancer onset, growth, and metastasis. Regarding ENPP1’s mechanism of action in primary tumors versus metastasis, a previous study postulated that the ENPP1/cGAMP/adenosine axis may contribute more to metastasis than to primary tumors using overexpression experiments (Li, Duran, Dhanota, Chatila, Bettigole, Kwon, Sriram, Humphries, Salto-Tellez, James, Hanna, Melms, Vallabhaneni, Litchfield, Usaite, Biswas, Bareja, Li, Martin, Dorsaint, Cavallo, Li, Pauli, Gottesdiener, DiPardo, et al., 2021). We formally tested this hypothesis by manipulating only the cGAMP hydrolysis activity of endogenous ENPP1 in Enpp1H362A mice. Our data showed that blocking STING signaling by endogenous ENPP1 alone is responsible for -mediated protection against metastasisENPP1’s role in tumor metastasis but less so in primary tumor growth. Together, our study provides a causal link between ENPP1’s cGAMP hydrolysis activity and its role in breast tumor metastasis, which is the dominant cause of breast cancer patient death. [0221] Previous studies on ENPP1’s roles in cancer focused on ENPP1 expressed on cancer cells (Takahashi et al., 2015; Hu et al., 2019; Bageritz et al., 2014, Lau et al., 2013; Li et al., 2021). Here, we discovered that tissue ENPP1 activity plays an equally if not more important role as cancer cell-intrinsic ENPP1. These findings highlight the fact that non-cancer cells in the TME are not passive bystanders but rather actively shape tumor development. Tissue ENPP1 activity vary in the human population. Here, we provide four mechanisms that affect ENPP1 activity. First, the common ENPP1 K173Q allele increases ENPP1 catalytic activity on the membrane, but not in serum. Second, the ENPP1 A84S SNP leads to increased membrane ENPP1 activity, but abolishes its secretion. Third, ENPP1 expression increases with cGAMP-STING signaling. Fourth, ENPP1 expression correlates with levels of four transcriptional regulators ETS1, AFF3, BACH2, and SATB1, and they are likely induced by STING signaling. The exact mechanism warrants future exploration. We predict that host tissue ENPP1 plays an important role in cancers that originate from tissues with high ENPP1 activity. We also hypothesize that the ENPP1 status of the tissue in which the cancer develops, either as the primary site or the site of metastasis, could dictate the extent of ENPP1’s role in tumor development and the efficacy of potential ENPP1 blockade therapy. Further work examining these questions in different cancer types and with different genetic backgrounds should clarify these points and contribute greatly to our understanding of which patients may benefit from ENPP1-targeted therapeutics. [0222] After uncovering that membrane tethered and secreted ENPP1 activity do not always track, we formally investigated the role of these two isoform in primary and metastasis progression as well as immune suppression. Indeed, we showed that ENPP1’s local control of cGAMP in the TME dictates its role in promoting primary tumor growth. This observation is in line with our understanding of paracrine extracellular cGAMP signaling as being short-ranged, as we have not detected biologically meaningful levels of cGAMP in the serum even after systemic viral infection or a lethal dose of whole-body radiation (Carozza et al., 2021). Compared to secreted ENPP1, cell surface-tethered ENPP1 is ideally poised to snatch a freshly exported or soon-to-be imported cGAMP molecule in physical vicinity to cGAMP transporters (Ritchie et al., 2019; Lahey et al., 2020; Cordova et al., 2021), thereby circumventing paracrine activation of the STING pathway within the TME. [0223] In contrast, secreted ENPP1 played a bigger role in promoting metastasis, perhaps because it helps with creating an immunosuppressive gradient along the trajectory of metastasis. In addition, cGAS in more activated in metastatic cancers (Bakhoum et al., 2018) and perhaps produces higher concentrations and longer range of cGAMP signaling, requiring longer range control of ENPP1. Our scRNAseq analysis of primary breast tumors as well as lung metastasis showed elevated levels of cGAS in metastatic cancer cells that are higher than surrounding immune cells. We observed high STING expression in infiltrating myeloid cells, and higher ISG expressions in these cells when infiltrated into tumors expressing enzymatically dead ENPP1 compared to in tumors expressing live or membrane retained ENPP1. Our analysis validated other previous known consequences of STING signaling activation, including increased immunosuppressive myeloid population and T cell exhaustion. In addition, we also discovered new immune cellular players and pathways in the primary TME shaped by intratumoral ENPP1, including B cell infiltration and cGAMP induced ENPP1 expression in normal tissues. Together, we bring forward a model that cancer cells export cGAMP and upregulate ENPP1 expression in self and surrounding stromal cells as a way to clear the cGAMP they produce, thereby circumventing canonical STING pathway activation along the trajectory of tumor metastasis. [0224] To further probe the transcriptional control of ENPP1, we turned to RNA-seq analysis of samples from breast cancer and lupus patients, which is another patient population where the STING-interferon pathway is known to be activated. Indeed, ENPP1 levels as well as its putative regulators ETS1, AFF3, BACH2, and SATB1are all elevated in non-lesions as well as lesions from lupus patients compared with samples from healthy donors. Elevated ENPP1 activity is even readily observable in serum from a breast cancer patient. Combining all pieces of evidence, we could tantalize the contribution of cGAMP signaling to Enpp1 upregulation along the oncogenic trajectory and in chronic inflammation or autoimmune conditions. Enpp1 upregulation is likely wired into a negative feedback loop to enable immune tolerance during autoimmunity but highjacked by cancers for immune evasion. [0225] Our data highlighted the notion that host-derived ENPP1 is not a passive bystander, but rather actively involved in shaping the tumor immune microenvironment. We hypothesize that the ENPP1 status of the tissue in which cancer develops, either as the primary site or the site of metastasis, and ENPP1 allele or expression level variations could dictate the extent of ENPP1’s role in tumor development. Furthermore, our I-SPY2 human clinical trial data elucidated ENPP1 as a potential prognostic biomarker: while ENPP1-low breast cancer patients would more likely benefit from anti-PD1 and PARPi therapies, ENPP1-high breast cancer patients may warrant combination therapy of ENPP1i with anti-PD1 or PARPi. [0226] In summary, our work pinpoints the cellular source, catalytic activity, enzyme isoform, and downstream pathways important for the pro-tumor and pro-metastasis effects of ENPP1 in several breast cancer models in mice. Future discovery of the regulatory mechanisms and genetic variants affecting ENPP1 activity, localization, and expression level in different tissues and cancer types will reveal additional strategies leading to immune evasion. As a central player dictating cancer-innate-adaptive immune communication through the STING pathway, ENPP1 is a promising target for cancer immunotherapy that may add to our arsenal of ICB therapeutics as a druggable innate immune checkpoint. [0227] Motivated by a lack of understanding of the role of paracrine cGAMP-STING signaling in cancer and contradictory mechanistic hypotheses of ENPP1’s role in cancer immunity, we aimed to uncover the causal molecular and cellular mechanisms by which ENPP1 impacts primary breast tumor growth and metastasis. While we previously identified ENPP1 as a cGAMP hydrolase, there has been significant debate as to whether its ability to deplete cGAMP and thereby dampen STING signaling is central to its pro-tumorigenic effects, as ENPP1 has other enzymatic activities toward ATP and other nucleotide triphosphates but also generates eADO – a cancer-associated metabolite – as a byproduct of its cGAMP hydrolase activity. Using an unbiased scRNA-seq approach, we systematically characterized the immunological impacts and signaling events upon overexpression of ENPP1’s catalytic activity in orthotopically implanted 4T1 cancer cells. We discovered that ENPP1-high cancer cells promote breast tumor growth by shunting the immunostimulatory cGAMP-STING pathway to the immunosuppressive eADO pathway, while fostering an angiogenic TME for tumor survival (Figure 3P). Using the Enpp1H362A variant that specifically abolishes ENPP1’s cGAMP hydrolysis activity and orthogonal molecular sponges to deplete extracellular cGAMP, we confirmed that cGAMP is the relevant substrate in in vivo cancer models in a manner dependent on downstream STING signaling. Together, our results demonstrate the importance of extracellular cGAMP-STING activation in antitumoral immunity and ascribe ENPP1 as an innate immune checkpoint of the extracellular cGAMP-STING pathway. [0228] ENPP1’s contribution to different stages of tumor development including initiation, progression, and metastasis was not well understood. Importantly, our work provides the first evidence that ENPP1 promotes breast cancer initiation (Figure 5B). Comparing between primary tumors and metastases, we noticed a stronger contribution of cGAMP-STING inhibition to the pro-metastatic phenotype of ENPP1 in our scRNA-seq analyses. We posit that this could be due to elevated cGAMP production along the oncogenic trajectory, as we showed that CIN-high pro- metastatic Kif2c+ cancer cells (Bakhoum et al., 2018b) expressed higher levels of Cgas (Figure 3A). While a previous study attributed the increasing role of cGAMP hydrolysis by ENPP1 in metastasis to it replacing ATP as the major source of eADO (Li et al., 2021), we raise an alternative explanation that direct dampening of cGAMP-STING activation is the culprit in metastasis. The causal link between ENPP1 levels in the TME and metastasis was established with the evidence that destroying ENPP1’s cGAMP hydrolysis activity phenocopied Enpp1 deletion in completely abolished metastasis. [0229] Prior studies supported a role for ENPP1 in promoting various cancer types, but largely focused on ENPP1 expressed on the cancer cells (Takahashi et al., 2015; Hu et al., 2019; Bageritz et al., 2014, Lau et al., 2013; Li et al., 2021). Our scRNA-seq analyses revealed that not only is ENPP1 expressed on many host responder cells, but that responder cell-derived ENPP1 has an outsized effect on blocking paracrine STING activation in those same cells. The importance of cancer- and responder cell- derived ENPP1 in tumor development is in line with our understanding of paracrine extracellular cGAMP signaling as being short-ranged. ENPP1 on the surface of cGAMP-producing and cGAMP-sensing cells would be ideally poised to snatch a freshly exported or soon-to-be imported cGAMP molecule in close proximity to cGAMP transporters (Ritchie et al., 2019; Lahey et al., 2020; Cordova et al., 2021), thereby circumventing paracrine activation of the STING pathway within the TME. As cGAS is rarely inactivated in cancer cells (Bakhoum et al., 2018b) and there is no known intracellular cGAMP hydrolase (Li et al., 2014), we bring forward a model that cancer cells export the high levels of cGAMP they produce, capitalizing on both cancer cell- and responder cell-derived ENPP1 for its extracellular clearance, to achieve immune evasion. [0230] While our study focused on ENPP1’s roles in breast cancers, our discoveries could potentially be generalized to other types of immunologically “cold” tumors. We hypothesize that ENPP1 also plays immunosuppressive roles in tumor types where either the cancer cells or the TME highly express ENPP1. We showed that ENPP1 is broadly expressed by previously known immunosuppressive immune and stromal cells such as the macrophages of the “M2” like phenotype myCAFs. In addition, we observed increased Enpp1 expression in myCAFs in both primary and metastatic niches of ENPP1T238A-OE 4T1 compared to ENPP1WT-OE 4T1, suggesting ENPP1 expression is inducible either directly or indirectly by extracellular cGAMP and or ATP. We therefore hypothesize ENPP1 should be targeted in tumors with high myeloid content and fibrosis. Future examination of ENPP1 in other mouse cancer models and patient cohorts are warranted to test our hypotheses. Future mechanistic studies on soluble factors, signaling pathways, and transcription factors that induce ENPP1 expression in the TME could lead to additional diagnostic and therapeutic insights. [0231] Together, our detailed understanding of the molecular and cellular mechanisms of ENPP1 and the paracrine cGAMP-STING pathway shed light into clinical translations. For example, our data highlight the notion that host-derived ENPP1 is not a passive bystander, but rather actively involved in shaping the tumor immune microenvironment. We hypothesize that the ENPP1 status of the tissue in which cancer develops, either as the primary site or the site of metastasis, together with ENPP1 allele or expression level variations, could dictate the extent of ENPP1’s role in tumor development and even the risk of tumor development altogether. Furthermore, the I-SPY2 human clinical trial data nominated ENPP1 as a potential companion diagnostic biomarker: ENPP1-low breast cancer patients are significantly more likely benefit from anti-PD- 1 and PARPi therapies than their ENPP1-high counterparts. Conversely, we predict that ENPP1- high breast cancer patients will greatly benefit from a combination of ENPP1 inhibition with anti-PD-1/anti-PD-L1 or PARPi treatments. 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[0234] Notwithstanding the appended claims, the disclosure is also defined by the following clauses: [0235] 1. A method for treating breast cancer in a subject, comprising: a) obtaining a sample from the subject; b) measuring ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in the sample from the subject, c) classifying the sample, wherein the sample has a high classification when its ENPP1 levels are increased; and d) if the sample has the high classification, then administering to the subject: i) an ENPP1 inhibitor and a PD1 inhibitor, when the breast cancer is responsive to a PD1 inhibitor; ii) an ENPP1 inhibitor and a PDL1 inhibitor, when the breast cancer is responsive to a PDL1 inhibitor, iii) an ENPP1 inhibitor and a PARP inhibitor, when the breast cancer is responsive to a PARP inhibitor, thereby treating the breast cancer in the subject. [0236] 2. The method of clause 1, further comprising: assessing that the breast cancer is responsive to a PD1 inhibitor when the breast cancer is responsive to a PD1 inhibitor; assessing that the breast cancer is responsive to a PDL1 inhibitor when the breast cancer is responsive to a PDL1 inhibitor; assessing that the breast cancer is responsive to a PARP inhibitor when the breast cancer is responsive to a PARP inhibitor. [0237] 3. The method of a preceding clause, wherein the treating is achieving a pathological complete response (pCR). [0238] 4. The method of a preceding clause, wherein the breast cancer is triple negative, microsatellite instability-high (MSI-H), or mismatch repair deficient (dMMR) when the breast cancer is responsive to a PD1 inhibitor; or the breast cancer has a BRCA1 mutation, a BRCA2 mutation, is HER2 negative, is locally advanced, or is metastatic when the breast cancer is responsive to a PARP inhibitor. [0239] 5. The method of a preceding clause, wherein the step d) further comprises iii) administering chemotherapy. [0240] 6. The method of clause 5, wherein the chemotherapy is paclitaxel. [0241] 7. The method of a preceding clause, wherein the sample is a breast cancer tissue sample. [0242] 8. The method of clause 7, wherein the measuring is selected from the group consisting of ENPP1 mRNA expression, ENPP1 immunohistochemical levels, ENPP1 gene amplification levels, and ENPP1 K173Q homozygosity. [0243] 9. The method of clause 8, wherein when the breast cancer is responsive to a PD1 inhibitor, the measuring is ENPP1 mRNA expression, and the high classification occurs when the breast cancer tissue sample ENPP1 mRNA expression has, relative to a reference, a value selected from the group consisting of 6.2 or above, 6.39 or above, 6.4 or above, 7.0 or above, 7.4 or above, 7.8 or above, 8.0 or above, 8.9 or above, 9.2 or above, 9.4 or above, 9.6 or above, 9.8 or above, 10.0 or above. [0244] 10. The method of clause 8, wherein when the breast cancer is responsive to a PARP inhibitor, the measuring is ENPP1 mRNA expression, and the high classification occurs when the breast cancer tissue sample ENPP1 mRNA expression has, relative to a reference, a value selected from the group consisting of 6.4 or above, 6.78 or above, 7.0 or above, 7.4 or above, 7.8 or above, 8.0 or above, 8.9 or above, 9.2 or above, 9.4 or above, 9.6 or above, 9.8 or above, 10.0 or above, 10.2 or above, 10.4 or above, and 10.5 or above. [0245] 11. The method of clause 9 or 10, wherein the reference is the ENPP1 mRNA expression in a population of breast cancer tissue samples. [0246] 12. The method of clause 8, wherein the measuring is ENPP1 immunohistochemical levels, and the high classification occurs when a percentage of cells positive for ENPP1 expression in the breast cancer tissue sample is 0.5 or above, 0.75 or above, 0.9 or above, 1 or above, 10 or above, or 50 or above. [0247] 13. The method of clause 8, wherein the measuring is ENPP1 gene amplification levels, and the high classification occurs when ENPP1/CEP6 probe ratio > 2, when ENPP1 copy number > 6, or when ENPP1 copies are > 6 if a single probe is used. [0248] 14. The method of clause 8, wherein the measuring is ENPP1 K173Q homozygosity, and the high classification occurs when the breast cancer tissue sample is homozygous for the K173Q mutation. [0249] 15. The method of a preceding clause, wherein the sample is a blood sample. [0250] 16. The method of clause 15, wherein the measuring is of ENPP1 activity through plasma ENPP1 cGAMP degradation half-life, and the high classification is when the cGAMP degradation half-life is less than 60 minutes, or less than 50 minutes or less than 70 minutes. [0251] 17. The method of clause 15, wherein the measuring is of ENPP1 plasma protein levels, and the high classification is when the ENPP1 plasma protein level is > 4.4 ^g/L, > 3.4 ^g/L, or > 5.4 μg/L. [0252] 18. The method of a preceding clause, wherein when the breast cancer is responsive to a PD1 inhibitor, then the PD1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, and toripalimab. [0253] 19. The method of clause 18, wherein the PD1 inhibitor is pembrolizumab. [0254] 20. The method of a preceding clause, wherein when the breast cancer is responsive to a PDL1 inhibitor, the PDL1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab. [0255] 21. The method of a preceding clause, wherein when the breast cancer is responsive to a PARP inhibitor, the PARP inhibitor is selected from the group consisting of veliparib, olaparib, rucaparib, niraparib, and talazoparib. [0256] 22. The method of clause 21, wherein the PARP inhibitor is veliparib. [0257] 23. The method of a preceding clause, wherein when the breast cancer is responsive to a PD1 inhibitor, the step d) further comprises administering a platinum complex selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin. [0258] 24. The method of clause 23, wherein the platinum complex is carboplatin. [0259] 25. The method of a preceding clause, wherein the ENPP1 inhibitor is described herein. [0260] 26. A method for treating a subject for a solid tumor, the method comprising: a) assessing mRNA expression in a solid tumor sample from the subject to obtain an mRNA expression result; and b) administering to the subject a therapy for the solid tumor based on the determined mRNA expression result; to treat the subject for the solid tumor. [0261] 27. The method of clause 26, wherein the step a) is assessing ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) mRNA expression in the solid tumor sample from the subject to obtain a subject ENPP1 mRNA expression result, wherein an increased subject ENPP1 mRNA expression result compared to reference value ranges for ENPP1 mRNA expression in a control solid tumor (“reference ENPP1 mRNA expression result”) indicates that the subject has high ENPP1 mRNA expression in the solid tumor sample, wherein a decreased subject ENPP1 mRNA expression result compared to the reference ENPP1 mRNA expression result indicates that the subject has low ENPP1 mRNA expression in the solid tumor sample. [0262] 28. The method of clause 27, wherein if the subject has the high ENPP1 mRNA expression in the solid tumor sample, then step b) is administering to the subject: i) an anti- ENPP1 therapy; and ii) a member selected from the group consisting of an anti-PD1 therapy, an anti-PDL1 therapy, and a PARP inhibitor, or a combination thereof, or if the subject has the low ENPP1 mRNA expression in the solid tumor sample, then step b) is administering to the subject a member selected from the group consisting of an anti-PD1 therapy, an anti-PDL1 therapy, and an anti-PARP therapy, or a combination thereof. [0263] 29. The method of clause 26, further comprising, prior to the step a): a') obtaining a solid tumor sample from the subject. [0264] 30. The method of clause 26, wherein the solid tumor is breast cancer. [0265] 31. The method of clause 30, wherein the breast cancer is: estrogen receptor positive (ER+); and/or human epidermal growth factor receptor 2 positive (HER2+). [0266] 32. The method of clause 27, wherein the anti-PD1 therapy is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, and toripalimab. [0267] 33. The method of clause 32, wherein the anti-PD1 therapy is pembrolizumab. [0268] 34. The method of clause 27, wherein the anti-PDL1 therapy is selected from the group consisting of atezolizumab, avelumab, and durvalumab. [0269] 35. The method of clause 27, wherein the anti-PARP therapy is selected from the group consisting of veliparib, olaparib, rucaparib, niraparib, and talazoparib. [0270] 36. The method of clause 35, wherein the anti-PARP therapy is veliparib. [0271] 37. The method of clause 35 or 36, wherein the anti-PARP therapy further comprises a platinum complex selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin. [0272] 38. The method of clause 37, wherein the platinum complex is carboplatin. [0273] 39. The method of clause 27, wherein the anti-ENPP1 therapy is described herein. [0274] 40. The method of a preceding clause, wherein the treating further comprises surgical resection of the solid tumor, radionuclide therapy, or chemotherapy. [0275] 41. A method for diagnosing a solid tumor in a subject, comprising: a) assessing mRNA expression in a solid tumor sample from the subject to obtain a subject mRNA expression result, thereby diagnosing the solid tumor in a subject. [0276] 42. The method of clause 41, wherein the diagnosing is for high ENPP1 mRNA expression in the solid tumor, wherein the step a) is assessing ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) mRNA expression in the solid tumor sample from the subject to obtain a subject ENPP1 mRNA expression result, wherein an increased subject ENPP1 mRNA expression result compared to reference value ranges for ENPP1 mRNA expression in a control solid tumor (“reference ENPP1 mRNA expression result”) indicates that the subject has high ENPP1 mRNA expression in the solid tumor sample, thereby diagnosing the solid tumor in a subject for high ENPP1 mRNA expression. [0277] 43. The method of clause 41, further comprising, prior to the step a): a') obtaining a solid tumor sample from the subject. [0278] 44. A method for treating a subject with a solid tumor with a K173Q mutation in ENPP1, comprising: a) obtaining a solid tumor sample from the subject; b) identifying the subject as having the K173Q mutation in ENPP1, c) if the subject has the K173Q mutation in ENPP1, administering an anti-ENPP1 therapy to the subject, thereby treating the subject with a solid tumor with a K173Q mutation in ENPP1. [0279] 45. A method for diagnosing a subject as having a solid tumor with a K173Q mutation in ENPP1, comprising: a) obtaining a solid tumor sample from the subject; b) identifying the subject as having the K173Q mutation in ENPP1, thereby diagnosing the subject with a solid tumor with a K173Q mutation in ENPP1. [0280] 46. A method for treating or preventing metastasis in a subject in need thereof, the method comprising administering to the subject an anti-ENPP1 therapy. [0281] 47. The method of clause 46, wherein the anti-ENPP1 therapy is administered to the subject individually as a single agent prophylaxis or therapy (monotherapy) or as a first therapy in combination with at least one additional therapy. [0282] 48. A method of predicting distant metastasis-free survival for a subject with breast cancer, comprising: a) obtaining a breast cancer tissue sample from the subject; b) measuring ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) mRNA expression in the breast cancer tissue sample from the subject; c) classifying the breast cancer tissue sample, wherein the breast cancer tissue sample has a high classification when its ENPP1 mRNA expression has, relative to a reference, a value which is 8.3 or above; or the breast cancer tissue sample has a low classification when its ENPP1 mRNA expression has, relative to a reference, a value which is 8.3 or below, and wherein the reference is the ENPP1 mRNA expression in a population of breast cancer tissue samples; and d) predicting a 100% chance of distant metastasis-free survival for up to 7 years with the low classification, and an 88% chance of distant metastasis-free survival at 4 years with the high classification, thereby predicting distant metastasis-free survival for a subject with breast cancer. [0283] In at least some of the previously described embodiments, one or more elements used in an embodiment can interchangeably be used in another embodiment unless such a replacement is not technically feasible. It will be appreciated by those skilled in the art that various other omissions, additions and modifications may be made to the methods and structures described above without departing from the scope of the claimed subject matter. All such modifications and changes are intended to fall within the scope of the subject matter, as defined by the appended claims. [0284] It will be understood by those within the art that, in general, terms used herein, and especially in the appended claims (e.g., bodies of the appended claims) are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.). It will be further understood by those within the art that if a specific number of an introduced claim recitation is intended, such an intent will be explicitly recited in the claim, and in the absence of such recitation no such intent is present. For example, as an aid to understanding, the following appended claims may contain usage of the introductory phrases “at least one” and “one or more” to introduce claim recitations. However, the use of such phrases should not be construed to imply that the introduction of a claim recitation by the indefinite articles “a” or “an” limits any particular claim containing such introduced claim recitation to embodiments containing only one such recitation, even when the same claim includes the introductory phrases “one or more” or “at least one” and indefinite articles such as “a” or “an” (e.g., “a” and/or “an” should be interpreted to mean “at least one” or “one or more”); the same holds true for the use of definite articles used to introduce claim recitations. In addition, even if a specific number of an introduced claim recitation is explicitly recited, those skilled in the art will recognize that such recitation should be interpreted to mean at least the recited number (e.g., the bare recitation of “two recitations,” without other modifiers, means at least two recitations, or two or more recitations). Furthermore, in those instances where a convention analogous to “at least one of A, B, and C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). In those instances where a convention analogous to “at least one of A, B, or C, etc.” is used, in general such a construction is intended in the sense one having skill in the art would understand the convention (e.g., “ a system having at least one of A, B, or C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or “B” or “A and B.” [0285] In addition, where features or aspects of the disclosure are described in terms of Markush groups, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group. [0286] As will be understood by one skilled in the art, for any and all purposes, such as in terms of providing a written description, all ranges disclosed herein also encompass any and all possible sub-ranges and combinations of sub-ranges thereof. Any listed range can be easily recognized as sufficiently describing and enabling the same range being broken down into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be readily broken down into a lower third, middle third and upper third, etc. As will also be understood by one skilled in the art all language such as “up to,” “at least,” “greater than,” “less than,” and the like include the number recited and refer to ranges which can be subsequently broken down into sub-ranges as discussed above. Finally, as will be understood by one skilled in the art, a range includes each individual member. Thus, for example, a group having 1-3 articles refers to groups having 1, 2, or 3 articles. Similarly, a group having 1-5 articles refers to groups having 1, 2, 3, 4, or 5 articles, and so forth. [0287] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. [0288] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims. [0289] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

WHAT IS CLAIMED IS: 1. A method for treating breast cancer in a subject, comprising: a) obtaining a sample from the subject; b) measuring ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) in the sample from the subject, c) classifying the sample, wherein the sample has a high classification when its ENPP1 levels are increased; and d) if the sample has the high classification, then administering to the subject: i) an ENPP1 inhibitor and a PD1 inhibitor, when the breast cancer is responsive to a PD1 inhibitor; ii) an ENPP1 inhibitor and a PDL1 inhibitor, when the breast cancer is responsive to a PDL1 inhibitor, iii) an ENPP1 inhibitor and a PARP inhibitor, when the breast cancer is responsive to a PARP inhibitor, thereby treating the breast cancer in the subject.
2. The method of claim 1, further comprising: assessing that the breast cancer is responsive to a PD1 inhibitor when the breast cancer is responsive to a PD1 inhibitor; assessing that the breast cancer is responsive to a PDL1 inhibitor when the breast cancer is responsive to a PDL1 inhibitor; assessing that the breast cancer is responsive to a PARP inhibitor when the breast cancer is responsive to a PARP inhibitor.
3. The method of claim 1, wherein the treating is achieving a pathological complete response (pCR).
4. The method of claim 1, wherein the breast cancer is triple negative, microsatellite instability-high (MSI-H), or mismatch repair deficient (dMMR) when the breast cancer is responsive to a PD1 inhibitor; or the breast cancer has a BRCA1 mutation, a BRCA2 mutation, is HER2 negative, is locally advanced, or is metastatic when the breast cancer is responsive to a PARP inhibitor.
5. The method of claim 1, wherein the step d) further comprises iii) administering chemotherapy.
6. The method of claim 5, wherein the chemotherapy is paclitaxel.
7. The method of claim 1, wherein the sample is a breast cancer tissue sample.
8. The method of claim 7, wherein the measuring is selected from the group consisting of ENPP1 mRNA expression, ENPP1 immunohistochemical levels, ENPP1 gene amplification levels, and ENPP1 K173Q homozygosity. 9. The method of claim 8, wherein when the breast cancer is responsive to a PD1 inhibitor, the measuring is ENPP1 mRNA expression, and the high classification occurs when the breast cancer tissue sample ENPP1 mRNA expression has, relative to a reference, a value selected from the group consisting of 6.2 or above, 6.39 or above, 6.4 or above, 7.0 or above, 7.4 or above, 7.8 or above, 8.0 or above, 8.9 or above, 9.2 or above, 9.4 or above, 9.6 or above, 9.8 or above, 10.0 or above. 10. The method of claim 8, wherein when the breast cancer is responsive to a PARP inhibitor, the measuring is ENPP1 mRNA expression, and the high classification occurs when the breast cancer tissue sample ENPP1 mRNA expression has, relative to a reference, a value selected from the group consisting of 6.4 or above, 6.78 or above, 7.0 or above, 7.4 or above, 7.8 or above, 8.0 or above, 8.9 or above, 9.2 or above, 9.4 or above, 9.6 or above,
9.8 or above, 10.0 or above, 10.2 or above,
10.4 or above, and 10.5 or above.
11. The method of claim 9 or 10, wherein the reference is the ENPP1 mRNA expression in a population of breast cancer tissue samples.
12. The method of claim 8, wherein the measuring is ENPP1 immunohistochemical levels, and the high classification occurs when a percentage of cells positive for ENPP1 expression in the breast cancer tissue sample is 0.5 or above, 0.75 or above, 0.9 or above, 1 or above, 10 or above, or 50 or above.
13. The method of claim 8, wherein the measuring is ENPP1 gene amplification levels, and the high classification occurs when ENPP1/CEP6 probe ratio > 2, when ENPP1 copy number > 6, or when ENPP1 copies are > 6 if a single probe is used.
14. The method of claim 8, wherein the measuring is ENPP1 K173Q homozygosity, and the high classification occurs when the breast cancer tissue sample is homozygous for the K173Q mutation.
15. The method of claim 1, wherein the sample is a blood sample.
16. The method of claim 15, wherein the measuring is of ENPP1 activity through plasma ENPP1 cGAMP degradation half-life, and the high classification is when the cGAMP degradation half-life is less than 60 minutes, or less than 50 minutes or less than 70 minutes.
17. The method of claim 15, wherein the measuring is of ENPP1 plasma protein levels, and the high classification is when the ENPP1 plasma protein level is > 4.4 ^g/L, > 3.4 ^g/L, or > 5.4 ^g/L.
18. The method of claim 15, wherein the measuring is ENPP1 K173Q homozygosity, and the high classification occurs when the breast cancer tissue sample is homozygous for the K173Q mutation.
19. The method of claim 1, wherein when the breast cancer is responsive to a PD1 inhibitor, then the PD1 inhibitor is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, and toripalimab.
20. The method of claim 19, wherein the PD1 inhibitor is pembrolizumab.
21. The method of claim 1, wherein when the breast cancer is responsive to a PDL1 inhibitor, the PDL1 inhibitor is selected from the group consisting of atezolizumab, avelumab, and durvalumab.
22. The method of claim 1, wherein when the breast cancer is responsive to a PARP inhibitor, the PARP inhibitor is selected from the group consisting of veliparib, olaparib, rucaparib, niraparib, and talazoparib.
23. The method of claim 22, wherein the PARP inhibitor is veliparib.
24. The method of claim 1, wherein when the breast cancer is responsive to a PD1 inhibitor, the step d) further comprises administering a platinum complex selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin.
25. The method of claim 24, wherein the platinum complex is carboplatin.
26. The method of claim 1, wherein the ENPP1 inhibitor is described herein.
27. A method for treating a subject for a solid tumor, the method comprising: a) assessing mRNA expression in a solid tumor sample from the subject to obtain an mRNA expression result; and b) administering to the subject a therapy for the solid tumor based on the determined mRNA expression result; to treat the subject for the solid tumor.
28. The method of claim 27, wherein the step a) is assessing ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) mRNA expression in the solid tumor sample from the subject to obtain a subject ENPP1 mRNA expression result, wherein an increased subject ENPP1 mRNA expression result compared to reference value ranges for ENPP1 mRNA expression in a control solid tumor (“reference ENPP1 mRNA expression result”) indicates that the subject has high ENPP1 mRNA expression in the solid tumor sample, wherein a decreased subject ENPP1 mRNA expression result compared to the reference ENPP1 mRNA expression result indicates that the subject has low ENPP1 mRNA expression in the solid tumor sample.
29. The method of claim 28, wherein if the subject has the high ENPP1 mRNA expression in the solid tumor sample, then step b) is administering to the subject: i) an anti-ENPP1 therapy; and ii) a member selected from the group consisting of an anti-PD1 therapy, an anti-PDL1 therapy, and a PARP inhibitor, or a combination thereof, or if the subject has the low ENPP1 mRNA expression in the solid tumor sample, then step b) is administering to the subject a member selected from the group consisting of an anti-PD1 therapy, an anti-PDL1 therapy, and an anti-PARP therapy, or a combination thereof.
30. The method of claim 27, further comprising, prior to the step a): a') obtaining a solid tumor sample from the subject.
31. The method of claim 27, wherein the solid tumor is breast cancer.
32. The method of claim 31, wherein the breast cancer is: estrogen receptor positive (ER+); and/or human epidermal growth factor receptor 2 positive (HER2+).
33. The method of claim 29, wherein the anti-PD1 therapy is selected from the group consisting of pembrolizumab, nivolumab, cemiplimab, dostarlimab, retifanlimab, and toripalimab.
34. The method of claim 33, wherein the anti-PD1 therapy is pembrolizumab.
35. The method of claim 29, wherein the anti-PDL1 therapy is selected from the group consisting of atezolizumab, avelumab, and durvalumab.
36. The method of claim 29, wherein the anti-PARP therapy is selected from the group consisting of veliparib, olaparib, rucaparib, niraparib, and talazoparib.
37. The method of claim 36, wherein the anti-PARP therapy is veliparib.
38. The method of claim 36 or 37, wherein the anti-PARP therapy further comprises a platinum complex selected from the group consisting of cisplatin, carboplatin, oxaliplatin, nedaplatin, triplatin tetranitrate, phenanthriplatin, picoplatin, and satraplatin.
39. The method of claim 38, wherein the platinum complex is carboplatin.
40. The method of claim 29, wherein the anti-ENPP1 therapy is described herein.
41. The method of a preceding claim, wherein the treating further comprises surgical resection of the solid tumor, radionuclide therapy, or chemotherapy.
42. A method for diagnosing a solid tumor in a subject, comprising: a) assessing mRNA expression in a solid tumor sample from the subject to obtain a subject mRNA expression result, thereby diagnosing the solid tumor in a subject.
43. The method of claim 42, wherein the diagnosing is for high ENPP1 mRNA expression in the solid tumor, wherein the step a) is assessing ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) mRNA expression in the solid tumor sample from the subject to obtain a subject ENPP1 mRNA expression result, wherein an increased subject ENPP1 mRNA expression result compared to reference value ranges for ENPP1 mRNA expression in a control solid tumor (“reference ENPP1 mRNA expression result”) indicates that the subject has high ENPP1 mRNA expression in the solid tumor sample, thereby diagnosing the solid tumor in a subject for high ENPP1 mRNA expression.
44. The method of claim 42, further comprising, prior to the step a): a') obtaining a solid tumor sample from the subject.
45. A method for treating a subject with a solid tumor with a K173Q mutation in ENPP1, comprising: a) obtaining a solid tumor sample from the subject; b) identifying the subject as having the K173Q mutation in ENPP1, c) if the subject has the K173Q mutation in ENPP1, administering an anti-ENPP1 therapy to the subject, thereby treating the subject with a solid tumor with a K173Q mutation in ENPP1.
46. A method for diagnosing a subject as having a solid tumor with a K173Q mutation in ENPP1, comprising: a) obtaining a solid tumor sample from the subject; b) identifying the subject as having the K173Q mutation in ENPP1, thereby diagnosing the subject with a solid tumor with a K173Q mutation in ENPP1.
47. A method for treating or preventing metastasis in a subject in need thereof, the method comprising administering to the subject an anti-ENPP1 therapy.
48. The method of claim 47, wherein the anti-ENPP1 therapy is administered to the subject individually as a single agent prophylaxis or therapy (monotherapy) or as a first therapy in combination with at least one additional therapy.
49. A method of predicting distant metastasis-free survival for a subject with breast cancer, comprising: a) obtaining a breast cancer tissue sample from the subject; b) measuring ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1) mRNA expression in the breast cancer tissue sample from the subject; c) classifying the breast cancer tissue sample, wherein the breast cancer tissue sample has a high classification when its ENPP1 mRNA expression has, relative to a reference, a value which is 8.3 or above; or the breast cancer tissue sample has a low classification when its ENPP1 mRNA expression has, relative to a reference, a value which is 8.3 or below, and wherein the reference is the ENPP1 mRNA expression in a population of breast cancer tissue samples; and d) predicting a 100% chance of distant metastasis-free survival for up to 7 years with the low classification, and an 88% chance of distant metastasis-free survival at 4 years with the high classification, thereby predicting distant metastasis-free survival for a subject with breast cancer.
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