WO2019016310A1 - Methods and compositions for treating cancers - Google Patents

Abstract

To investigate whether pharmacological inhibition of both glutamino lysis and aspartate/glutamate exchange are relevant therapies for cancer, inventors performed combination treatments in the highly metastatic orthotopic syngenic 4T1 breast cancer mouse model. Namely, whereas either CB839 or TFB-TBOA treatment alone decreases intratumoral aspartate and glutamate concentration, combined treatment led to a greater inhibition. Such metabolic effects further decreased stromal activation as reflected by a-SMA and decreased tumor cell proliferation, as reflected by in situ staining of the proliferation marker PCNA. Consistent with these observations and their in vitro results, CB839 or TFB-TBOA, treatments inhibited 4T1 tumor progression as quantified by tumor volume and lung and liver metastasis. Combined treatment led to greater inhibition of tumor growth and invasion and further improved survival outcomes. Accordingly, the present invention relates to a method for treating cancer in a subject in need thereof comprising a step of administering the subject with a therapeutically effective amount of GLS1 and SLC1A3 inhibitors.

Description

METHODS AND COMPOSITIONS FOR TREATING CANCERS

FIELD OF THE INVENTION:

The invention is in the field of oncology. More particularly, the invention relates to methods and compositions for treating cancers with a combination of inhibitors of GLSl and SLC1A3.

BACKGROUND OF THE INVENTION:

Non-transformed cell types within the tumor microenvironment continuously co-evolve with tumor cancer cells to promote tumorigenesis (Hanahan and Weinberg, 2011; Kalluri, 2016; Quail and Joyce, 2013). Evidence has indicated that fibroblasts are among the first cells to be recruited by tumor cells; however, it is widely accepted that normal fibroblasts generally suppress tumor formation (Kalluri, 2016). To promote tumorigenesis, normal fibroblasts are thought to interact with tumor cells and are converted to Cancer-Associated Fibroblasts (CAF). Once accomplished, CAF promote extensive tissue remodelling (or tumor niche formation). Further establishment of a complex, dynamic network of cytokines, chemokines, growth factors, and matrix remodelling enzymes ultimately changes the physical and chemical properties of the tumor (Lu et al., 2012). Indeed, tumors exhibit altered tissue-level and cell mechanics, including extracellular matrix (ECM) remodelling and stiffening (Kai et al., 2016). Experimental models demonstrate that enhancing ECM stiffness promotes malignancy, and, conversely, inhibiting matrix stiffening reduces tumor incidence and improves treatment (Levental et al., 2009). While genetic modifications in tumor cells undoubtedly initiate and drive malignancy (Watson et al, 2013), cancer progresses within a dynamically evolving ECM that modulates virtually every behavioral facet of both the tumor cells and cancer-associated stromal cells, including sustained proliferation, evasion of growth suppression, death resistance, induced angiogenesis and initiation of invasion (Pickup et al, 2014). Nevertheless, the processes that link ECM mechanotransduction (i.e., the processes that enable cells to sense and adapt to external mechanical forces) to the molecular mechanisms that influence cell behavior and modulate malignancy are just beginning to be defined.

Tumors alter their metabolic program to maintain cell autonomous proliferation in the nutrient-poor conditions of tumor microenvironment (Vander Heiden and DeBerardinis, 2017). Some of the most striking changes of tumor cellular bioenergetics include Warburg metabolism (i.e., a chronic shift in energy production from mitochondrial oxidative phosphorylation to glycolysis) and increases in glutamino lysis, amino acid and lipid metabolism, flux through the pentose phosphate pathway, macromolecule biosynthesis, and mitochondrial biogenesis (Ben- Sahra and Manning, 2017; Sullivan et al., 2016; Vander Heiden and DeBerardinis, 2017).

Prior mechanistic studies in cancer utilized hypoxia exposure to investigate this metabolic shift. Yet, numerous tumors are also characterized by profound metabolic dysregulations in the absence of obvious hypoxic stress (Hensley et al, 2016). Data are emerging regarding the molecular regulators of metabolic dysfunction operating independent of outright hypoxic stress. Extracellular protein can provide nutrients to the starved cancer cells (Davidson et al., 2017), thus there is a need to understand the mechanical role of ECM in the provision of nutrients to proliferation and development of the tumor.

SUMMARY OF THE INVENTION:

The invention relates to a method for treating cancer in a subject in need thereof comprising a step of administering the subject with a therapeutically effective amount of GLS1 and SLC1A3 inhibitors. In particular, the invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

CAF are a predominant cell type in the squamous cell carcinoma (SCC) stroma and are important mediators of the desmoplastic response (Quail and Joyce, 2013). Their abundance suggests that their communication with cancer cells may alter tumor cell metabolism. Consequently, in this study inventors investigated whether a metabolic response to tumor niche stiffness controls tumor progression. Specifically, inventors aimed to determine whether ECM stiffness directly modulates both cancer cell and CAF metabolism and coordinates nutrient availability within the tumor niche to support the metabolic needs of tumor progression. They elucidate the interconnection between mechanotransduction and tumor metabolic reprogramming, and show a stiffness-dependent tumor progression promoted by amino acid crosstalk between stromal and cancer cells. These results place glutamino lysis and aspartate/glutamate exchange as a central mediator of the extracellular environment's effects on tumor cell functions, and provide evidence that targeting the YAP/TAZ-GLS1-SLC1A3 axis will affect both tumor and stroma. More particularly, the inventors performed a combination treatment in the highly metastatic orthotopic syngenic 4T1 breast cancer mouse model. Namely, whereas either CB839 or TFB-TBOA treatment alone decreases intratumoral aspartate and glutamate concentration, combined treatment led to a greater. Such metabolic effects further decreased stromal activation as reflected by a-SMA staining and decreased tumor cell proliferation, as reflected by in situ staining of the proliferation marker PCNA. Consistent with these observations and our in vitro results, CB839 or TFB-TBOA, treatments inhibited 4T1 tumor progression as quantified by tumor volume and lung and liver metastasis. Combined treatment led to greater inhibition of tumor growth and invasion and further improved survival outcomes.

Accordingly, the present invention relates to a method for treating cancer in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of GLS1 and SLC1A3 inhibitors.

As used herein, the terms "treating" or "treatment" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

As used herein, the term "subject" refers to any mammals, such as a rodent, a feline, a canine, and a primate. Particularly, in the present invention, the subject is a human afflicted with or susceptible to be afflicted with a cancer. As used herein, the term "cancer" refers to an abnormal cell growth with the potential to invade or spread to other parts of the body. In the context of the invention, the cancer is a solid cancer. The term "solid cancer" has its general meaning in the art and refers to solid cancer selected from the group consisting of, but not limited to, head and neck squamous cell carcinoma (HNSCC), adrenal cortical cancer, anal cancer, bile duct cancer (e.g. periphilar cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochrondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating lobular carcinoma, lobular carcinoma in situ, gynecomastia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenocanthoma, papillary serous adnocarcinoma, clear cell), esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer, skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer, testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma).

As used herein, the term "GLSl" also known as glutaminase, L-glutaminase or glutamine amino hydrolase refers to a phosphate-activated amidohydrolase that catalyzes the hydrolysis of glutamine to glutamate and ammonia. This enzyme in human is encoded by GLSl gene. The naturally occurring human GLSl gene has a nucleotide sequence as shown in Genbank Accession numbers NM 014905.4 and the naturally occurring human GLSl protein has an aminoacid sequence as shown in Genbank Accession numbers NP 055720.3. The naturally occurring murine GLS1 gene has a nucleotide sequence as shown in Genbank Accession numbers NM 001081081.2 and the naturally occurring murine GLS 1 protein has an aminoacid sequence as shown in Genbank Accession numbers_NP_001074550.1.

As used herein, the term "SLC1A3" refers to solute carrier family 1, member 3. It is a member of a high affinity glutamate transporter family. It is a protein that, in humans, is encoded by the SLC1A3 gene. This gene functions in the termination of excitatory neurotransmission in central nervous system. The naturally occurring human SLC1A3 gene has a nucleotide sequence as shown in Genbank Accession numbers NM_001166696.2 and the naturally occurring human SLC1A3 protein has an aminoacid sequence as shown in Genbank Accession numbers NP 001160168.1. The naturally occurring murine SLC1A3 gene has a nucleotide sequence as shown in Genbank Accession numbers NM_148938.3 and the naturally occurring murine GLS1 protein has an aminoacid sequence as shown in Genbank Accession numbers NP_683740.1.

As used herein, the term "GLS1 and SLC1A3 inhibitors" refers to a natural or synthetic compounds that have a biological effect to inhibit the activity or the expression of GLS1 and SLC1A3. More particularly, such compound is capable of inhibiting the catalyses activity of GLS1 (and thus the transport of the glutamate).

In a particular embodiment, the GLS1 and SLC1A3 inhibitors are peptide, petptidomimetic, small organic molecules, antibodies, aptamers, siRNA or antisense oligonucleotides. The term "peptidomimetic" refers to a small protein-like chain designed to mimic a peptide. In a particular embodiment, the inhibitor of GLS1 and SLC1A3 is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

In a particular embodiment, the GLS1 and SLC1A3 inhibitors are small organic molecules. The term "small organic molecule" refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. In a particular embodiment, the GLS1 and SLC1A3 inhibitors are CB839 and TFB- TBOA. As used herein, the term CB839 refers to an inhibitor of human glutaminase, this small molecule is developed by Calithera and is ongoing on Phase lb. The CAS number of this molecule is 1439399-58-2. This molecule has the following formula and structure in the art

As used herein, the term ' FB-TBOA" refers to (2S, 3S)-3-[3-[4- (trifluoromethyl)benzoylamino]benzyloxy]aspartate, is a glutamate transport activity inhibitor. The CAS number of this molecule is 480439-73-4. This molecule has the following formula and structure in the art C19H17F3N2O6:

In some embodiments, the GLS1 and SLC1A3 inhibitors are an antibodies. As used herein, the term "antibodies" is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv- scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody-based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al, 2004; Reff & Heard, 2001 ; Reiter et al, 1996; and Young et al, 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a "chimeric" antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A "human antibody" such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388. In a particular embodiment, the inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.

In a particular, the GLS1 and SLC1A3 inhibitors is an intrabody having specificity for GLS1 and SLC1A3. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term "single domain antibody" (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called "nanobody®". According to the invention, sdAb can particularly be llama sdAb.

In some embodiments, the GLS1 and SLC1A3 inhibitors is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of GLS1 and SLC1A3. In a particular embodiment, the inhibitor of GLS1 and SLC 1 A3 expression is siRNA. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA- induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide- long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Anti- sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 1 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno- associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

In some embodiments, the inhibitor of GLS1 and SLC1A3 expression is an endonuclease. In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homo logy-directed repair (HDR).

In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term

"CRISPR-cas" has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffmi, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trap. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al, 2014 Cell Res. doi: 10.1038/cr.2014.1 1.), bacteria (Fabre et al, 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al, 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al, 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534/genetics.l 13.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al, 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al, 2014, Cell Res. doi: 10.1038/cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al, 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. ("Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., inhibitors of GLS1 and SLC1A3) into the subject, such as by mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

More particularly, the two inhibitors as described above are administered in combination to the subject suffering from a cancer. The inhibitors are administered as a combined preparation. More particularly, the GLS1 and SLC1A3 inhibitors are administered simultaneously, separately or sequentially.

As used herein, the term "administration simultaneously" refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term "administration separately" refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term "administration sequentially" refers to an administration of 2 active ingredients at different times, the administration route being identical or different.

A "therapeutically effective amount" is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The GLS1 and SLC1A3 inhibitors as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile- filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

The invention thus relates to a pharmaceutical composition comprising the inhibitors of GLS1 and SLClA3.

In a particular embodiment, the pharmaceutical composition according to the invention, wherein the inhibitors of GLS1 and SLC1A3 are TBOA and CD839.

Throughout the specification, several terms are employed and are defined in the following paragraphs. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1: The aspartate/glutamate transporter SLC1A3 is needed to sustain CAF and SCC pro-tumoral activities. RT-qPCR analyses of SLC1A family expression in SCC12, fibroblast and CAF cells reveal that SLC1A3 was increased by stiffness and overexpressed in CAF compared to fibroblast. Quantification of PCNA fluorescent labellingrevealed that supplementation by aspartate sustained proliferation of SCC 12, even when GLS 1 was knocked down. However, inhibition of GLS1 and SLC1A3 blunted proliferation, even when aspartate was added.Traction force microscopy revealed that supplementation by glutamate sustained CAF-dependant ECM contraction, even when GLS 1 was knocked down. However, inhibition of GLS1 and SLC1A3 blunted CAF-dependant ECM contraction, even when glutamate was added. F-H) In three-dimensional co-culture assay ,GLS1 or SLC1A3 knockdown either in CAF or SCC12 slightly decreased cell invasion while inhibition of GLS1 and SLC1A3 either in CAF or SCC 12 more substantially inhibited cell invasion. In patient-derived spheroids, pharmacological inhibition of CB839 and, to a greater extent, inhibition of both CB839 and TFB-TBOA reduced cancer cells invasion. In all panels, mean expression in control groups (si- NC or vehicle cultivated on soft matrix) was assigned a fold change of 1, to which relevant samples were compared. Data are expressed as the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001) of at least 3 independent experiments performed in triplicate. Paired samples were compared by 2-tailed Student's t test, while 1-way ANOVA and post-hoc Tukey's tests were used for group comparisons.

Figure 2: Additive effects between pharmacological inhibitors of metabolic reprogramming. Following 4T1 cells implantation, mice were treated with daily i.p. injections of CB839 (n=10); with daily i.p. injections of TFB-TBOA (n=10); with daily i.p. injections of both TFB-TBOA and CB839 (n=10) or vehicle (n = 10). A-B) Intratumoral aspartate (A) and glutamate (B measurement revealed a decrease of both aspartate and glutamate level in mice treated with CB839 and CB839+TFB-TBOA as compared to control (Vehicle Coimmuno fluorescence microscopy and quantification revealed a decrease of stromal activation as reflected by D-SMA stain as well as a decrease of PCNA-positive cells in CB839- and CB839+TFB-TBOA-treated mice compared with vehicle control. C) CB839+TFB-TBO A drastically reduced breast cancer progression as quantified by tumor volume (C), lung metastasis (D), liver metastasis (E) and survival outcome (F). Similar trends were observed upon CB839 or TFB-TBOA treatment. G-M) In the HNSCC PDX mouse model, following tumor implantation, mice were treated with daily i.p. injections of CB839 (N=3; n=6); with daily i.p. injections of TFB-TBOA (N=3; n=6); with daily i.p. injections of both TFB-TBOA and CB839 (N=3; n=6) or vehicle (N=3; n = 6). Intratumoral aspartate and glutamate (G) measurement revealed a decrease of both aspartate and glutamate level in mice treated with CB839 and CB839+TFB-TBOA as compared to control (Vehicle). Coimmuno fluorescence microscopy and quantification (J) revealed a decrease of stromal activation as reflected by□ - SMA stain as well as a decrease of PCNA-positive cells in CB839-, in TFB-TBOA-, and , to a greater extent, in CB839+TFB-TBOA-treated mice compared with vehicle control. CB839+TFB-TBOA more substantially reduced HNSCC progression as quantified by tumor volume ( -M). Similar trends were observed upon CB839 or TFB-TBOA treatment. Data are expressed as the mean ± SEM (*P < 0.05, **P < 0.01, ***P < 0.001) of at least 3 independent experiments performed in triplicate. Paired samples were compared by 2-tailed Student's t test, while 1-way ANOVA and post-hoc Tukey's tests were used for group comparisons.

EXAMPLE:

Material & Methods

Cell Culture and cell culture reagents.

Human cancer cell lines including head and neck cancer cell lines CAL27 and CAL33, lung cancer cell lines A427 and A549, breast cancer lines MDA-MB-231 and MDA-MB-468 and Human primary Dermal Fibroblasts (hDF), and human HEK293Tcells as well as murin breast cancer cell lines 67NR, 410.4 and 4T1 were purchased from ATCC and maintained in DMEM supplemented with 10% FCS (fetal calf serum) and 2mM glutamine. Carcinoma associated fibroblasts (CAFs) isolated from patients with head and neck, and breast cancer were cultured in DMEM supplemented with 10% FCS, 2mM glutamine and insulin-trans ferrin- selenium (Invitrogen). SCC12 skin cancer cell line was cultured in FAD media, as described previously (Gaggioli et al, 2007). All cells were grown in collagen-coated plastic (50ug/mL) at 37oC in a humidified 5% C02 atmosphere. Experiments were performed at passages 3-10. Collagen-coated hydrogels were purchased from Matrigen.

The following inhibitors were used in this study: BPTES (Sigma Aldrich), CB839 (Selleckchem), TFB-TBOA (Selleckchem) and Y27632 (Sigma Aldrich) at 10 μΜ, Verteporfm (Sigma Aldrich) and PF573228 (Sigma Aldrich) at 2 μΜ.

Glutamate was purchased from Sigma Aldrich and used at concentration of 2mM; aspartate was purchased from Sigma Aldrich and used at concentration of lOmM, consistent with prior in vitro studies linking cancer cell proliferation to glutamine metabolism and aspartate levels (Birsoy et al., 2015; Sullivan et al., 2015).

Oligonucleotides and Transfection

On Target Plus siRNAs for YAP (J-012200-07 and J-012200-05,), TAZ (WWTR1; J- 016083-05 and J-016083-06), GLS1 (J-004548-09; J-004548-10), SLC1A3 (J-007427-05; J- 007427-07) and scrambled control D-001810-01 and D-001810-02) were purchased from Thermo Scientific/Dhermacon. Cells were plated in collagen-coated plastic (50ug/mL) and transfected 24h later at 70-80% confluence using siRNA (25nM) and Lipofectamine 2000 reagent (Life Technologies), according to the manufacturers' instructions. Eight hours after transfection, cells were trypsinized and re-plated on hydrogel or used for spheroid assay.

Plasmids

The following antisens sequences Control

(CCGGCAACAAGATGAAGAGCACCAACTCGAG

TTGGTGCTCTTCATCTTGTTGTTTTTG) Gls 1 (CCGGAAGTTCCTTTTTGTCTTCAGTCTCG

AGACTGAAGACAAAAAGGAACTTTTTTTG) and Slcla3

(CCGGCTTTCAAGTTTTTGGTGT

AACCTCGAGGTTACACCAAAAACTTGAAAGTTTTTG) were sub-cloned in the pL O- Tet-On (Wiederschain et al, 2009) using EcoRI and Agel restriction sites. Stable expression of these constructs in 4T1 cells, and BalB/c mouse CAFs was achieved by lentiviral transduction. All cloned plasmids were confirmed by DNA sequencing.

Lentivirus production

HEK293T cells were transfected using Lipofectamine 2000 (Life Technologies) with lentiviral plasmids along with packaging plasmids (pPACK, System Biosciences), according to the manufacturer's instructions. Virus was harvested, sterile filtered (0.45 um), and utilized for subsequent infection of 410.4 and Balb/c mouse CAFs (24-48 hour incubation) for gene transduction.

Targeted LC-MS/MS

Metabolite extraction was performed essentially as described with minor modifications (Oldham et al., Cell Metabo, 2016). Briefly, metabolites were extracted from cultured cells on dry ice using 80% aqueous methanol precooled at -80°C. Supernatants were extracted with 4 volumes of 100% methanol precooled at -80°C for 4 hours at -80°C. An internal standard, [13C4]-2-oxoglutarate ([13C4]-20G) (Cambridge Isotope Laboratories), was added during metabolite extraction. Insoluble material from both cell and supernatant extractions was removed by centrifugation at 20,000 g for 15 minutes at 4°C. The supernatant was analyzed by targeted LC-MS/MS as previously described (Oldham et al., Cell Metabo, 2016). Metabolites were separated using a ZIC-HILIC stationary phase (150 mm x 2.1 mm x 3.5 mm; Merck). The MS parameters were optimized using a glutamine standard solution. Monitored mass transitions were 87 to 87 (pyruvate), 132 to 88 (aspartate), 145 to 101 (20G), 145 to 127 (glutamine), 146 to 128 (glutamate), and 149 to 105 ([13C4J-20G). Mass transitions and retention time windows were confirmed by the analysis of neat and matrix-spiked standards. Peak areas were quantified by Xcalibur Software (Thermo Fisher Scientific) and manually reviewed.

Messenger RNA extraction

Cells were homogenized in 1 ml of QiaZol reagent (Qiagen). Total RNA content, was extracted using the miRNeasy kit (Qiagen) according to the manufacturer's instructions. Total RNA concentration was determined using a ND-1000 micro-spectrophotometer (NanoDrop Technologies).

Quantitative RT-PCR of messenger RNAs

Messenger RNAs were reverse transcribed using the Multiscript RT kit (Life

Technologies) to generate cDNA. cDNA was amplified via fluorescently labeled Taqman primer sets using an Applied Biosystems 7900HT Fast Real Time PCR device. Fold-change of RNA species was calculated using the formula (2-□□ Ct), normalized to RPLP0 expression.

Matrix production

Cell-derived matrices were generated as described (Beacham et al., Current protocol in cell biology 2006). Briefly, normal fibroblast or CAF were seeded at a density of 200,000 cells per well in a 6-well plate. When confluent, cells were cultured for a further 10 days, with medium being changed every 48 h to complete medium supplemented with 50 mg.ml-l ascorbic acid (Sigma- Aldrich) to ensure collagen cross-linking. Mature matrices were then denuded of cells using lysis buffer (PBS containing 20 mM NH40H and 0.5 % (vol/vol) Triton X-100). Following PBS washes, matrices were incubated with 10 mg.ml-1 DNase I (Roche) at 37 °C for 30 min. Matrices were then stored in PBS containing 1% (vol/vol) penicillin/streptomycin at 4 °C before use.

Matrix remodeling assay

For gel contraction assay, 25.103 cells were embedded in ΙΟΟμΙ of matrix gel and seeded in triplicate into 96 wells plate. After lh at 37°C, matrix gels were overlaid with ΙΟΟμΙ of 0,5% FCS medium (with indicated cytokines or inhibitors) and changed every two days. At day 6 the relative diameter of the well and the gel were measured using ImageJ. The percentage of gel contraction was calculated using the formula 100 * (well diameter - gel diameter) / well diameter.

Traction force microscopy

Contractile forces exerted by CAF on different stiffness gels were assessed by traction force microscopy essentially as described (Liu et al, 2016). Briefly, polyacrylamide substrates with shear moduli of 1, 8, or 50 kPa conjugated with fluorescent ed latex microspheres (0.5 μιτι, 505/515 ex/em) were purchased from Matrigen. CAF were plated on fluorescent bead- conjugated discrete stiffness gels and grown for 24 hours, at which time they were treated with the indicated treatments for 1 hour before traction force measurements. Images of gel surface- conjugated fluorescent beads were acquired for each cell before and after cell removal using a Axiovert 200M motorized microscope stand (Zeiss) and a x32 magnification objective. Tractions exerted by CAF were estimated by measuring bead displacement fields, computing corresponding traction fields using Fourier transform traction microscopy, and calculating root- mean-square traction using the PIV 5particle Image velocity) and TFM (Traction force microscopy) pakage on ImageJ (Tseng et al, 2012). To measure baseline noise, the same procedure was performed on a cell- free region.

Cell counting assays.

SCC12 cells transfected with the indicated siR A - or not- were plated in triplicate in 6 well plates at 30 000 cells per well. After overnight incubation for cells to adhere, 6 wells were counted to determine initial count at time of treatments (glutamate, aspartate, BPTES, CB-839 or TFB-TBOA). After 1 day, 2 days or 3 days, the entire contents of the well was trypsinized and counted and proliferation rate was calculated.

Cell tracking experiments

SCC12 cells treated with the indicated siRNA or pharmacological inhibitors were plated in 6 well plates at 30 000 cells per well. After 6 hours incubation for cells to adhere, 6 wells were imaged every 10 minutes during 10 hours using an Axiovert 200M motorized microscope stand (Zeiss) and a xlO magnification objective. Images were analyzed to determine the distance travelled by cells using the TrackMate package on ImageJ.

Three-dimensional co-culture invasion assay

Cancer cells (SCC12 or MDA-MB-468) and CAF cells were removed from the cell culture dishes with trypsin and re-suspended in DMEM 10% FCS. The solution contained a 1 :1 ratio of cancer cells and CAF cells at a concentration of 1 x 106 cells per mL. Fifty-micro litre droplets were plated onto the underside of a 10 cm culture dish and allowed to form spheroids in a 37 °C incubator 24 hours. The spheroids were then embedded in a collagen I/Matrigel gel mix at a concentration of approximately 4 mg ml-l collagen I and 2 mg ml-l Matrigel (BD Bioscience) in 24-well glass-bottomed cell culture plates (MatTek). The gel was incubated for at least 45 min at 37 °C with 5% C02. The gel was covered with DMEM media. Forty-eight hours later, the spheroids were imaged with an inverted at a magnification of x4 and xlO. Invasion was quantified using ImageJ.

Immunoblotting and antibodies

Cells were lysed in Laemmeli buffer. Protein lysate were resolved by SDS-PAGE and transferred onto a PVDF membrane (Biorad). Membranes were blocked in 5% non-fat milk in TN buffer (50 mM Tris-HCl pH 7.4, 150 mM NaCl) or 5% BSA in TN buffer and incubated in the presence of the primary and then secondary antibodies. After washing in TN buffer containing 0.1% Tween, immunoreactive bands were visualized with the ECL system (Amersham Biosciences). Primary antibody for YAP1 (#4912; 1/1000) and YAP/TAZ (#8418; 1/1000) were obtained from Cell Signaling. Primary antibodies for GLS1 (abl56876; 1/1000), and LDHA (ab47010; 1/1000) were obtained from Abeam. Primary antibody for Tubulin (T4026; 1/5000) was obtained from Sigma Aldrich. Primary antibody for SLC1A3 (sc-7757; 1/200) was obtained from Santa Cruz Biotechnology. Appropriate secondary antibodies (anti- rabbit, anti-mouse and anti-goat) coupled to HRP were used (Dako).

Immunofluorescence

After the different treatment cells were fixed with PBS/PFA 4% for 10 min and permeabilized with PBS/Triton 100X 0.1% for lOmin. Then cells were incubated with anti- PCNA (#4912; 1/100; Invitrogen) at room temperature for 2 hours. Secondary antibodies coupled with Alexa-594 (Thermo Scientific) were used at 1 :500. Nuclei were counterstained with DAPI (Sigma- Aldrich).

Inhibition of YAP and Lox in breast cancer mouse model

Metastatic mouse (Balb/c) 4T 1 breast cancer cell line (Yang et al., 2004) were implanted into the right fourth mammary fat pad in 10 μΐ Matrigel of 8 weeks old female Balb/c mice. After 10 days, mice with palpable tumor (5-10mm3) were randomly assigned to treatment groups and underwent i.p. injection daily with 20mg/kg of Verteporfin (Tocris Bioscience) or vehicle control. In parallel but in separate mouse cohort, β-aminopropionitrile (BAPN; 100 mg/kg/d; Sigma- Aldrich) was administered in drinking water. Tumor dimensions were measured using digital calipers, and tumor volume was calculated as (small diameter)2 x (large diameter)/2.. For survival analyses, mice were monitored daily for breast cancer progression and euthanized according to a standard body condition score, taking into account initial signs of moribund state and discomfort associated with the progression of breast cancer. Mice were also euthanized when total tumor burden exceeded 1,500 mm3 in volume. Postmortem, the lungs, and livers were harvested and examined for the presence of macroscopic lesions.

Genetic inhibition of GLS1 and/or SLC1A3 in 4T1 breast cancer mouse model Metastatic mouse (Balb/c) 4T1 (5.104) breast cancer cell line stably transfected with either doxycycline inducible sh-NC (Control) or shGLSl or shSLClA3 or shGLSl and shSLClA3 were implanted into the right fourth mammary fat pad in 10 μΐ Matrigel of 8 weeks old female Balb/c mice. After 11 days, mice with palpable tumor (5-10mm3) were treated with lmg/mL doxycycline (Sigma) 5% sucrose in dirking water. Tumor dimensions were measured using digital calipers, and tumor volume was calculated as (small diameter)2 x (large diameter)/2.. For survival analyses, mice were monitored daily for breast cancer progression and euthanized according to a standard body condition score, taking into account initial signs of moribund state and discomfort associated with the progression of breast cancer. Mice were also euthanized when total tumor burden exceeded 1,500 mm3 in volume. Postmortem, the lungs were harvested and examined for the presence of macroscopic lesions.

Genetic inhibition of GLS1 and/or SLC1A3 in 67NR breast cancer mouse model

Non invasive mouse (Balb/c) 67NR (5.105) breast cancer cell line were co-implanted with CAF (1.106) isolated from Balb/c mammary tumor and stably transfected with either doxycycline inducible sh-NC (Control) or shGLSl or shSLClA3 or shGLSl and shSLClA3 were implanted into the right fourth mammary fat pad in 10 μΐ Matrigel of 8 weeks old female Balb/c mice. After 1 days, mice were treated with lmg/mL doxycycline (Sigma) 5% sucrose in dirking water. Mice were killed 35 days post injection, tumors were removed. After excision and 12 h of fixation in 3.7% neutral-buffered formalin at 25 °C, tumors were paraffin-embedded. For invasion analyses, 5-μιη paraffin sections were made and stained with haematoxylin and eosin or Picrosirius Red. Local invasion was determined by observation under light microscopy.

Pharmacological inhibition of GLS1 and/or SLClAl-3 in breast cancer mouse model

Metastatic mouse (Balb/c) 4T1 breast cancer cell line were implanted into the right fourth mammary fat pad in 10 μΐ Matrigel of 8 weeks old female Balb/c mice. After 10 days, mice with palpable tumor (5-10mm3) were randomly assigned to treatment groups and underwent i.p. injection daily with 20mg/kg of CB839 (Tocris Bioscience) or with 20mg/kg of TFB-TBOA or with 20mg/kg CB839 and 20mg/kg TFB-TBOA or vehicle control. Tumor dimensions were measured using digital calipers, and tumor volume was calculated as (small diameter)2 x (large diameter)/2.. For survival analyses, mice were monitored daily for breast cancer progression and euthanized according to a standard body condition score, taking into account initial signs of moribund state and discomfort associated with the progression of breast cancer. Mice were also euthanized when total tumor burden exceeded 1,500 mni3 in volume. Postmortem, the lungs, and livers were harvested and examined for the presence of macroscopic lesions.

Generation of patient derived xenograft

Tumor specimens were obtained at initial surgery (Face and Neck University Institute, Nice, France) from primary diagnosed HNSCC. None of the patient received neoadjuvant chemotherapy and/or radiotherapy. Written informed consent was obtained from each patient and the study was approved by the hospital ethics committee. Patient tumor material was collected in culture medium and partially digested during 1 hour at room temperature in RPMI1640 with 1 mg/ml Collagenase IV, 1 mg/ml Dispase and 1 mg/ml Hyaluronidase. Approximately 20-30 mg tissue fragments in 50 % Matrigel were implanted subcutaneously into the flank region of NMRI-nu (RjOrkNMRI-Foxnlnu /Foxnlnu) mice. The first passage PDX were dissociated in a collagenase/dispase mixture and cells were cultured in low serum conditions (2 %FBS/F12/DMEM/1XB27) in presence 5 ng/ml EGF. Subsequently, 75.104 cells in 50 % Matrigel were implanted subcutaneously into the flank region of NMRI-nu (RjOrl:NMRI-Foxnlnu /Foxnlnu) mice. One week after tumors engraftment, to avoid any interference with tumors uptake, mice were treated with the corresponding inhibitors. Verteporfm (20mg/kg), CB838 (20mg/kg), TFB-TBOA (20mg/kg) or a combination of CB839 (20mg/kg) and TFB-TBOA (20mg/kg) were injected intraperitoneally every days; BAPN (100 mg/kg/day) was dissolve in drinking water. The dose in drinking water was determined using average daily water intake (4 ml) and mouse weight. Tumor volume was measured every day from the beginning of the treatment with the following formula: (small diameter)2 x (large diameter)/2.

Glutaminase activity assay

According to the manufacturer instructions (Glutaminase Microplate Assay Kit, Cohesion Biosciences), flash frozen tissue (O.lg/sample) was homogenized in lmL of assay buffer on ice and centrifuged at 8000g 4°C for 10 min. Protein concentration was determined by Bradford assay. Samples, normalized to total protein (100μg), were incubated with kit reagents for 1 hr at 37°C, and absorbances were measured at 420nm.

LOX activity assay

Lox activity was measured using the Lysyl Oxidase Activity Assay Kit (Abeam; Abl 12139), following the manufacturer instructions and as we previously described. Briefly, 5μg of total protein extracts from whole tumor, as described above, were analyzed. Extracts were incubated for 30min in presence of 50uL of reaction mixture +/- 500μΜ BAPN. Fluorescence was monitored with a fluorescence plate reader at Ex/Em = 540/590 nm and fluorescence (a measure of LOX activity) was plotted, where 0 = sample + 500μΜ BAPN (complete LOX inhibition).

Determination of intratumoral aspartate or glutamate concentration

Aspartate and glutamate concentration were measured using the Aspartate colorimetric assay kit (BioVision; 552) and the Glutamate Colorimetric Assay Kit (Bio vision; K629) following the manufacturer instructions. Briefly, 10μg of total protein extracts from whole tumor, as described above, were analysed (Glutamate assay) or were pretreated to remove interfering substances using the serum clean-up Mix and deproteinized by centrifuging 10 min with a 10 kDa spin filter (Aspartate assay). Extracts (glutamate assay) or filtrates (aspartate assay) were incubated with kit reagents for 30 min at 37°C and absorbances were measured at 450 nm and 70nm respectively.

Ininiunohistochemical staining and quantification methods of human samples 48 head and neck tumor biopsies were fixed (3.7% formaldehyde in PBS) for 4 h and transferred to 70% ethanol (24 h), embedded in paraffin wax and sectioned at 5 μιη. After deparaffination, microwave antigen retrieval was performed in a-citrate buffer (lOmM, pH6; 5min at 900W, lOmin at 150W and 30 min at room temperature). Sections were washed three times in PBS (5min per wash). After incubation in blocking buffer for one hours (PBS 3%BSA; 10% serum; 0.3% Triton X100), sections were incubated with primary antibody for D-SMA (ab21027; 1/300), SLC1A3 (sc-7757; 1/100) and GLS 1 (abl56876; 1/100) staining diluted in blocking buffer overnight at 4°C. After three washes in PBS/0,1% Tween 20, sections were incubated with secondary antibody diluted 1 :400 in blocking buffer for 1 hour at room temperatue and washed 3 times in PBS/0,1% Tween 20. Nuclei were then stained with DAPI and mounted in Montant, Permafluor (Thermo Scientific). Two authors, blinded to each other's assessment, scored the slides using the Quick Score method (as described in (Albrengues et al., 2014) to determine SLC1A3, GLS1 , and D-SMA status within the tumor.

Immunohistochemistry and immunofluorescence of mammary tumor sections Mammary tumor sections (5μηι) were deparaffmized and high temperature antigen retrieval was performed, followed by blocking in TBS/BSA 5%, 10% donkey serum and exposure to primary antibody and biotinylated secondary antibody (Vectastain ABC kit, Vector Labs) for immunohistochemistry or Alexa 488, 568 and 647-conjugated secondary antibodies (Thermo Fisher Scientific) for immunofluorescence. Primary antibodies against, GLS 1 (abl56876; 1/100), and D-SMA (ab32575; 1/1000 and ab21027; 1/300) were purchased from Abeam. A primary antibody against D-SMA (A2547; 1/300) was purchased from Sigma. A primary antibody against SLC1A3 (sc-7757; 1/100), was purchased from Santa Cruz Biotechnology. A primary antibody against PCNA (13-3900, 1/100) was purchased from Thermo Fisher Scientific. In most cases, color development was achieved by adding streptavidin biotinylated alkaline phosphatase complex (Vector Labs) followed by Vector Red alkaline phosphatase substrate solution (Vector Labs). Levamisole was added to block endogenous alkaline phosphatase activity (Vector Labs). Pictures were obtained using an Olympus Bx51 microscope or ZEISS LSM Exciter confocal microscope. Intensity of staining was quantified using ImageJ software (NIH). All measurements were performed blinded to condition.

Atomic force microscopy

Mice tumors were embedded in OCT, frozen on liquid nitrogen vapor and store at -80°C. Tumor slices (10 μιη thickness) were cut out from their glass slide and the fragment of glass containing the sample was glued on the bottom of a 50 mm dish (Willco Glass Bottom Dish). Before measurements the sample was first rinsed and after covered with 4 ml of PBS lx. The mechanical properties of the samples were studied using a BioScope Catalyst atomic force microscope (Bruker) coupled with and optical microscope (Leica DMI6000B) that enables, by phase contrast, to pinpoint the areas of interest. For each sample, at least 3 areas were analyzed using the "Point and Shoot" method, collecting from 80 to 100 force-distance curves at just as many discrete points. The experiments of nano indentation were performed in PBS using a probe with a Borosilicate Glass spherical tip (5 um of diameter) and a cantilever with a nominal spring constant of 0.06 N/m (Novascan). Indentations were carried out using a velocity of 6.5 μητ/s, in relative trigger mode and by setting the trigger threshold to 2 nN. The apparent Young's (elastic) modulus was calculated using the NanoScope Analysis 1.8 software (Bruker), fitting the force curves to the Hertz spherical indentation model and using a Poisson's ratio of 0.5. To avoid large indentation, a minimum and a maximum Force Fit Boundary of 5% and 25% respectively of the whole force curve was taken into account for the fit.

Picrosirius Red stain and quantification

Picrosirius Red stain was achieved through the use of D m paraffin sections stained with 0.1% Picrosirius Red (Direct Red80, Sigma- Aldrich) and counterstained with Weigert's hematoxylin to reveal fibrillar collagen. The sections were then serially imaged using with an analyzer and polarizer oriented parallel and orthogonal to each other. Microscope conditions (lamp brightness, condenser opening, objective, zoom, exposure time, and gain parameters) were constant throughout the imaging of all samples. A minimal threshold was set on appropriate control sections for each experiment in which only the light passing through the orthogonally-oriented polarizers representing fibrous structures (i.e., excluding residual light from the black background) was included. The threshold was maintained for all images across all conditions within each experiment. The area of the transferred regions that was covered by the thresholded light was calculated and at least five 20x field per condition were averaged together (Image J software).

Patient-derived spheroids

Following excision biopsy samples were directly transferred to freshly prepared culture medium containing DMEM/F12-medium, 10 % fetal calf serum, as well as a mixture of antibiotic/antifungal compounds (0.26 μΜ Amphotericin B, Ampicillin 0.14 mM, Ciprofloxacin 7.54 μΜ). Fresh tumor tissue samples were mechanically and enzymatically (Collagenase 200μg/mL in PBS; Roche) digested to generate a single-cell suspension. After determination of cell viability using the trypan-blue exclusion test, the single cell suspension was directly processed into spheroids. No red blood cell lysis was performed. Briefly, fifty- microlitre droplets containing 25 to 50.103 cells were plated onto the underside of a 10 cm culture dish and allowed to form spheroids in a 37 °C incubator 48 hours. The spheroids were then embedded in a collagen I/Matrigel gel mix at a concentration of approximately 4 mg ml-1 collagen I and 2 mg ml-l Matrigel (BD Bioscience) in 24-well glass-bottomed cell culture plates (MatTek). The gel was incubated for at least 45 min at 37 °C with 5% C02. The gel was covered with DMEM/F12 media. Forty-eight hours later, the spheroids were imaged with an inverted at a magnification of ^x4 and x 10. Invasion was quantified using ImageJ.

Human subjects

Informed consent was obtained for all study procedures. For formalin- fixed paraffin- embedded HNSCC samples, human specimens were collected from the Pasteur hospital tissues biobank and the protocol for staining was approved by the local ethic committee of the Nice University Hospital. All the observations on tumor samples were performed by independent double-blind examiners. The Quick Score method was used to score invasiveness status in the tumor samples by quantification of the numbers of invasive clusters within the tuomr stroma. Tumor samples were classified for high, middle and low invasiveness corresponding to a QS <7, 8<QS<11 and 12<QS<16 respectively. GLS1, SLC1A3 and D-SMA staining were quantified on low, middle and high invasiveness samples as determined by measuring the mean value with imageJ software.

Statistics Cell culture experiments were performed at least three times and at least in triplicate for each replicate. The number of animals in each group was calculated to measure at least a 20% difference between the means of experimental and control groups with a power of 80% and standard deviation of 10%>. The number of unique patient samples for this study was determined primarily by clinical availability. In situ expression/histologic analyses of both mouse and human tissue were performed in a blinded fashion. Numerical quantifications for in vitro experiments using cultured cells or in situ quantifications of transcript expression represent mean ± standard deviation (SD). Numerical quantifications for physiologic experiments using mouse or human reagents represent mean ± standard error of the mean (SEM). Immunoblot images are representative of experiments that have been repeated at least three times. Micrographs are representative of experiments in each relevant cohort. Paired samples were compared by a 2-tailed Student's t test for normally distributed data, while Mann- Whitney U non-parametric testing was used for non-normally distributed data. For comparisons among groups, one-way ANOVA and post-hoc Tukey testing was performed. A P-value less than 0.05 was considered significant. Correlation analyses were performed by Pearson correlation coefficient calculation. The Mantel-Cox log-rank test was used for statistical comparisons in survival analyses.

Study approval

All animal experiments were approved by the local committee of the host institute and by the Institutional Animal Care and Use Committee (CIEPAL AZUR committee, MESR number 2015051917125051) at the University Cote d'Azur, Nice, France. All experimental procedures involving the use of human tissue included the relevant receipt of written informed consent and were approved by institutional review boards at Nice hopsital University. Ethical approval for this study and informed consent conformed to the standards of the Declaration of Helsinki.

Results

Mechanical stimuli regulate metabolic reprogramming and coordinate nonessential amino-acids exchanges within the tumor niche.

ECM stiffness activates SCC and CAF pro-tumoral activities, as reflected by inducing proliferation and generating contractile forces (assessed by traction force microscopy (TFM). In order to sustain these energy-requiring activities, cells adapt their metabolism accordingly. To determine whether mechanical/physical cues conveyed by ECM stiffness modulate tumor cell metabolism, we performed metabolic screening of carcinoma cells and CAF cultivated on soft or stiff matrix (data not shown). Via liquid chromatography-tandem mass spectrometry (LC-MS/MS) candidate intracellular amino acids and metabolites were assessed to determine the activity of glycolysis, anaplerosis, and anabolic biosynthesis (data not shown) in SCC12 (data not shown) and CAF (data not shown). As reflected respectively by the lactate/pyruvate ratio, glutamine/glutamate ratio and aspartate production, stiff ECM increased glycolysis, glutamino lysis and activated anabolic biosynthesis in both SCC and CAF cells (data not shown). Similar observations were made on SCC 12 cultivated on ECM synthetized by CAF compared to ECM synthesized by non-activated fibroblast (data not shown). Consistent with increased glycolysis and glutamino lysis in stiff conditions, we observed an accumulation of extracellular lactate (data not shown) as well as decreased extracellular glutamine (data not shown) in conditioned media of both SCC12 and CAF. Importantly, ECM stiffening also increased release of glutamate and uptake of aspartate in SCC 12 cells while increasing aspartate release and decreasing glutamate release of CAF cells (data not shown). Levels of two key enzymes in SCC, lactate dehydrogenases A (LDHA) and glutaminase 1 (GLS1), which are implicated in both glycolysis and glutamino lysis, were elevated in stiff matrix (data not shown). As above, similar results were obtained for CAF (data not shown). Thus, matrix stiffness acts as a mechanical stimulus to increase glycolysis and glutaminolysis as well as to modulate flux of extracellular amino acids.

Cells within the tumor niche cooperate to promote tumorigenesis (Quail and Joyce, 2013). To assess whether there exists a crosstalk between SCC and CAF at a metabolic level, we performed a series of metabolomic studies. Specifically, we sought to identify molecules that were over-represented in CAF medium (and therefore secreted by CAF); under-represented in the CAF medium after contact with SCC cells (removed by SCC cells); and over-represented inside SCC cells treated with the CAF medium (taken up by SCC cells). Based on our previous results that identified complementary secretion and uptake of aspartate and glutamate between CAF and SCC cells, we focused specifically on those amino acids. Indeed, we found that aspartate was secreted by CAF and appeared to be taken up by SCC (data not shown). Moreover, we found that glutamate is increased in SCC medium, decreased in the SCC medium after contact with CAF cells, and increased inside CAF cells treated with the SCC medium (data not shown). Thus, these results indicate that metabolic crosstalk between SCC and CAF cells may rely upon dynamic alterations of amino acid flux. Taken together, stiff conditions sustain the metabolic demands of tumor microenvironement cells by activating glycolysis and glutaminolysis and coordinate cell to cell metabolic communication. Increased GLSl expression and glutaminolysis in both SCC and CAF cells are critical for metabolic reprogramming and sustaining pro-tumoral activity in stiff environment.

In order to determine whether GLSl is critical for stiffness-induced metabolic reprogramming in both SCC and CAF, cells were cultivated on stiff matrix and exposed to known pharmacologic inhibitors of GLSl, BPTES (Bis-2-(5-phenylacetamido-l,3,4- thiadiazol-2-yl)ethyl sulfide) and CB839 (data not shown) or siR A (siGLSl). As quantified by LC-MS/MS, inhibition of GLSl in both SCC and CAF cells blunted the stiffness induced processes of glutamine consumption, glutamate production, and aspartate production as well as blunted the secretion of glutamate by the SCC and the secretion of aspartate by CAF (data not shown). GLSl inhibition also decreased glycolysis in stiff matrix, as indicated by decreased lactate/pyruvate ratio (data not shown).

We next investigated whether GLSl inhibition affected cell proliferation and migration through its effects on glutamate and aspartate metabolism (data not shown). Consistent with prior observations (Gross et al, 2014), GLSl inhibition, achieved via siRNA (data not shown) or pharmacologic means (data not shown), inhibited SCC proliferation and migration as assessed by cell count (data not shown), PCNA staining (data not shown) and microsopic cell migration tracking (data not shown). Importantly, in cells with decreased GLSl activity, cellular proliferation and migration were restored by glutamate and/or aspartate supplementation (data not shown).

It is known that CAF cells actively remodel the ECM to promote tumor progression (Gaggioli et al., 2007; Sanz-Moreno et al., 2011). We investigated whether GLSl inhibition controls CAF-dependent ECM remodelling and whether increased glutamate and aspartate levels are central to the actions of GLS 1 (data not shown). GLS 1 inhibition, achieved via siRNA (data not shown) or pharmacologic means (data not shown), inhibited CAF-dependent ECM production and remodelling as well as generation of contractile forces as assessed by gel contraction assays (data not shown), and traction force microscopy (data not shown). Importantly, in cells with diminished GLSl ECM remodelling activity and generation of contractile forces were at least partially restored by glutamate supplementation but not aspartate (data not shown). Thus, these results demonstrate that GLSl and its control of glutamate and aspartate production by glutaminolysis are essential for metabolic reprogramming and consequent pro-invasive phenotypes of tumor microenvironment cells.

To interrogate whether such metabolic cooperation between CAF and SCC is crucial for tumor progression and cell invasion, we performed three-dimensional co-culture invasion assays (spheroid) where GLSl inhibition was achieved either in CAF, SCC cells or both (data not shown). GLSl inhibition in both CAF cells and SCC cells inhibited cellular invasion, while inhibition of GLSl in either CAF or SCC was not sufficient to block invasion. Collectively, these results argue for a crucial role of aspartate and glutamate exchange between CAF and SCC in order to shape a pro-invasive tumor niche and sustain pro-tumoral activities.

SLC1A3 enables aspartate/glutamate exchange within the tumor niche to promote tumor progression.

To demonstrate that aspartate/glutamate exchange between CAF cells and SCC cells is a key step for tumor progression we sought to identify a transporter responsible for these exchanges (data not shown). Specifically, we hypothesized that such a transporter should be expressed in both CAF and SCC cells, over-expressed in SCC cultivated on stiff matrix (data not shown), and over-expressed in CAF compared to normal fibroblasts (data not shown). RT- qPCR screening of the glutamate/aspartate transporter family members (SLClAl-7) identified SLC1A3 as the only one that displayed this pattern of expression. We next investigated whether SLC1A3 is critical to sustain SCC (data not shown) and CAF (data not shown) pro-tumoral cellular activities. Consistent with our prior findings, GLSl inhibition, achieved via siRNA (data not shown) or pharmacologic means (data not shown), inhibited SCC proliferation and migration which was rescued by aspartate and/or glutamate supplementation. However, SLC1A3 inhibition alone, achieved via siRNA (data not shown) or TFB-TBOA (data not shown), a pharmacologic inhibitor of SLClAl-3 family, did not significantly affect SCC proliferation and slightly decreased SCC migration. Importantly, simultaneous inhibition of GLSl and SLC1A3 prevented glutamate/aspartate rescue of SCC proliferation and migration (data not shown). Similar to SCC cells, inhibition of SLC1A3 in CAF cells did not alter the generation of contractile forces and slightly decreased ECM remodelling (data not shown). Importantly, and consistent with our hypothesis that glutamate exchange between SCC and CAF is critical to sustain CAF activity, pharmacologic or genetic inhibition of GLSl and SLC1A3 together in CAF blunted glutamate rescue of CAF pro-tumoral responses (data not shown).

We next interrogated whether inhibition of metabolic crosstalk between CAF and SCC is sufficient to blunt tumor progression and invasion. Consistent with our prior findings, in 3D co-culture assay, siRNA knockdown of GLSl or SLC1A3 alone in CAF or SCC is not sufficient to impair cellular invasion (data not shown). In contrast, siRNA knockdown of SLC1A3 and GLSl together blunted cancer cell invasion (data not shown). Next, we assessed whether pharmacological inhibition of both SLC1A3 and GLSl could impair tumor progression more robustly than a pharmacological inhibitor of GLS1 alone (data not shown). As above, 3D co- cultures were treated with either vehicle control, CB839, BPTES, TFB-TBOA or a combination of these drugs in the presence or absence of glutamate/aspartate (data not shown). In the absence of glutamate/aspartate, inhibition of GLS1 alone was sufficient to blunt cancer cell invasion, however, in the presence of glutamate/aspartate inhibition, both GLS1 and SLC1A3 were required to block tumor cell invasion efficiently.

Based on these findings, we wanted to determine whether humans suffering from SCC may also be sensitive to these combined metabolic therapies. As observed in cell lines, in patient-derived spheroids either CB839 or TFB-TBOA treatment alone decreased intracellular aspartate and glutamate concentration (data not shown) and blunted tumor cell invasion. Combined treatment led to a greater inhibition (Fig.l). Collectively, these data demonstrate that aspartate and glutamate act through SLC1A3 as fuel sources for tumor microenvironment cells. As such, both SLC1A3 and glutamate/aspartate are necessary to sustain tumor microenvironment cellular activity in the absence of glutamine.

Modulation of mechanotransduction controls metabolism reprogramming of tumor niche cells.

Given our prior findings that mechanotransduction coordinates glycolysis and glutamino lysis in response to mechanical stress in the lung vasculature (Bertero et al., 2016), we next investigated whether manipulation of the mechanotransduction cascade (data not shown) affects the metabolic reprogramming and consequent behavior of tumor niche cells. In SCC, pharmacologic inhibition of FAK (PF573228), ROCK (Y27632), or YAP/TAZ (verteporfm) decreased the lactate/pyruvate ratio, indicative of a decrease of glycolysis (data not shown). Inhibition of the mechanotransduction cascade also blunted the effects of tumor niche stiffening on glutamino lysis and aspartate production (data not shown). Notably, the same pathways of glycolysis and glutamino lysis mechanically controlled by stiff ECM in CAF were activated (data not shown). Correspondingly, GLS 1 and LDHA expression were downregulated in SCC (data not shown) and CAF cells (data not shown) upon pharmacological and siRNA knockdown of YAP/TAZ (data not shown). Importantly, these metabolic changes were accompanied by decreased SCC cell proliferation and migration as well as decreased CAF matrix remodelling (data not shown). Consistently, inhibition of the mechanotransduction cascade in 3D co-culture assay decreased GLS activity (data not shown) and blunted cell invasion (data not shown). Importantly, similar results were obtained using patient-derived spheroids (data not shown). We next interrogated whether metabolic crosstalk between CAF and SCC is also affected by mechanotransduction. In the first instance, we determined whether inhibition of mechanotransduction altered SLC1A3 expression. In both CAF and SCC, inhibition of mechanotransduction decreased SLC1A3 expression (data not shown). By LC-MS/MS analysis of cell media at different experimental time points, we found that inhibition of mechanotransduction decreased aspartate secretion and glutamate in CAF, while inhibiting glutamate secretion and aspartate uptake in SCC cells (data not shown).

To test this hypothesis further, we performed rescue experiments. SCC and/or CAF cells were treated with siRNA YAP/TAZ in the presence or absence of glutamate/aspartate (data not shown). Consistent with our results (data not shown) and prior findings (Aragona et al., 2013; Calvo et al., 2013; Cordenonsi et al, 2011), siYAP/TAZ decreased SCC proliferation and CAF dependent ECM remodelling while addition of aspartate/glutamate resulted in at least partial rescue of these effects (data not shown). Importantly, in 3D co-culture assay, inhibition of YAP/TAZ in CAF or SCC was not sufficient to fully impair cell invasion (data not shown). However, inhibition of YAP/TAZ in both SCC and CAF impaired cell invasion and was slightly improved by aspartate/glutamate supplementation (data not shown). Considering all of these points together, we conclude that the FAK-ROCK- YAP/TAZ cascade is integral to the stiffness-induced metabolic reprogramming of the tumor niche.

The mechanotransduction cascade controls metabolism reprogramming of tumor niche cells in vivo.

To establish definitively whether tumor niche remodelling and stiffening modulate tumor cells metabolism and behaviour in vivo, we tested whether alteration of mechanotransduction cascades directly controls glutamino lysis and tumor progression in an orthotopic syngenic mouse model of a highly metastatic breast cancer (data not shown). First, using the well established Balb/c mammary tumor cell lines 67NR, 410.4 and 4T1, we determined whether ECM-dependent metabolic changes were conserved (data not shown). As reflected by LC-MS/MS analysis, ECM stiffening increased glycolysis, glutaminolysis and aspartate production as well as increased GLS1, LDHA, and SLC1A3 expression (data not shown).

Next, using a known pharmacologic inhibitor (β-aminopropionitrile, BAPN) of lysyl- oxidase (Lox), the enzyme responsible for collagen cross-linking and consequent matrix stiffening (Mouw et al., 2014), we determined whether inhibition of ECM stiffening could prevent metabolic changes and tumor progression in mice using a disease reversal dosing protocol. BAPN treatment decreased breast tumor Lox activity (data not shown) and consequent ECM stiffening, as assessed by atomic force microscopy (data not shown). Consistent with our in vitro results, reduction of ECM stiffening by BAPN led to a decrease of GLS1 and SLC1A3 expression, and downstream GLS activity (data not shown), as reflected by direct enzymatic activity measurement (data not shown). Such metabolic effects further decreased stromal activation as reflected by□ -SMA staining (data not shown), decreased tumor cell proliferation, as reflected by in situ staining of the proliferation marker PCNA (data not shown), blunted tumor progression as reflected by tumor volume (data not shown) and quantitation of lung (data not shown) and liver metastasis (data not shown), and increased survival outcome (data not shown).

In a parallel experiment, verteporfin, a known pharmacologic inhibitor of YAP (Park and Guan, 2013), was used to interrogate whether YAP is also essential for activating glutamino lysis and metabolic changes to sustain tumor progression (data not shown). As expected, verteporfin decreased YAP-dependent gene expression (data not shown). Consequently, in a similar fashion to BAPN, verteporfin improved the downstream metabolic (GLS land SLC1A3 expression, and GLS activity), proliferative (data not shown), and end- stage manifestations of breast cancer, including reductions of tumor volume, lung and liver metastasis, and survival (data not shown). Notably, verteporfin also decreased tumor stiffness and stromal activation (data not shown ), consistent with prior report of YAP-dependent control of ECM remodelling (Bertero et al., 2015a; Calvo et al., 2013).

As a result, these data provide causative evidence in vivo that tumor niche stiffening relies on mechanotransduction pathways in order to induce tumor cell glutamino lysis and glycolysis, proliferation, invasion, and overall survival outcome.

On the basis of these findings in mice, we wanted to determine whether humans suffering from SCC may also display signs of increased ECM stiffness and consequent alterations in glutamino lysis and aspartate/glutamate transport (data not shown). We first studied a cohort of 48 patients with pathological diagnosis of head and neck squamous cell carcinoma (HNSCC) resulting in the generation of 3 cohorts based on tumor invasiveness (Low, N=15; middle, N=l l; high N=22). Correlating with increased collagen remodelling in highly invasive cases (data not shown), a concurrent upregulation of GLS1 (data not shown) and SLC1A3 (data not shown) as well as stromal activation (D-SMA staining) were observed (data not shown). Finally, a patient-derived xenograft (PDX) model of HNSCC expansion was tested in vivo (data not shown). Three independent F1NSCC tumors were subcutaneously engrafted in the flanks of nude mice. One week later, to avoid any interference with tumor uptake, mice were treated with BAPN or veteporfm. Both BAPN and verteporfin treatment improved the downstream metabolic (GLS1 and SLC1A3 expression, and GLS activity), and proliferative (data not shown), manifestations of HNSCC, including reductions of stromal activation (□- SMA staining; data not shown) as well as tumor volume (data not shown). Together, these results support the notion that tumor niche stiffening activates mechanotransduction in order to induce a metabolic switch and tumor progression across both mouse and human cancers in vivo.

Aspartate/glutamate exchanges within the tumor niche are crucial to sustain tumor progression in vivo.

We next determined whether amino acid exchange was occurring in the tumor microenvironment in vivo. We developed a co-injection system in which CAF could be manipulated genetically and then implanted alongside cancer cells into the mammary fat pads of syngenic mice. Although our previous study demonstrated that co-injection of CAF with non- invasive 67NR cells promotes tumor growth and local invasion (Albrengues et al, 2015), the contribution of metabolic cross-talk to this effect has not been explored. Therefore, we performed co-injection studies with 67NR cells along with CAF with conditional knockdown of SLC1A3 and GLS1, separately or together. Fifteen day after cells injections mice were treated with doxycyline in order to induce depletion of GLS1 and/or SLC1A3 in stromal fibroblast (data not shown). Consistent with the in vitro 3D assay data , while GLS1 or SLC1A3 knockdown slightly decreases intratumoral aspartate (data not shown).) and glutamate (data not shown) concentration, combined treatment led to a greater inhibition. Such metabolic effects further decreased stromal activation as reflected by D-SMA staining (data not shown) and decreased stromal-dependant ECM remodelling as quantified by picrosirius red staining (data not shown). Moreover, these treatments decreased tumor cell invasion (data not shown) and tumor cell proliferation (data not shown), as reflected by in situ staining of the proliferation marker PCNA.

We next investigated whether the reciprocal mechanism was also effective in vivo.

Consistent with our findings and a previous report (Yang et al., 2004), 4T1 cells promoted tumor invasion by converting resident fibroblasts into CAF. However, the contribution of metabolic cross-talk to this effect has not been explored. Thus, we injected 4T1 cells conditionally knock-down for SLC1A3, GLS1 or both SLC1A3 and GLS1 into the mammary fat pads of syngenic mice (data not shown). Namely, whereas either GLS1 or SLC1A3 knockdown alone decreased intratumoral aspartate and glutamate concentration, combined treatment led to a greater inhibition (data not shown). Such metabolic effects further decreased stromal activation as reflected by D-SMA staining (Fig.6J-K) and decreased tumor cell proliferation, as reflected by in situ staining of the proliferation marker PCNA (data not shown). Consistent with these observations and our in vitro results, GLS1 or SLC1A3 knockdown in epithelial cancer cell inhibited 4T1 tumor progression, as quantified by tumor volume (data not shown) and lung metastasis (data not shown). Combined treatment led to greater inhibition of tumor growth and invasion and further improved survival (data not shown).

In sum, these data provide evidence for two-way intratumoral metabolic cross-talk, in which cancer cells release glutamate that results in the activation of CAF and the release of aspartate, thereby used by cancer cell to proliferate and invade.

Pharmacological inhibition of glutaminolysis and aspartate/glutamate exchange synergizes to block tumor progression in vivo.

To investigate whether pharmacological inhibition of both glutaminolysis and aspartate/glutamate exchange are relevant therapies for cancer, we performed combination treatments in the highly metastatic orthotopic syngenic 4T1 breast cancer mouse model (Fig.2A). Namely, whereas either CB839 or TFB-TBOA treatment alone decreases intratumoral aspartate and glutamate concentration, combined treatment led to a greater inhibition (Fig.2B-C). Such metabolic effects further decreased stromal activation as reflected by D-SMA staining (data not shown) and decreased tumor cell proliferation, as reflected by in situ staining of the proliferation marker PCNA (data not shown). Consistent with these observations and our in vitro results, CB839 or TFB-TBOA, treatments inhibited 4T1 tumor progression as quantified by tumor volume (data not shown) and lung and liver metastasis (Fig.2D). Combined treatment led to greater inhibition of tumor growth and invasion and further improved survival outcomes (Fig 2F).

Finally, on the basis of these findings in mice, we wanted to determine whether humans suffering from SCC may also be sensitive to these combined therapies. A PDX model of FTNSCC expansion was tested in vivo (Fig.2 G-M). Three independent FTNSCC tumors were subcutaneously engrafted in the flanks of nude mice. One week later, to avoid any interference with tumor uptake, mice were treated with either vehicle control, CB839, BPTES, TFB-TBOA or a combination of these drugs. As observed in vitro and in vivo, in PDX model of FTNSCC (Fig.2 G-M) either CB839 or TFB-TBOA treatment alone decreased intracellular aspartate and glutamate concentration (Fig.2 G), decreased stromal activation as reflected by D-SMA staining (Fig.2 J), decreased tumor cell proliferation as reflected by in situ staining of the proliferation marker PCNA (Fig.2 J), and blunted tumor growth (Fig.2 K-M). Combined treatment led to a greater inhibition (Fig. G-M).

Taken together, these results directly implicate glutaminolysis and amino acid crosstalk within the tumor niche, two processes dependent on ECM stiffening, as critical metabolic mediators necessary for sustaining tumor cells activation and cancer progression. Importantly, combining CB839 treatment with TFB-TBOA led to significantly greater SCC suppression than each treatment alone. Our findings from combined pharmacologic, molecular, and genetic studies in diverse preclinical models of SCC establish the YAP/TAZ-GLS1-SLC1A3 axis as a promising therapeutic target that robustly suppresses SCC progression through the regulation of aspartate and glutamate— key metabolites of SCC tumor expansion and metastasis.

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Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A method for treating cancer in a subject in need thereof comprising a step of administering the subject with a therapeutically effective amount of GLSl and SLC1A3 inhibitors.

2. The method according to claim 1, wherein, the GLSl and SLC1A3 inhibitors are small organic molecules.

3. The method according to claims 1 to 2, wherein, the GLSl and SLC1A13 inhibitors are TBOA and CD839.

4. The method according to claim 1 to 3, wherein, the the GLSl and SLC1A3 inhibitors are administered as a combined preparation.

5. The method according to claims 1 to 4, wherein, the GLSl and SLC1A3 inhibitors are administered simultaneously, separately or sequentially.

6. A pharmaceutical composition comprising the inhibitors of GLSl and SLC1A3.

7. The pharmaceutical composition according to claim 6, wherein the inhibitors of GLSl and SLC1A3 are TBOA and CD839.