WO2025210123A1 - Methods and pharmaceutical composition for treating cancers - Google Patents

Abstract

Inventors demonstrated that an epigenetic regulator CBX3 antagonizes IFNγ signalling via directly repressing the transcription of two key interferon-stimulated immune genes, STAT1 and PD-L1. The key role of CBX3 in repressing STAT1 and PD-L1 transcription suggests that CBX3 is an important checkpoint to control immune genes' activation in response to different ligands' stimulation, such as the important immune modulator IFNγ, which placed CBX3 in a key position to keep colon immune homeostasis. This role placed also CBX3 in a key position to keep colon immune homeostasis. These studies have started to reveal a new role of CBX3 in controlling the colon epithelium inflammatory response, in addition to its classical role in the formation and stabilization of heterochromatin. Particularly, low CBX3 expression is associated with better CRC patients' overall survival. Corresponding to this result, CBX3 depletion makes IFNγ-insensitive CRC cells dramatically regain IFNγ sensitivity, which significantly increases CRC cells' chemosensitivity under IFNγ stimulation. Accordingly, the invention relates to a Chromobox protein homolog 3(CBX3) inhibitor for use in the treatment of cancer in a subject in need thereof.

Description

METHODS AND PHARMACEUTICAL COMPOSITION FOR TREATING

CANCERS

FIELD OF THE INVENTION:

The present invention is in the field of medicine, in particular oncology.

BACKGROUND OF THE INVENTION:

The signal transducer and activator of transcription 1 (STAT1) is a transcription factor that is encoded by the STAT1 gene in humans and activated by JAK kinase. STAT1 is mainly activated by IFN (a, P, y). In addition, other cytokines, including, IL-2, IL-6, platelet-derived growth factor (PDGF), epidermal growth factor (EGF), hepatocyte growth factor, and angiotensin II etc. can also activate STAT1. The JAK/STAT signaling pathway, in response to different ligands’ stimulation, has a profound influence on malignancies and autoimmune diseases.

Interferon y (IFNy or IFNG) is an essential cytokine in orchestrating both innate and adaptive immune responses. IFNy signaling is activated by binding of IFNy to its receptors (IFNyRl and IFNyR2). This binding forms the IFNGR protein complex and subsequently activates JAK1/JAK2, which further phosphorylates and dimerizes the transcription factor STAT1 (signal transducer and activator of transcription). The phospho-STATl dimer then migrates to the nucleus and activates the inflammation response by promoting interferon- stimulated genes (ISGs) transcription, including STAT1 itself and Interferon regulatory factor 1 (IRF1). The latter, in turn promotes CD274 (Programmed cell death ligand 1, PD-L1) transcription to downregulate the magnitude of the inflammation response. Therefore, as an important immune stimulator and modulator, IFNy, along with its effectors STAT1 and PD LI, plays very crucial roles in balancing immune homeostasis and inflammatory response. The aberrant IFNy signaling is often associated with oncogenesis (such as colorectal cancer) (Kak, Raza, and Tiwari 2018) (Yi et al. 2018) (Du et al. 2022) (Alspach, Lussier, and Schreiber 2019).

Chemotherapy is the most used treatment strategy in colon and breast cancer. Chemotherapy induces immunogenic cell death which stimulates antigen-specific T cells to produce IFNy; which then influence chemotherapeutic efficacy either by regulating immune effect, such as driving Thl rather than Th2 differentiation, activating NK cells and increasing antigen presentation, or by exerting anti-proliferative, anti-angiogenic, and pro-apoptotic effects on cancer cells in response to genotoxic damage (Minn 2015) (Coffelt and de Visser 2015). Drug resistance to chemotherapy is a main problem that limits the efficacy of conventional cancer therapy. Given the important role of IFNy/STATl/PD-Ll axis in innate and adaptive immune responses, targeting the IFNy pathway is a rational and novel management in colorectal and breast cancer (Du et al. 2022).

The heterochromatin protein 1 (HP1) family are epigenetic regulators, which are readers of the H3K9me2/3 histone modifications and play an important role in the formation and maintenance of heterochromatin. HP1 family includes CBX1 (HPip), CBX5 (HPla) and CBX3 (HPly) in mouse and human.

Nowadays, CBX3 has obtained widespread attention as a potential biomarker in several cancers (Niu H 2022). However, CBX3 was largely unexplored as a therapeutic target in cancer, more particularly in different cancer therapies, such as chemotherapy.

SUMMARY OF THE INVENTION:

The invention relates to a Chromobox protein homolog 3(CBX3) inhibitor for use in the treatment of cancers.

In a particular embodiment, the invention relates to i) a Chromobox protein homolog 3(CBX3) inhibitor, ii) an activator of IFNy/STATl/PD-Ll axis and iii) a classical treatment, as a combined preparation for use in the treatment of a cancer in a subject in need thereof.

The invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION:

In this study, inventors demonstrated that an epigenetic regulator CBX3 directly represses the transcription of two key immune genes, STAT1 and PD-L1, which could finally antagonize IFNy signaling activation. Likewise, upon IFNy stimulation, CBX3 binding is decreased from the promoters of STAT1 and CD274, concomitant with their increased gene expression. The key role of CBX3 in repressing STAT1 and PD-L1 transcription suggests that CBX3 is an important checkpoint to control immune genes’ activation in response to different ligands’ stimulation, such as the important immune modulator IFNy, which placed CBX3 in a key position to keep colon immune homeostasis.

These studies have started to reveal a new role of CBX3 in controlling the colon epithelium inflammatory response, in addition to its classical role in the formation and stabilization of heterochromatin.

Particularly, low CBX3 expression is associated with better CRC patients’ overall survival. Corresponding to this result, CBX3 depletion makes IFNy-insensitive CRC cells dramatically regain IFNy sensitivity, which significantly increases their chemosensitivity under IFNy stimulation in vitro with CRC cells and in vivo with a syngeneic mouse tumor model. Altogether, this work identifies a new antagonist interplay between CBX3 and IFNy signalling to regulate colon immune response and chemosensitivity of colorectal cancer, which highlights CBX3 as a potential target to improve the treatment of colorectal cancer.

Furthermore, for breast cancer, the clinical analysis revealed also low CBX3 but high IFNy expression associated with better patients’ overall survival, and high CBX3 expression combined with low STAT1 and CD274 expression associated with worse overall survival. At the same time, the deletion of CBX3 in breast cancer cell lines increased also STAT1 and PD- L1 expression, which suggested the same antagonist mechanism between CBX3 and IFNy also existed in breast cancer.

In a first aspect, the present invention relates to a Chromobox protein homolog 3(CBX3) inhibitor for use in the treatment of cancer in a subject in need thereof.

More particularly, the present invention relates to a method for treating cancer in a subject in need thereof comprising a step of administrating the subject with a therapeutically effective amount of Chromobox protein homolog 3(CBX3) inhibitor.

As used herein, the term “subject” denotes a vertebrate such as mammal, bird, fish, amphibian or reptile. In some embodiments, the vertebrate is a warm-blooded vertebrate (i.e. mammal, bird, fish). In some embodiments, the subject is a mammal, such as a rodent, a feline, a canine (e g. a dog), an equine (e.g. horse), a bovine (e.g. a beef), a sheep or a primate. More particularly, the subject according to the invention is a human.

In some embodiments, the subject suffers from a cancer.

In some embodiments, the subject suffers from a cancer in which the CBX3 expression and/or activity is increased abnormally.

In some embodiments, the subject suffers from STAT1 related cancer.

In some embodiments, the subject suffers from IFNY related cancer.

In some embodiments, the subject suffers from colon cancer.

In some embodiments, the subject suffers from breast cancer.

As used herein, the term “cancer” refers to liquid or solid cancer.

More particularly, in the context of the invention, the cancer wherein the expression and/or activity of CBX3 is increased abnormally.

In a particular embodiment, the cancer is selected from the group consisting of but not limited to: Colorectal cancer (CRC), breast cancer, osteosarcoma, melanoma, glioblastoma, glioma cancer (Brain lower grade glioma), neuroblastoma, lung cancer(Lung squamous cell carcinoma, Lung Adenocarcinoma), ovarian cancer, gastric cancer (Stomach adenocarcinoma), prostate cancer(Prostate adenocarcinoma), pancreatic cancer(Pancreatic adenocarcinoma), endometrial cancer, uterine cancer (Uterine corpus endometrial carcinoma), bladder cancer, cholangiocarcinoma, esophageal cancer, head and neck squamous cell cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors, kidney cancer (Kidney renal clear cell carcinoma, Kidney renal papillary cell carcinoma), liver cancer, thyroid cancer, Cervical squamous cell carcinoma and endocervical adenocarcinoma, myeloma cancer (Acute myeloid leukemia), B-cell lymphoma cancer (Lymphoid neoplasm diffuse large B-cell lymphoma), leukaemia (like acute myeloid leukaemia, acute lymphoid leukaemia, chronic myelomonocytic leukemia (CMML)...), lymphoma and myelodysplastic syndrome (MDS), Hodgkin's disease, laryngeal and hypopharyngeal cancer, thymus cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, penile cancer, pituitary cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer testicular cancer (e g. seminoma, nonseminoma germ cell cancer), vaginal cancer, vulvar cancer.

In a particular embodiment, the cancer is colorectal cancer.

In a particular embodiment, the cancer is breast cancer.

In another embodiment, the cancer is resistant cancer.

In another embodiment, the cancer is metastatic cancer.

As used herein, the term “metastatic cancer” refers to the spread of cancer from one organ or tissue to another location. The term also refers to tumor tissue that forms in a new location as a result of metastasis. A "metastatic cancer" is a cancerthat spreads from its original, or primary, location, and may also be referred to as a "secondary cancer" or "secondary tumor". Generally, metastatic tumors are named for the tissue of the primary tumor from which they originate.

As used herein, the term “resistant cancer” refers toa cancer which does not respond to a treatment. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment. The resistance to drug leads to rapid progression of metastatic cancer. The resistance of cancer for the medication is caused by mutations in the gene which are involved in the proliferation, divisions or differentiation of cells. In a particular embodiment, the resistant cancer is resistant to IFNy related therapy.

In a further embodiment, the cancer is a cancer in which IFNy signalling pathway deeply affects the development and treatment of the cancer. As used herein, the term STAT 1 -related cancer refers to all cancers in which STAT1- related signalling pathway is involved. In the context of the invention, Interferon y, a major STAT1 activator, is focused as an important example to validate the great potential of controlling STAT1 expression and activation in improving the efficacity of diverse cancer treatments.

As used herein, the term “STAT1” refers to Signal transducer and activator of transcription 1 (STAT1). STAT1 is a transcription factor which in humans is encoded by the STAT1 gene.

As used herein, the term “IFNy related cancer” refers to all cancer in which IFNy signalling pathway is involved.

As used herein, the term “IFN” refers to interferon (IFN). IFN genes and proteins have been identified, and typically categorized into three classes: Type I IFN (IFN-a and IFN-0), Type II IFN (IFN-y), and Type III IFN (IFN-k). IFN-y is a notably different member characterized by unique receptor activity and distinct intracellular signalling pathway: type I IFNs depends on the interferon-stimulated gene factor-3 (ISGF3) complex containing STAT1/STAT2/IRF9, which binds to interferon-sensitive response elements (ISREs), whereas the phosphorylated signal transducer and activator of transcription 1 (STAT1) homodimer downstream of IFN-y binds to interferon-gamma activation sites (GASs).

Interferon y (IFNy or IFNG) is an essential cytokine in orchestrating both innate and adaptive immune responses. IFNy signaling is activated by binding of IFNy to its receptors (IFNyRl and IFNyR2). This binding forms the FFNGR protein complex and subsequently activates JAK1/JAK2, which further phosphorylates and dimerizes the transcription factor STAT1 (signal transducer and activator of transcription). Inventors have demonstrated that an epigenetic regulator CBX3 antagonizes IFNy signaling via directly repressing the transcription of two key interferon-stimulated immune genes, ST ATI and PD-L1.

As used herein, the term "Chromobox protein homolog 3” (CBX3) also called as heterochromatin protein 1 (HPly) refers to highly conserved proteins, which have important functions in the cell nucleus. HP1 is considered to be a major constituent of heterochromatin important for gene silencing, HP1 is now known to be a dynamic protein that also functions in transcriptional elongation, centromeric sister chromatid cohesion, telomere maintenance and DNA repair.

The naturally occurring human CBX3 gene has a nucleotide sequence as shown in Genbank Accession number NM_007276 and the naturally occurring human SOX21 protein has an aminoacid sequence as shown in Genbank Accession numbers NP_009207. The naturally occurring mouse CBX3 gene has a nucleotide sequence as shown in Genbank Accession number NM_007624.

The human CBX3 has the following nucleotide sequence in the art SEQ ID NO: 1 :

1 gcgccagccg ctgaggctgc caagcagaaa agccaccgct gaggagactc cggtcactgt

61 cctcgccccg cctccccctt ccctcccctt ggggaccacc gggcgccacg ccgcgaacgt 121 aatagctctt caagtctgca ataaaaaatg gcctccaaca aaactacatt gcaaaaaatg 181 ggaaaaaaac agaatggaaa gagtaaaaaa gttgaagagg cagagcctga agaatttgtc 241 gtggaaaaag tactagatcg acgtgtagtg aatgggaaag tggaatattt cctgaagtgg 301 aagggattta cagatgctga caatacttgg gaacctgaag aaaatttaga ttgtccagaa 361 ttgattgaag cgtttcttaa ctctcagaaa gctggcaaag aaaaagatgg tacaaaaaga 421 aaatctttat ctgacagtga atctgatgac agcaaatcaa agaagaaaag agatgctgct 481 gacaaaccaa gaggatttgc cagaggtctt gatcctgaaa gaataattgg tgccacagac 541 agcagtggag aattgatgtt tctcatgaaa tggaaagatt cagatgaggc agacttggtg 601 ctggcgaaag aggcaaatat gaagtgtcct caaattgtaa ttgcttttta tgaagagaga 661 ctaacttggc attcttgtcc agaagatgaa gctcaataat tgttcacatt gttcttttat 721 atatatttat atatatatat aaaaattggg tcttagattt tgatttacta gtgtgacaaa 781 ataactacat cctaatgaaa atcaagtttg atatgtttgt tttgaaagta gcgttggaag 841 agttgttggg ggttttttgc atccatagca ctggttactt tgaacaaata aataaaagct 901 ttctgtagtt gcttccttta tcagaaaaga acatttgata ccatggtata tcatttcctc 961 ttcattaaag aacagctttt ctaaatgttg ggggaaatgt ccatagtcat tactcagtca 1021 aaacttgtgt tctcatgagc ctaaggacca ttctagattt attacgtgtt ttttgtgtgt 1081 gtgtgtgtgt gtgtgtgtgt gtgtatccat aaaatgcata tgtaaatttt tttttgtttt 1141 taagcattca cccaaacaaa aaaatcacag gtaaacccat gtttctgaga tgccattatt 1201 ccaagcaaaa taagagataa tcccttcaag ttaaattgaa aattttcctg aaaccataca 1261 tttcaagtga aataagtaat tctagatagg acaatttaaa ttggataatt ttaaagtgtc 1321 tataattgca gtggtttatt tgcaaaattc ctaaaaggaa aaattttatc actgccatca 1381 cagcaggttt cctcatccag atgaggaaac tagacaaatg ctagtgtgtt ttaactagct 1441 aaacaaaact aagttaaatg aacatttaaa agtttcccta gcgggccatt ccttagcaaa 1501 atgttggaat ccctgttgct acattgacta aaaggtcatg atgaatggaa tatgtaagac 1561 ttggctcata gaaacctaat cagatggtta gaggtgttgg cagtttagga cctgctgtca 1621 taaatgtgtg aacaaccttt tgtaacctaa cctattgacc tgcatgtttt ttctttaccc 1681 caattcatta catggaggct caatcttgag tttgctttac tggttcagca aaagccagga 1741 agaacaactt tgtagtaatc aaaatgttat ccaactgtat attgtttact ttattgtaaa 1801 tactggtgaa cagtggttaa taaatagttt tatattcctt tatgcaatta ttagactttt 1861 ttctttattt gatatgcctt tacagtagaa atagaaatgc ccacactcat tggattatct 1921 ttgtttataa gttagatgat accagtaagg cattacagta catatcctag atcttttgag 1981 cttacgagtt ttaaacttga atatgtattt ccacaggaat gtttccacag ttgggaaata 2041 aaagtttcat gtgatgccta gggtcaattg tctcattaaa atgaggtttt aaattctg

As used herein, the term "treatment" or "treat" refers to both prophylactic or preventive treatment as well as curative or disease-modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects 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 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., disease manifestation, etc.]).

As used herein, the term “CBX3 inhibitor” denotes a molecule that partially or totally inhibits CBX3 biological activity or expression. The term also encompasses a molecule able to decrease or inhibit CBX3 gene expression. In order to test the functionality of a putative agent for CBX3 protein expression, a test is necessary. For that purpose, to identify an agent for CBX3 protein expression, a western blot analysis can be run on cell extracts to test the effect of the putative agent for CBX3 protein expression on the level of CBX3. If the level of CBX3 is decreased, the putative agent for CBX3 protein expression will have the desired effect.

In some embodiments, the CBX3 inhibitor is selected from the group consisting of but not limited to: small organic molecule, peptide, peptidomimetic, antibody, aptamers, siRNA or antisense oligonucleotide.

CBX3 inhibitors are well known in the state of the art and include those described in : Rani R et al . , 2019, RNA Biol Yan W et al., 2019, Helicobacter Chen Y et al., 2021, Front Microbiol Maeda R et al . , 2022, EMBO Rep Porschke D et al., 1999, JMB

In the context of the present invention, “CBX3 inhibitor” is an inhibitor which neutralizes, blocks, inhibits, abrogates, reduces or interferes with the biological activity of CBX3. In a particular embodiment, the CBX3 inhibitor according to the invention is a low molecular weight compound, e. g. a small organic molecule (natural or not).

The term "small organic molecule" refers to a molecule (natural or not) 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 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da. In some embodiments, the CBX3 inhibitor is a compound selected in Ian A. MacDonald et al 2019 (DOI:https://doi.org/10.1177/2472555219849838).

In a particular embodiment, the CBX3 inhibitor is compound I as described below:

Compound I (formula I)

In a further embodiment, the CBX3 inhibitor is a compound as described in W02019/006322A1.

In some embodiments, the inhibitor of CBX3 is an antibody. As used herein, the term “antibody” 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 0368 684, WO 06/030220 and WO 06/003388.

In a particular embodiment, the inhibitor of CBX3 is scFv fragments as described in Ilaria Filesi et al 2002 (doi.org/10.1242/jcs.115.9.1803).

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 inhibitor is an intrabody having specificity for CBX3. 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 inhibitor of CBX3 expression is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of PSMD14. In a particular embodiment, the inhibitor of CBX3 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. In a particular embodiment, the inhibitor of PSMD14 expression is an anti-sense oligonucleotides (ASO). 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 15 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 CBX3 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 homology-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. Trop. Dis., Vol. 8:e267E), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038/cr.2014.11.), 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. H3.160713), 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).

Combined preparation

Inventors have demonstrated that low CBX3 expression associated with better CRC patients’ overall survival. Corresponding to this result, CBX3 depletion makes IFNy-insensitive CRC cells dramatically regain IFNy sensitivity, which significantly increases CRC cells’ chemosensitivity under IFNY stimulation.

Altogether, this work identifies a new antagonist interplay between CBX3 and IFNy signalling to regulate colon immune response and chemosensitivity of colorectal cancer, which highlights CBX3 as a potential target to improve the treatment of UC and colorectal cancer.

Accordingly, in a second aspect, the invention relates to i) a Chromobox protein homolog 3(CBX3) inhibitor and ii) a classical treatment, as a combined preparation for use in the treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) a Chromobox protein homolog 3(CBX3) inhibitor, ii) an activator of IFNy/STATl/PD-Ll axis, and iii) a classical treatment as a combined preparation for use in the treatment of a cancer in a subject in need thereof.

As used herein, the term “classical treatment” refers to treatments well known in the art and used to treat a cancer. In the context of the invention, the classical treatment refers to an activator of IFNy/STATl/PD-Ll axis, targeted therapy, radiation therapy, immunotherapy, chemotherapy or ferroptosis therapy. In a particular embodiment, the invention relates to i) a Chromobox protein homolog 3(CBX3) inhibitor and ii) an activator of IFNy as a combined preparation for use in the treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) a Chromobox protein homolog 3(CBX3) inhibitor and ii) an activator of STAT1 as a combined preparation for use in the treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) a Chromobox protein homolog 3(CBX3) inhibitor, ii) an activator of IFNy/STATl/PD-Ll axis and iii) a classical treatment, as a combined preparation for use in the treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) a Chromobox protein homolog 3(CBX3) inhibitor, ii) an activator of IFNy and iii) a classical treatment, as a combined preparation for use in the treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) a Chromobox protein homolog 3(CBX3) inhibitor, ii) an activator of STAT1 and iii) a classical treatment, as a combined preparation for use in the treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) a Chromobox protein homolog 3(CBX3) inhibitor, ii) an activator of IFNy/STAT l/PD-Ll axis, iii) a ferroptosis inducer and iv) a classical treatment, as a combined preparation for use in the treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) CBX3 inhibitor, ii) an activator of IFNy/STATl/PD-Ll axis and iii) a classical treatment for use by simultaneous, separate or sequential administration in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) CBX3 inhibitor, ii) an activator of IFNy/STATl/PD-Ll axis and iii) a classical treatment for use by simultaneous, separate or sequential administration in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) CBX3 inhibitor and ii) immunotherapy for use by simultaneous, separate or sequential administration in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to a method for enhancing the efficacy of classical treatment by increasing the sensitivity of cancer cells to IFNy/STATl/PD-Ll axis comprising a step of administrating an inhibitor of CBX3 in a subject in need thereof. In a particular embodiment, the invention relates to a method for enhancing the efficacy of classical treatment by increasing the sensitivity of cancer cells to IFNy comprising a step of administrating an inhibitor of CBX3 in a subject in need thereof.

In a particular embodiment, the invention relates to a method for enhancing the efficacy of classical treatment by increasing the sensitivity of cancer cells to STAT1 comprising a step of administrating an inhibitor of CBX3 in a subject in need thereof.

In a particular embodiment, the invention relates to a method for enhancing the efficacy of classical treatment by increasing the expression or activation of STAT1 in response to different molecules, which could activate JAK/STAT1 signaling and increase the sensitivity of cancer cells to different treatments.

More particularly, the invention relates to a method for sensitizing cancer cells to IFNy/STATl/PD-Ll axis stimulation in order to improve the efficacy to a classical treatment of cancer in a subject in need thereof.

More particularly, the invention relates to a method for sensitizing cancer cells to IFNY stimulation in order to improve the efficacy to a classical treatment of cancer in a subject in need thereof.

More particularly, the invention relates to a method for sensitizing cancer cells to STAT1 related signalling in order to improve the efficacy to a classical treatment of cancer in a subject in need thereof.

As used herein, the term “cancer” refers to liquid or a solid cancer as described above. More particularly, in the context of the invention, the cancer is a cancer in which the expression and/or activity of CBX3 is increased abnormally.

In a particular embodiment, the cancer is selected from the group consisting of but not limited to: Colorectal cancer (CRC), breast cancer, osteosarcoma, melanoma, glioblastoma, glioma cancer (Brain lower grade glioma), neuroblastoma, lung cancer(Lung squamous cell carcinoma, Lung Adenocarcinoma), ovarian cancer, gastric cancer (Stomach adenocarcinoma), prostate cancer(Prostate adenocarcinoma), pancreatic cancer(Pancreatic adenocarcinoma), endometrial cancer, uterine cancer (Uterine corpus endometrial carcinoma), bladder cancer, cholangiocarcinoma, esophageal cancer, head and neck squamous cell cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors, kidney cancer (Kidney renal clear cell carcinoma, Kidney renal papillary cell carcinoma), liver cancer, thyroid cancer, Cervical squamous cell carcinoma and endocervical adenocarcinoma, myeloma cancer (Acute myeloid leukemia), B-cell lymphoma cancer (Lymphoid neoplasm diffuse large B-cell lymphoma), leukaemia (like acute myeloid leukaemia, acute lymphoid leukaemia, chronic myelomonocytic leukemia (CMML) . ), lymphoma and myelodysplastic syndrome (MDS), Hodgkin's disease, laryngeal and hypopharyngeal cancer, thymus cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, penile cancer, pituitary cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer testicular cancer (e g. seminoma, nonseminoma germ cell cancer), vaginal cancer, vulvar cancer.

In a particular embodiment, the cancer is colon cancer.

In a particular embodiment, the cancer is colorectal cancer.

In a particular embodiment, the cancer is breast cancer.

In another embodiment, the cancer is resistant cancer.

In another embodiment, the cancer is metastatic cancer.

In a further embodiment, the cancer is a cancer in which STAT1 expression and activation pathway deeply affect the development and treatment of the cancer.

In a further embodiment, the cancer is a cancer in which IFNy signalling pathway deeply affects the development and treatment of the cancer.

As used herein, the term “IFNy related cancer” refers to all cancer in which IFNy signalling pathway is involved.

In a further embodiment, the cancer is resistant to IFNy related therapy.

In some embodiments, the subject suffers from IFNy related cancer.

In some embodiments, the subject suffers from a resistant cancer to IFN therapy.

In some embodiments, the subject suffers from a metastatic cancer.

As used herein, the term “enhance the sensibility of cells to IFNy stimulation” refers to increase the cytotoxic effects of classical therapy to treat a cancer in a subject in need thereof.

As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be a bi-therapy combining a CBX3 inhibitor and a chemotherapeutic agent.

In a further embodiment , the combined therapy may be a tri -therapy combining a CBX3 inhibitor, an IFNy activator and a chemotherapeutic agent.

In a further embodiment, the combined therapy may be a tri-therapy combining a CBX3 inhibitor, an IFNy activator and a radiation therapy.

In a further embodiment, the combined therapy may be a tri-therapy combining a CBX3 inhibitor, an IFNy activator and a ferroptosis inducer. In a further embodiment, the combined therapy may be a tri-therapy combining a CBX3 inhibitor, a STAT1 activator and a chemotherapeutic agent.

In a further embodiment, the combined therapy may be a tri-therapy combining a CBX3 inhibitor, a STAT1 activator and a radiation therapy.

In a further embodiment, the combined therapy may be a tri-therapy combining a CBX3 inhibitor, a STAT1 activator and a ferroptosis inducer.

In a further embodiment, the combined therapy may be a combination of: i) a CBX3 inhibitor, ii) a ferroptosis inducer (such as RSL-3), iii) an immunotherapy (such as anti-PD-Ll) and iv) an IFNy activator.

As used herein, the term “administration simultaneously” refers to administration of at least 2 or 3 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 at least 2 or 3 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of at least 2 or 3 active ingredients at different times, the administration route being identical or different.

In a particular embodiment, the classical treatment refers to a combined treatment to treat a cancer.

In a particular embodiment, the invention relates to i) a CBX3 inhibitor according to the invention, ii) an activator of IFNy/STATl/PD-Ll axis and iii) a targeted therapy used as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) a CBX3 inhibitor according to the invention, ii) an activator of IFNy and iii) a targeted therapy used as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 ii) an activator of IFNy and iii) a chemotherapeutic agent as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to to i) an inhibitor of CBX3, ii) an activator of IFNy and iii) a chemotherapeutic agent for use by simultaneous, separate or sequential administration in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) a CBX3 inhibitor according to the invention, ii) an activator of STAT1 and iii) a targeted therapy used as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof. In a particular embodiment, the invention relates to i) an inhibitor of CBX3 ii) an activator of STAT1 and iii) a chemotherapeutic agent as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to to i) an inhibitor of CBX3, ii) an activator of STAT1 and iii) a chemotherapeutic agent for use by simultaneous, separate or sequential administration in the prevention and/or treatment of a cancer in a subject in need thereof.

As used herein, the term “activator of IFNy” refers to a molecule that partially or totally activates/enhances of IFNy biological activity or expression. The term also encompasses a molecule able to increase or activate the IFNy gene expression. In order to test the functionality of a putative agent for IFNy protein expression a test is necessary. For that purpose, to identify agent for IFNy protein expression, a western blot analysis can be run on cell extracts to test the effect on the putative agent for IFNy protein expression on the level of IFNy. If the level of IFNy is increased, the putative agent for IFNy protein expression will have the desired effect.

In a particular embodiment, the activator of IFNy is a recombinant human IFN-y.

In a particular embodiment, the activator of IFNy is Actimmune® as described in US6936695 and US6936694.

In a particular embodiment, the IFNy is pegylated interferon (PEG-IFN). Example of such pegylated IFNy are: Pegylated interferon-alpha-2a, Pegylated interferon-alpha-2b or Pegylated interferon beta-la.

As used herein, the term “activator of STAT1” refers to a molecule that partially or totally activates/enhances of STAT1 biological activity or expression. The term also encompasses a molecule able to increase or activate the STAT1 gene expression. In order to test the functionality of a putative agent for STAT1 protein expression a test is necessary. For that purpose, to identify agent for STAT1 protein expression or activation, a western blot or cytometry analysis can be run on cell extracts or fixed cells to test the effect on the putative agent for STATlprotein expression or activation on the level of STAT1 or phospho-STATl . If the level of STAT1 is increased, the putative agent for STAT1 expression or activation will have the desired effect.

In a particular embodiment, the activator of STAT1 is: IFNa, IFNP, IFNco, IFNy or IFNX, IL2, IL3, IL6, IL9-IL12, IL15, IL17, IL22, IL21, IL26, IL27, EGF(epidermal growth factor), VEGF (vascular endothelial growth factor), FGF (fibroblaste growth factor), HGF (hepatocyte growth factor), GH (growth hormone), Angiotensine, or Oncostatine M (OSM). As used herein, the term “targeted therapy” refers to drugs which attack specific genetic mutations within cancer cells, such as Colorectal cancer or breast cancer while minimizing harm to healthy cells. Typically, the targeted therapy for Colorectal cancer or breast cancer refers to use of classical treatment of the art.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of IFNy/STATl/PD-Ll axis and ii) a radiation therapy used as a combined preparation for use in the prevention and/or treatment of IFN related disease in a subject in need thereof.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of IFNy, and ii) a radiation therapy used as a combined preparation for use in the prevention and/or treatment of IFNy related disease in a subject in need thereof.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of STAT1, and ii) a radiation therapy used as a combined preparation for use in the prevention and/or treatment of IFNy related disease in a subject in need thereof.

As used herein, the term “radiation therapy” or “radiotherapy” have their general meaning in the art and refers the treatment of cancer with ionizing radiation. Ionizing radiation deposits energy that injures or destroys cells in the area being treated (the target tissue) by damaging their genetic material, making it impossible for these cells to continue to grow. One type of radiation therapy commonly used involves photons, e.g. X-rays. Depending on the amount of energy they possess, the rays can be used to destroy cancer cells on the surface of or deeper in the body. The higher the energy of the x-ray beam, the deeper the x-rays can go into the target tissue. Linear accelerators and betatrons produce x-rays of increasingly greater energy. The use of machines to focus radiation (such as x-rays) on a cancer site is called external beam radiation therapy. Gamma rays are another form of photons used in radiation therapy. Gamma rays are produced spontaneously as certain elements (such as radium, uranium, and cobalt 60) release radiation as they decompose, or decay. In some embodiments, the radiation therapy is external radiation therapy. Examples of external radiation therapy include, but are not limited to, conventional external beam radiation therapy; three-dimensional conformal radiation therapy (3D-CRT), which delivers shaped beams to closely fit the shape of a tumor from different directions; intensity modulated radiation therapy (IMRT), e.g., helical tomotherapy, which shapes the radiation beams to closely fit the shape of a tumor and also alters the radiation dose according to the shape of the tumor; conformal proton beam radiation therapy; image-guided radiation therapy (IGRT), which combines scanning and radiation technologies to provide real time images of a tumor to guide the radiation treatment; intraoperative radiation therapy (IORT), which delivers radiation directly to a tumor during surgery; stereotactic radiosurgery, which delivers a large, precise radiation dose to a small tumor area in a single session; hyperfractionated radiation therapy, e.g., continuous hyperfractionated accelerated radiation therapy (CHART), in which more than one treatment (fraction) of radiation therapy are given to a subject per day; and hypofractionated radiation therapy, in which larger doses of radiation therapy per fraction is given but fewer fractions.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of IFNy/STATl/PD-Ll axis, and iii) a chemotherapy used as a combined preparation for use in the prevention and/or treatment of cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of IFNy stimulation, and iii) a chemotherapy used as a combined preparation for use in the prevention and/or treatment of IFNy related disease in a subject in need thereof.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of STAT1 and iii) a chemotherapy used as a combined preparation for use in the prevention and/or treatment of IFNy related disease in a subject in need thereof.

As used herein, the term “chemotherapy” refers to use of chemotherapeutic agents to treat a subject. As used herein, the term "chemotherapeutic agent" refers to chemical compounds that are effective in inhibiting tumor growth.

Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, trietylenephosphoramide, triethylenethiophosphaorarnide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a carnptothecin (including the synthetic analogue topotecan); bryostatin; cally statin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CBI-TMI); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlomaphazine, cholophosphamide, estrarnustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimus tine, trofosfamide, uracil mustard; nitrosureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as the enediyne antibiotics (e.g. calicheamicin, especially calicheamicin (11 and calicheamicin 211, see, e.g., Agnew Chem Inti. Ed. Engl. 33: 183-186 (1994); dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antiobiotic chromomophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, canninomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6- diazo-5-oxo-L-norleucine, doxorubicin (including morpholino- doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalarnycin, olivomycins, peplomycin, potfiromycin, puromycin, quelamycin, rodorubicin, streptomgrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate and 5 -fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti- adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophospharnide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraxate; defo famine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamol; nitracrine; pento statin; phenamet; pirarubicin; podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; rhizoxin; sizofiran; spirogennanium; tenuazonic acid; triaziquone; 2, 2', 2"- trichlorotriethylarnine; trichothecenes (especially T-2 toxin, verracurin A, roridinA and anguidine); urethan; vindesine; dacarbazine; mannomustine; mitobromtol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxoids, e g. paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.].) and doxetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6- thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisp latin and carbop latin; vinblastine; platinum; etoposide (VP- 16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbine; novantrone; teniposide; daunomycin; aminopterin; xeloda; ibandronate; CPT-1 1 ; topoisomerase inhibitor RFS 2000; difluoromethylomithine (DMFO); retinoic acid; capecitabine; and pharmaceutically acceptable salts, acids or derivatives of any of the above. Also included in this definition are antihormonal agents that act to regulate or inhibit honnone action on tumors such as anti-estrogens including for example tamoxifen, raloxifene, aromatase inhibiting 4(5)-imidazoles, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018, onapristone, and toremifene (Fareston); and anti-androgens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin; and pharmaceutically acceptable salts, acids or derivatives of any of the above.

In a particular embodiment, the chemotherapeutic agent is 5 -fluorouracil (5-FU).

Accordingly, in a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of IFNy/STATl/PD-Ll axis and iii) 5-FU as a chemotherapy agent as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

Accordingly, in a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of IFNy, and iii) 5-FU as a chemotherapy agent as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of STAT1, and iii) 5-FU as a chemotherapy agent as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of IFNy/STATl/PD-Ll axis and iii) an immunotherapy used as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of IFNy and iii) an immunotherapy used as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of STAT1 and iii) an immunotherapy used as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

As used herein, the term “immunotherapy” refers to the treatment disease by activating or suppressing the immune system. Immunotherapies are designed to elicit or amplify an immune response.

In a particular embodiment, the immunotherapy is performed with immune checkpoint inhibitors (ICI). It has become an important armamentarium against cancers in recent years, which can specifically activate immune cells by targeting immune checkpoints. Immune checkpoints are a type of immunosuppressive molecules expressed on immune cells, which can regulate the degree of immune activation and avoid autoimmune responses. Over the past few decades, numerous immune checkpoint molecules have been identified, including but not limited to PD-1/PD-L1, CTLA-4, lymphocyte-activation gene 3 (LAG-3), T-cell immunoglobulin and mucin-domain containing-3 (TIM-3). Currently, FDA-approved checkpoint inhibitors block CTLA4, PD-1 and PD-L1. PD1 and its ligand PDL1 are two important targets for ICI therapy.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of IFNy/STATl/PD-Ll axis and iii) an immune checkpoint inhibitor used as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of IFNy and iii) an immune checkpoint inhibitor used as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of STAT1 and iii) an immune checkpoint inhibitor used as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

As used herein, the term "immune checkpoint inhibitor" refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins. As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules).

Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480- 489). Examples of stimulatory checkpoint include CD27 CD28 CD40, CD122, CD137, 0X40, GITR, and ICOS. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. The Adenosine A2A receptor (A2AR) is regarded as an important checkpoint in cancer therapy because adenosine in the immune microenvironment, leading to the activation of the A2a receptor, is negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine. B7-H3, also called CD276, was originally understood to be a co-stimulatory molecule but is now regarded as co-inhibitory. B7-H4, also called VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumour escape. B and T Lymphocyte Attenuator (BTLA) and also called CD272, has HVEM (Herpesvirus Entry Mediator) as its ligand. Surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumor-specific human CD8+ T cells express high levels of BTLA. CTLA-4, Cytotoxic T-Lymphocyte-Associated protein 4 and also called CD152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation. IDO, Indoleamine 2,3-dioxygenase, is a tryptophan catabolic enzyme. Another important molecule is TDO, tryptophan 2,3-dioxygenase. IDO is known to suppress T and NK cells, generate and activate Tregs and myeloid-derived suppressor cells, and promote tumour angiogenesis. KIR, Killercell Immunoglobulin-like Receptor, is a receptor for MHC Class I molecules on Natural Killer cells. LAG3, Lymphocyte Activation Gene-3, works to suppress an immune response by action to Tregs as well as direct effects on CD8+ T cells. PD-1, Programmed Death 1 (PD-1) receptor, has two ligands, PD-L1 and PD-L2. This checkpoint is the target of Merck & Co.'s melanoma drug Keytruda, which gained FDA approval in September 2014. An advantage of targeting PD- 1 is that it can restore immune function in the tumor microenvironment. TIM-3, short for T-cell Immunoglobulin domain and Mucin domain 3, expresses on activated human CD4+ T cells and regulates Thl and Thl7 cytokines. TIM-3 acts as a negative regulator of Thl/Tcl function by triggering cell death upon interaction with its ligand, galectin-9. VISTA, Short for V-domain Ig suppressor of T cell activation, VISTA is primarily expressed on hematopoietic cells so that consistent expression of VISTA on leukocytes within tumors may allow VISTA blockade to be effective across a broad range of solid tumors. Tumor cells often take advantage of these checkpoints to escape detection by the immune system. Thus, inhibiting a checkpoint protein on the immune system may enhance the anti-tumor T-cell response.

In some embodiments, an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. Inhibition includes reduction of function and full blockade. In some embodiments, the immune checkpoint inhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands.

In a particular embodiment, the immune checkpoint inhibitor is an antibody.

Typically, antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, PD-L1, LAG-3, TIM-3 or VISTA. In a particular embodiment, the immune checkpoint inhibitor is an anti-PD-1 antibody such as described in WO2011082400, W02006121168, W02015035606, W02004056875, W02010036959, W02009114335, W02010089411, WO2008156712, WO2011110621, WO2014055648 and WO2014194302. Examples of anti-PD-1 antibodies which are commercialized: Nivolumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK).

In some embodiments, the immune checkpoint inhibitor is an anti-PD-Ll antibody such as described in WO2013079174, W02010077634, W02004004771, WO2014195852, W02010036959, WO2011066389, W02007005874, W02015048520, US8617546 and WO2014055897. Examples of anti-PD-Ll antibodies which are on clinical trial: Atezolizumab (MPDL3280A, Genentech/Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS).

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention and ii) an inhibitor of PD-L1, as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154.

In the context of the invention, the immune checkpoint inhibitor inhibits Tim-3 or its ligand.

In a particular embodiment, the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, W02011155607, WO2015117002, W02010117057 and WO2013006490.

In a particular embodiment, the immunotherapy is performed with an anti-PD-Ll.

In a particular embodiment, the immunotherapy is performed with Avelumab.

Accordingly, in a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of IFNy/STATl/PD-Ll axis and iii) Avelumab, as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

Accordingly, in a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of IFNy, and iii) Avelumab, as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of STAT1, and iii) Avelumab as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof. In some embodiments, the immune checkpoint inhibitor is a small organic molecule.

The term "small organic molecule" as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, 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.

Typically, the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, small organic molecules interfere with transduction pathway of PD-1 and Tim-3. For example, they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway.

In a particular embodiment, the small organic molecules interfere with Indoleamine- pyrrole 2, 3 -di oxygenase (IDO) inhibitor. IDO is involved in the tryptophan catabolism (Liu et al 2010, Vacchelli et al 2014, Zhai et al 2015). Examples of IDO inhibitors are described in WO 2014150677. Examples of IDO inhibitors include without limitation 1-methyl-tryptophan (IMT), P- (3-benzofuranyl)-alanine, P-(3-benzo(b)thienyl)-alanine), 6-nitro-tryptophan, 6- fluoro-tryptophan, 4-methyl-tryptophan, 5 -methyl tryptophan, 6-methyl-tryptophan, 5- methoxy-tryptophan, 5 -hydroxy-tryptophan, indole 3-carbinol, 3,3'- diindolylmethane, epigallocatechin gallate, 5-Br-4-Cl-indoxyl 1,3-diacetate, 9- vinylcarbazole, acemetacin, 5- bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a P- carboline derivative or a brassilexin derivative. In a particular embodiment, the IDO inhibitor is selected from 1-methyl-tryptophan, P-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and P-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof.

In a particular embodiment, the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to -N-(3-bromo-4- fluorophenyl)-N' -hydroxy -4-{[2-(sulfamoylamino)-ethyl]amino}-l, 2, 5-oxadiazole-3 carboximidamide : In a particular embodiment, the inhibitor is BGB324, also called R428, such as described in W02009054864, refers to lH-l,2,4-Triazole-3,5-diamine, l-(6,7-dihydro-5H- benzo[6,7]cyclohepta[l,2-c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(l-pyrrolidinyl)- 5H-benzocyclohepten-2-yl]- and has the following formula in the art:

In a particular embodiment, the inhibitor is CA-170 (or AUPM-170): an oral, small molecule immune checkpoint antagonist targeting programmed death ligand-1 (PD-L1) and V- domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015). Preclinical data of CA-170 are presented by Curis Collaborator and Aurigene on November at ACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics.

In some embodiments, the immune checkpoint inhibitor is an aptamer.

Typically, the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, aptamers are DNA aptamers such as described in Prodeus et al 2015. A major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration. Thus, aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG). In a particular embodiment, the aptamer is an anti-PD-1 aptamer. Particularly, the anti-PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015.

In a particular embodiment, the invention relates to i) an inhibitor of CBX3 according to the invention, ii) an activator of IFNy and iii) a ferroptosis inducer, used as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

As used herein, the term “ferroptosis inducer” denotes a compound able to increase ferroptosis occurrence. Ferroptosis inducers are well-known in the art. As example, the ferroptosis inducer may be APR-246, Ras Synthetic Lethal 3 (RSL3), ML162, ML210, acrolein, erastin, Imidazole Ketone Erastin (IKE), Piperazine Erastin (PE), sulfasalazine, sorafenib, Ferroptosis Inducer 56 (FIN56), Ferroptosis inducer endoperoxide (FIN02), Caspase- Independent Lethal 56 (CIL56), mevalonate-derived coenzyme Q10, buthionine sulfoximine (BSO), amentoflavone, dihydroartemisinin (DHA), typhaneoside, artesunate, Withaferin A (WA), auranofin.

In a particular embodiment, the ferroptosis inducer is Ras Synthetic Lethal 3 (RSL3).

Typically the CBX3 inhibitor alone and/or in combination with a classical treatment (targeted therapy, radiation therapy, immunotherapy, chemotherapy or ferroptosis therapy) according to the invention as described above are administered to the subject in a therapeutically effective amount.

By a "therapeutically effective amount" of CBX3 inhibitor alone and/or in combination with a classical treatment (targeted therapy, radiation therapy, immunotherapy or chemotherapy) of the present invention as above described is meant a sufficient amount of the CBX3 inhibitor for treating a cancer at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood, however, that the total daily usage of the CBX3 inhibitor 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 the CBX3 protein or fragment thereof and/or of the agent for CBX3 inhibitor 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 CBX3 inhibitor employed; the duration of the treatment; drugs used in combination or coincidental with the CBX3 inhibitor 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 CBX3 inhibitor 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 CBX3 inhibitor of the present invention 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 CBX3 inhibitor of the present invention, preferably from 1 mg to about 100 mg of the CBX3 inhibitor of the present invention. 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. In a particular embodiment, the CBX3 inhibitor according to the invention may be used in a concentration between 0.01 pM and 20 pM, particularly, the CBX3 inhibitor of the invention may be used in a concentration of 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 20.0 pM. According to the invention, the CBX3 inhibitor of the present invention is administered to the subject in the form of a pharmaceutical composition. Thus, the invention also relates to a therapeutic composition comprising the CBX3 inhibitor for use in promoting skeletal muscle hypertrophy in a subject in need thereof

Pharmaceutical composition

The inhibitors of CBX3 as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

Accordingly, in a third aspect, the invention relates to a pharmaceutical composition comprising inhibitors of CBX3 and pharmaceutically acceptable excipients.

In a particular embodiment, the invention relates to a pharmaceutical composition comprising an inhibitor of CBX3 according to the invention and ii) a classical treatment, as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to a pharmaceutical composition comprising an inhibitor of CBX3 according to the invention and ii) an immunotherapy, as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In a particular embodiment, the invention relates to a pharmaceutical composition comprising an inhibitor of CBX3 according to the invention and ii) an inhibitor of PD-1, as a combined preparation for use in the prevention and/or treatment of a cancer in a subject in need thereof.

In some embodiments, the invention relates to pharmaceutical composition comprising an inhibitor of CBX3, an activator of IFNy/STATl/PD-Ll axis and a classical treatment and a pharmaceutically acceptable excipient.

In some embodiments, the invention relates to pharmaceutical composition comprising an inhibitor of CBX3, an activator of IFNy and a classical treatment and a pharmaceutically acceptable excipient.

In some embodiments, the invention relates to pharmaceutical composition comprising an inhibitor of CBX3, an activator of STAT1 and a classical treatment and a pharmaceutically acceptable excipient.

In some embodiments, the invention relates to pharmaceutical composition comprising i) a Chromobox protein homolog 3(CBX3) inhibitor, ii) an activator of IFNy/STATl/PD-Ll axis, iii) a ferroptosis inducer and iv) a classical treatment, and a pharmaceutically acceptable excipient.

In some embodiments, the invention relates to pharmaceutical composition according to the invention, for use in the treatment a cancer in a subject in need thereof.

In a particular embodiment, the inhibitor of CBX3 is compound II as described above, the activator of IFNy is recombinant IFNy and the classical treatment is 5-FU.

In a particular embodiment, the inhibitor of CBX3 is compound II as described above, the activator of IFNy is recombinant IFNy and the classical treatment is RSL3.

As used herein, the terms "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 inj ectable 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 isopropyl amine, 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 vacuumdrying 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.

Method of screening

A further object of the present invention relates to a method of screening a drug suitable for the treatment of a cancer comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the activity and/or expression of CBX3.

Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit the activity of CBX3. In some embodiments, the assay first comprises determining the ability of the test compound to bind to CBX3. In some embodiments, a population of cells is then contacted and activated so as to determine the ability of the test compound to inhibit the activity of CBX3. In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression. It is to be understood that test compounds capable of inhibiting the activity of CBX3, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, peptidomimetics, small organic molecules, aptamers or nucleic acids. For example, the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules.

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: CBX3 targets STAT1 and PD-L1 genes to repress in vivo and ex vivo their expression. RT-qPCR revealed that organoids derived from the Cbx3 KO colon expressed higher levels of Statl and Pd-11 under basal culture state and under additional 40U IFNy stimulation (3 mice in each group, two-sided t test). The error bar represented SEM.

Figure 2: CBX3 deficiency significantly increases STAT1 in vitro expression upon IFNy stimulation. A and B) RT-qPCR and Western blot were performed to check CBX3 mRNA and protein levels after CRISPR/Cas9-mediated CBX3 deletion in two CRC cell lines, HT29 and SW480. n=3 independent experiments, two-sided t test for RT-qPCR results. The error bar represented SEM. C and D) RT-qPCR (left), Western-blot (middle) and immunofluorescence (right) analysis revealed that IFNY stimulation dramatically increased STAT1 expression in CBX3 KO cells compared to WT controls. n=3 independent experiments, two-sided t test for RT-qPCR results. The error bar represented SEM. Immunofluorescence was performed with anti-HPly (CBX3) antibody (red), anti-STATl (green) and DAPI (blue) for WT and KO cells without or with IFNy stimulation (200U). Scale bar: 20pM.

Figure 3: CBX3 deficiency significantly increases PD-L1 in vitro expression upon IFNy stimulation. A) PD-L1 mRNA expression strongly increased in HT29 and SW480 CBX3 KO cells compared to WT upon IFNy stimulation. B) 3 independent experiments, two-sided Student’s t-test. The error bar represented SEM. B)Different histograms displays the mean values of PD-L1 APC fluorescence intensity of WT and KO cells under different experimental conditions. 3 independent experiments, two-sided Student’s t test. The error bar represented SEM. C) Knock down STAT1 by shRNA in HT29 cells significantly deceased CD274 expression either in basal condition or under IFNy stimulation. 3 independent experiments, two- sided Student’s t test. The error bar represented SEM.

Figure 4 : Clinical analysis revealed the negative correlation and opposite prognostic significance of CBX3 and STAT1/PD-L1 expression for CRC patients. A) Clinical co-expression analysis in CRC patients was based on mRNA expression z-scores relative to diploid samples (RNA Seq V2 RSEM, 592 samples). The high and low categories are cut on the median of the expression level of CBX3 and IFNy, each group is composed of 296 tumor samples. The analysis showed that high IFNy expression correlated to high expression of STAT1 or CD274, while high CBX3 expression associated with low expression of STAT1 or CD274. Two-sided t test was applied for statistical analysis. Box of boxplot represent interquantile (IQR) Q1-Q3 (50 percent of samples) and error bar represent 1.5 times IQR allowing exclusion of outliers. B and C) Clinical co-expression analysis based on Colorectal Adenocarcinoma revealed that IFNy correlated positively to JAK1, JAK2 and STAT2, while CBX3 correlated negatively to JAK1, JAK2 and STAT2. Two-sided t test was applied for statistical analysis. Box of boxplot represent interquantile (IQR) Q1-Q3 (50 percent of samples) and error bar represent 1.5 times IQR allowing exclusion of outliers (n=296). D) The Optimal cut points of expression with overall survival as outcome were determined by R software for CBX3, STAT1 and CD274 expression in CRC tumors (RNA-sequencing transcriptome experiments from the TCGA colorectal cohort). E) Left: Overall survival analysis was stratified based on combined CBX3 and STAT1 expression thresholds. Colorectal cancer patients with CBX3-high STATl-low samples exhibited worse overall survival. Right: Overall survival analysis was stratified based on combined CBX3 and CD274 expression thresholds. Low CBX3 expression indicates a better overall survival for the majority CRC patients who exhibit a low expression of CD274. Time is represented in months. The patients’ number associated with each group is showed in the under table. Tables under Kaplan Meier graph represent patients still at risk for the distinct times of follow (0,50,100,150 months) according the molecular group stratification.

Figure 5 : A and B) Flow Cytometry with anti-Ki67 revealed that CBX3 KO led to higher cycling cell in HT29 cells but 72 hours of IFNy incubation reversed this effect. These effects are not observed in SW480 cells. The error bar represented SEM (4 independent experiments, two-sided t test).

Figure 6 : CBX3 KO sensitizes CRC cells to chemotherapy through regained IFNy sensitivity. A and B) CBX3 KO significantly increased the chemosensitivity of HT29 and SW480 cells to 5-FU and Irinotecan upon to IFNy stimulation. 200U IFNy was administered 24 hours before 48 hours lOOpM drug treatment. The whiskers go down to the smallest value and up to the largest, the line in the box is plotted at the median. The box extends from the 25th to 75th percentiles computed by the GraphPad Prism (4 independent experiments, two-sided t test). C and D) The ratio calculated by comparing the cell viability under drug alone or with IFNy is clearly decreased for CBX3 KO CRC cells but almost did not change in WT cells. n=3 or 4 independent experiments, 2-sided t test. The data are presented as means ± SEM. E) Cbx3 KO increased the sensitivity of MC38 cells to the treatment combined 5-FU and IFNy. The whiskers go down to the smallest value and up to the largest, the line in the box is plotted at the median, the box extends from the 25th to 75th percentiles computed by the GraphPad Prism (6 independent experiments, two-sided t test). F) Tumor weights at D22 of 6 different treatment groups. The tumors from Cbx3KO treated with 5-FU combined IFNy are significantly lighter than the tumors from WT group following the same treatment. The whiskers go down to the smallest value and up to the largest, the line in the box is plotted at the median, the box extends from the 25th to 75th percentiles computed by the GraphPad Prism (n=6 or 7, two-sided t test). G-H) Left: MC38Cbx3 KO tumors significantly more sensitize to 5-FU+IFNy treatment compared to WT tumors. Middle: Cbx3 KO induce a tendency but do not significantly sensitize the MC38 to 5-FU treatment. Right: Compare to WT tumor which do not presented a significant difference between 5-FU and 5-FU+IFNy treatment, Cbx3KO tumor exhibit a significant difference between 5-FU and 5-FU+IFNy treatment. 2way Anova was applied for statistical analysis, n=5 to 8 mice. The data are presented as means ± SEM.

Figure 7: A-B) The Optimal cut points of expression with overall survival as outcome were determined by R software for CBX3 (7A), STAT1 and CD274 (7B) expression in breast tumors (RNA-sequencing transcriptome experiments from the TCGA breast cohort). C-D) Overall survival analysis was based on CBX3 (7C), STAT1 (7C) and CD274 (7D) expression thresholds. Low CBX3 expression, but high STAT1 or high CD274 expression indicates better overall survival. Time is represented in months. The patients’ number associated with each group is showed in the under table. Tables under Kaplan Meier graph represent patients still at risk for the distinct times of follow (0,50,100,150 months) according the molecular group stratification.

Figure 8: A) Overall survival analysis was stratified based on combined CBX3 and CD274 expression thresholds. C L¥3-high CD274 ow breast cancer patients indicate a worse overall survival compared to others. B) Overall survival analysis was stratified based on combined CBX3 and STAT1 expression thresholds. Breast cancer patients with CBAS-high STATI-k w samples exhibited worse overall survival. Time is represented in months. The patients’ number associated with each group is shown in the table. Tables under Kaplan Meier graph represent patients still at risk for the distinct times of follow (0, 100, 200 months) according to the molecular group stratification.

Figure 9: A) The Optimal cut points of expression with overall survival as outcome were determined by R software for fFNy expression in breast tumors (RNA-sequencing transcriptome experiments from the TCGA breast cohort). B) Overall survival analysis was based on IFNy expression thresholds. High fFNy expression patients show better overall survival. C) Overall survival analysis was stratified based on combined CBX3 and IFNy expression thresholds. Breast cancer patients with C2?A3-high IFNy -low samples exhibited worse overall survival. Time is represented in months. The patients’ number associated with each group is shown in the table. Tables under Kaplan Meier graph represent patients still at risk for the distinct times of follow (0, 100, 200 months) according to the molecular group stratification.

Figure 10: A) Top and Middle: Compare to WT tumors (Top) which do not present a significant difference between RSL3 and RSL3+IFN treatment, Cbx3¥J3 tumors (Middle) exhibit a significant difference between RSL3 and RSL3+IFNy treatment. Low: MC38 Cbx3 KO tumors were significantly more sensitive to RSL3 + IFNy treatment compared to WT tumors. Statistical analysis was performed using two-way ANOVA, with n = 6 to 10 mice per group. Data are presented as means ± SEM. B) Representative images of tumors from six treatment groups at day 22 (D22). C) Tumor weights at D22 for the six different treatment groups showed that tumors from Cbx3 KO mice treated with RSL3 combined with IFNy were significantly lighter than tumors from WT mice given the same treatment. Statistical analysis was performed using one-way ANOVA, with n = 9 to 13 mice per group. Data are presented as means ± SEM.

EXAMPLES:

Example 1:

Material & Methods

Cbx3KO Mouse model

The Villin-creERT2:Cbx3-/- mouse model and Tamoxifen administration were produced as previously described (Mata-Garrido et al, 2022). Briefly, Tamoxifen (0.5mg/mouse) diluted in 20% clinOleic acid was administrated by oral gavage, at 3 doses every 5 days in Cbx3Flox/Flox;Tg(Villin- CreERT2) mice, noted as Cbx3 Villin-Cre or Cbx3 KO mice in the later text. Control Cbx3 Villin-Cre mice received 20% clinOleic acid alone by oral gavage. Additional controls using Cbx3Flox/Flox mice that do not express the Cre recombinase were identically treated with Tamoxifen (data not shown). Animal studies were approved by the ethical committee of Paris Descartes University (authorization number 17-022). The mice were housed in a maximum of 5 ventilated cages in accordance with their social needs, with water and food provided ad libitum. To ensure the best possible housing conditions for the animals, cardboard houses and tunnels, as well as wooden sticks for gnawing, were added to the cage to reduce stress and anxiety for the mice. The mice were monitored daily.

Colon crypts isolation and organoids culture

Colon crypt was isolated as previously described at 7 days and 12 months posttamoxifen induction (Mata-Garrido et al, 2022). Cbx3 KO and WT mice colon organoids were cultured in Matrigel and after three days of culture, the mouse 40U/ml IFNy (Miltenyi) was added to the culture medium for an additional 24h before RT-qPCR analysis.

Cell culture and CBX3 KO cell lines construction

Human SW480 and HT29 cells were originated from ATCC (CCL-228 and HTB-38). MC38 cell was kindly provided by Dr. Eliane Piaggio. The cells were cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with GlutaMAX (GIBCO, Life Technology) and 10% fetal bovine serum (FBS, Hyclone) with 5% CO2. No mycoplasma contamination was detected. To generate CRISPR/Cas9-mediated CBX3 KO cell line, HT29, SW480 and MC38 cells were transfected in 6-well plates at around 80% confluency in the presence of Lipofectamine 2000 (Life Technologies). I pg Cas9+sgRNA plasmid (Mata-Garrido et al, 2022) was used for each well. Single GFP+ cells were sorted by cytometry to 96 wells plates 48h after transfection. CBX3 deletion in CRISPR/Cas9 CBX3 KO cell lines derived from different single cell clones were confirmed by Western blot and RT-qPCR before experiments.

For STAT1 knockdown, HT29 cells were seeded in a 48-well plate, and then transfected for 24h with 50ul lentiviral particles (SIGMA, NM_007315 / TRCN0000004265) when cells arrived at 70% confluence. The cells stay in DMEM, 10% FBS without puromycin for two days and then transplanted in a six-well plate. After the cell adhesion, 2 pg/mL puromycin (Thermo Fisher) was added in the culture milium for one week. The stable shRNA STAT1 cell line was used for further analysis.

RT-qPCR

RT-qPCR for cells was performed as previously described (Zhang et al, 2020). For organoids RT-qPCR analysis, the organoid culture dish was incubated 20 mins on ice to dissolve Matrigel before collecting the sample. Primers used for samples are listed in Table EVI. 36B4 or Gapdh were used as a reference for target gene quantification with the “delta delta Ct” (AACt) method.

Western blot

Western blot were performed with the CRC cells samples as previously described (Zhang et al, 2020). Mouse colon crypts were isolated and lysed in RIPA buffer (50 mM HEPES 0.1% SDS, 1% Triton X-100,1 mM EDTA ,0.5% Sodium deoxycholate, 150 mM NaCl) with proteinase inhibitor (cOmplete™, EDTA-free Protease Inhibitor Cocktail, Roche). Western blots were performed with following antibodies: Anti-STATl (14994, Cell signaling, 1:1000), Anti -Phosphorylated STAT1 (9167, Cell signaling, 1 :200 or 1 : 1000), Anti-HPly (IG-2M0D- 1G6-AS, Euromedex, 1:2000), Anti-PD-Ll (4059, Prosci, 1: 1000), Anti-P-actin (A3854, Sigma, 1 :25000 or 1:50000), Anti-Tubulin (4D11, Thermo Scientific, 1 :1000). Immunofluorescence and immunochemistry

Immunofluorescence (IF) were performed as previously described (Zhang et al, 2020) (Mata-Garrido et al, 2022). The organoids were grown on coverslip and fixed with 4% paraformaldehyde at 4°C overnight before experiments. IF were performed with the following antibodies: Anti-STATl (14994, cell signaling, 1 : 100), Anti-ZO-l(Invitrogen, 33-9100, 1:100) and Anti-HPly (IG-2MOD-1G6-AS, Euromedex, 1 :100).

For immunochemistry, slides were processed on the automaton Leica Bond RX and unmasked at pH 6 (except pH 9 for CD68) before being incubated 30 minutes with an anti-CD8 antibody (Abeam, ab98941, 1:200) or anti-CD4 (Abeam, Abl83685, 1 :500), anti-Ly6G (Biolegend 127605, 1:400), anti CD68 (Abeam, Abl2512, 1 :400) anti Ki67 (Abcaml5580, 1:200) and anti-BrdU (Sigma B8434, 1:1000) then washed. BrdU (Sigma) was injected intraperitoneally at 100 pg/g animal body weight, Ih prior to sacrifice. The revelation system (“Bond Polymere Refine” kit, DS9800, Leica) included a secondary antibody HRP (Cell Signaling, 98941, 1 : 1000 to 1:3000). Hematoxylin (blue) counterstaining allows the visualization of cell nuclei. CD4+ or CD8+ T cells, CD68+ macrophage, Ly6G I neutrophil, Ki67+ and Brdu+ cells were counted with 4pm thickness colon tissue section with around 10- 20 sections from 4 mice of each group.

ChlP-qPCR

ChIP experiments were performed as previously described (Batsche et al, 2006) (Saint- Andre et al, 2011). Clarified samples were incubated overnight with HPly (CBX3) antibodies (Sigma-Aldrich, 05-690) or mouse nonimmune IgG as negative control. Immunoprecipitated DNA was quantified by qPCR with primer sets specific for the indicated CBX3 binding domain (Table EV2). Reference background levels were estimated by ChIP with nonimmune IgG. qPCR with primer sets aside from for indicated CBX3 binding domain were used as negative control. The relative quantification (Enrichment) was calculated with the ratio between ChIP with CBX3 and with nonimmune IgG.

ChlP-seq and RNA-seq integrative analysis

The CBX3 ChlP-seq dataset on HCT116 (GSE28115) (Data ref: Smallwood et al, 2012) was mapped onto the human genome (hg38) using the Binding and Expression Target Analysis (BETA) python algorithm. This allowed for the identification of proximal binding sites around -3000 and +500 base pairs from the Transcription Stating Sites (TSS) of human genes (Wang et al, 2013). Gene set enrichment analysis on RNA-seq from GSE192800 ( Data ref: Mata- Garrido et al, 2022) was performed by using GSEA version 4.3.0 standalone application (Subramanian et al, 2005). During the integrative analysis, CBX3 targets identified by ChlP- seq and upregulated in the RNA-seq were used as input for network analysis in the STRING web tools version 11.5 (Szklarczyk et al, 2021).

Flow cytometry analysis

For cell surface labeling, cells were detached with Trypsin-EDTA (Thermo Fisher, MA, USA), centrifuged and suspended in PBS containing 0.5% BSA, 2 mM EDTA and APC antihuman CD274 (Biolegend, 329708, 1:20) at 4°C for 30 min. For apoptosis analysis, the APC Annexin V Apoptosis Detection Kit with 7-AAD (Biolegend, 640930, APC Annexin V, 1:20) was used with following the manufacturer’s instructions. For Ki67 and p-STATl labeling, cells were harvested, then fixed with cold 80% ethanol, incubated at -20°C for minimum 2 hours, then labeled either with APC Ki67 (Milteny Biotec, 130100330,1:50) and PI (50 ug/mL) or with PE anti-STATl Phospho (Tyr701) (Biolegend, 666403, 1 : 10), respectively. After labelling, cells were washed and analyzed by LSR Fortessa™ cell analyzer (Becton Dickinson, NJ, USA).

Bioinformatics clinical survival analyses and co-expression analyses based on colorectal cancer RNA-seq transcriptome

TCGA Matrix of colorectal and breast cancer RNA-sequencing V2 Z score was downloaded on Cbioportal website (https://www.cbioportal.org/) (Cerami et al, 2012) (Gao et al, 2013) (Liu et al, 2018) and aggregated with clinical data of the respective patients. This cohort of colorectal cancer comprised of 592 tumor samples. This cohort of breast cancer comprised of 1082 tumor samples. Overall survival time in months and status were integrated with the RNA-seq data to perform survival analyses in R software environment version 4.1.3 with survival R-package version 3.2-13 and Survminer R-package version 0.4.9. The optimal cut point of CBX3, STAT1, and CD274 expressions were selected using the Survminer package based on standardized log-rank statistics and Kaplan-Meier plot. The log-rank test was stratified based on their respective expression threshold.

The co-expression correction between different genes was based on mRNA expression z-scores relative to diploid samples (RNA-Seq V2 RSEM, 592 samples), which are classified in two categories defined on the median of IFNy or CBX3 mRNA expression level.

Drug Sensitivity Assay

20x103 HT29 cells or 40x 103 SW480 cells per well were seeded in 12-well plate. After 72h of culture, 200U/ml human IFNy was added in culture medium. 24h later, lOOpM Irinotecan or lOOpM 5-FU was added for an additional 48h before analysis. The living cells with different treatments were counted by using a cell counter (Bio-Rad TC20TM) as Trypan blue negative cells or by an LSR flow cytometer (BD) as Propidium Iodide (PI) negative cells. MC38 Syngeneic Mouse Tumor Models

C57BL/6 male mice (7 weeks) were from Janvier-Labs. Animal studies were approved by the ethical committee of Paris Descartes University (authorization number DAP 23-036). The mice were housed and elevated as previously described. After 1 week of settling down, a total of 0.5x 106 MC38 WT and Cbx3 KO cells were injected subcutaneously into C57BL/6 mice at their fourth inguinal nipple. After 1 week, mice started treatment respectively three times per week with DPBS (Control), 25mg/kg Fluorouracil (5-FU, F0250000, Sigma-Aldrich,) and 25mg/kg 5-FU combined with 5000U/mouse of recombinant marine IFNy (315-05, Pepro Tech). For the mice treated with 5-FU and IFNy, the first injection of IFNy is 6 hours before the 5-FU injection and the following injections are done at the same time. Tumor size was measured three times per week in two dimensions, the longest tumor dimension (L, length) and the value at right angles to it (W, width), with a caliper. Tumor volume was calculated as an ellipse (tumor volume = (width2 * length)/2). After 16 days treatment, the mice were sacrificed and the tumors were separated and weight.

Statistical analysis

For biological experiments, paired, unpaired or multiple two-sided t-test and two-way Anova were performed with the GraphPad Prism based on at least three independent experiments or mice samples according to different experiment conditions. P-value inferior to 0.05 was considered as significant. For clinical analysis, sample size of the original TCGA COAD transcriptome cohort was taken in account with exclusion of samples comporting missing data which represent less than 0.3 percent of the total cohort. No randomization was performed during the observational studies.

Data availability

The datasets and computer code produced in this study are available in the following databases:

RNA-Seq data: https://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSEl 92800 ChlP-seq data: https://www.ncbi. nlm.nih.gov/geo/query/acc.cgi?acc=GSE28115 Results

Conditional knockout of Cbx3 in the mice gut epithelium induces a chronic inflammatory state in the colon

Different gut inflammation criteria were assessed in Villin-creERT2:Cbx3-/- mice (assigned as Cbx3 KO mice in later text) 7 days and 12 months post-tamoxifen induction (data not shown) (U. et al, 2014). Firstly, we checked immune cells infiltration in colon epithelium. CD4+ T cells, LyG6+ neutrophil cells and CD68+ macrophages were all significantly increased in the proximal and distal colon of the Cbx3 KO mice at day 7 post-tamoxifen induction. CD8+ T cells significantly increased in the proximal but not in the distal colon of the Cbx3 KO mice at day 7 post-tamoxifen induction, (data not shown).

Remarkably, infiltration of CD4+ T cells, CD68+ macrophage and LyG6+ neutrophil cells in the proximal colon was long-lasting, as shown by the increased detection in the Cbx3 KO mucosa at 12 months post-tamoxifen induction (data not shown). Furthermore, the size of lymphatic nodules became larger in Cbx3 KO mice as compared to WT mice 12 months post- tamoxifen induction (data not shown).

Moreover, Periodic Acid Schiff (PAS) staining revealed a clear reduction of goblet cells in both 7 days and 12 months Cbx3 KO colon (data not shown). At the same time, the crypt length was significantly increased in Cbx3 KO colon (data not shown). Likewise, the Ki67 and BrdU positive cells are also increased in 7 days Cbx3 KO colon (data not shown), which suggested an increased cell cycling in the Cbx3KO colon crypt. GSEA analysis based on RNA seq data from day 7 post-tamoxifen induction colon epithelium reflected the enrichment of G2/M checkpoint, mitotic spindle as well as E2F targets in Cbx3 KO mice (data not shown). Together, all the data indicate increased cell proliferation in colon crypt and epithelial hyperplasia.

Overall, these data indicate that CBX3 deficiency triggers long-lasting colon mucosal inflammation, demonstrated by goblet cell reduction, crypt length alterations, epithelial hyperplasia and increased immune cells infiltration.

CBX3 targets Statl and Cd274 genes to repress their expression in vivo in the mice colon epithelium

Integrative analysis was applied to identify the direct gene target(s) of CBX3 (HPly) in the colon epithelium. We crossed the RNA-seq data obtained from purified Cbx3 KO mice colon epithelium (GSE192800) (Data ref: Mata-Garrido et al, 2022), with a publicly available CBX3 Chromatin immunoprecipitation-sequencing (ChlP-seq) performed in HCT116, a frequently used colorectal cancer cell line (GSE28115) (Data ref: Smallwood et al, 2012).

Over 1100 genes’ expression exhibited an over 1.5 times modification (increase or decrease) in RNA sequencing in comparing the colon epithelium of Cbx3KO mice to which of WT mice. 4942 genes revealed by ChlP-seq whose proximal promoters’ regions (from 3000bp upstream to 500bp downstream of their Transcription Starting Site) possess at least a CBX3 binding site. By crossing these two databases, 68 genes were sorted as potential CBX3 target genes (data not shown). Interestingly, among the up-regulated genes, the STRING network analysis revealed a cluster composed of 10 CBX3 direct targets implicated in the immune response. Among them, Statl and Cd274, two important components of the IFNy signaling pathway (data not shown), were recognized as CBX3 direct targets in colon epithelium. On these two genes, ChlP-seq evidenced the peaks of CBX3 associations at their promoter regions (data not shown) and RNA-seq revealed that their transcription was significantly up-regulated upon Cbx3 inactivation (data not shown). In parallel, GSEA analysis revealed significant enrichments of genes involved in the inflammatory and IFNy responses in the Cbx3 KO mice colon epithelium (data not shown).

The Statl and PD-L1 that appeared from bioinformatic analysis could be just considered as potential targets of CBX3, further analysis is required to validate the bioinformatics analysis. Firstly, the expressions of STAT1 and PD-L1 were examined with colon epithelium from Cbx3 WT and Cbx3 KO mice at 7 days and 12 months post-tam oxifen induction. The increased STAT1 and PD-L1 protein levels were detected in Cbx3 KO colon epithelium (data not shown). In parallel, RT-qPCR showed that deletion of Cbx3 durably increased Statl and Cd274 mRNA levels in the colon epithelium even after 12 months of tamoxifen induction (data not shown). Likewise, immunofluorescence studies revealed a more intense STAT1 nucleus labeling in Cbx3 KO colon epithelium (data not shown). Overall, these results spotlight in vivo, Cbx3 deletion increased STAT1 and PD-L1 expressions in the colon epithelium.

CBX3 deficiency upregulates STAT1 and PD-L1 ex vivo expressions in colon organoid

In vivo results revealed that both STAT1 and PD-L1 expressions were increased in Cbx3 KO colon epithelium. However, these transcriptional inductions could be due to higher IFNy production in Cbx3 KO colon resulting from increased T cell infiltration (data not shown). To circumvent this possibility and establish a direct link between Cbx3 KO and increased STAT1 and PD-L1 expressions, we generated colon organoids derived from WT or Cbx3 KO mice and stimulated them in vitro with mouse IFNy (data not shown). Importantly, deletion of Cbx3 resulted in enhanced expression of Statl and Cd274, both under basal culture state and after 24 hours fFNy stimulation (Figure 1). CBX3 deficiency thus increased Statl and Cd274 expression under the same IFNy concentration, suggesting CBX3 depletion is a direct and sufficient condition for enhancing Statl and Cd274 expression.

CBX3 deficiency dramatically increases STAT1 and PD-L1 in vitro expression upon IFNy stimulation

We then tried to study in vitro the interaction between CBX3 and IFNy on STAT1/ PD- L1 expression by generating CRISPR/Cas9 CBX3 KO cells with SW480 and HT29, two commonly used CRC cell lines. The editing efficiency of the CRISPR/Cas9 was verified by RT-qPCR and Western blot analyses (Figure 2A-B). A compensatory mechanism revealed by an increased mRNA expression of CBX5 and CBXlwas found in SW480 but not HT29 CRISPR/Cas9 CBX3 KO cells (data not shown).

Remarkably, IFNy stimulation limitedly increased STAT1 expression in WT cells, but strongly increased its mRNA and protein expression levels in two different CBX3 KO cell lines (Figure 2C-D). Western blot analysis using a phospho-STATl antibody further evidenced the increased active STAT1 specifically in CBX3 KO cells under IFNy stimulation (data not shown). Finally, immunofluorescence analysis visually confirmed the dramatically increased STAT1 expression in CBX3 KO cells upon IFNy stimulation (data not shown). Moreover, we further applied cytometry analysis by using an anti-STATl Phospho (Tyr701) PE antibody to confirm the increased p-STATl level in CBX3 KO cells. As shown, the IFNy stimulation increased significantly p-STATl level in two different types CBX3KO cells compared to their WT control, but this increase is more evident in HT29 CBX3 KO cells than in SW480 CBX3KO cells (data not shown).

PD-L1 expression followed a similar trend. Upon IFNy stimulation, both mRNA and cell surface protein level of PD LI much more increased in the two CBX3 KO cell lines compared to WT control, as shown by RT-qPCR (Figure 3A) and flow cytometry analyses (Figure 3B).

Finally, knock down STAT1 by shRNA in HT29 cells significantly deceased CD274 expression in basal condition or under IFNy stimulation, which confirmed the existence of IFNy/STATl/PD-Ll axis in CRC cells (Figure 3C).

Hence, similar to the phenotype observed in the ex vivo model of colon organoid, CBX3 deficiency is sufficient for priming STAT1 and PD-L1 expressions in the CRC cells. Particularly, contrary to colon organoids which answer well to IFNy, the CRC cells (HT29 and SW480) limitedly respond to IFNy stimulus. However, CBX3 deficiency ultimately made these IFNy-insensitive CRC cells becoming extremely sensitized to IFNy stimulation.

IFNy stimulation reduces the binding of CBX3 to the promoters of STAT1 and CD274

We next investigated the association of CBX3 to its target genes STAT1 and CD274. CBX3 ChlP-Seq data revealed three CBX3 association peaks in STAT1 and CD274 genes (peaks 51235, 51236 for STAT1 and peak 91126 for CD274) which should be localized at their promoter regions. Accordingly, the UCSC Genome Browser confirms that these CBX3 binding sites (data not shown) are located exactly in the promoter regions of STAT1 and CD274 genes, which are enriched with H3K4Me3, H3K27Ac, CpG island regions, located just upstream of their transcription start codon (data not shown).

ChIP assays for CBX3 were performed in the HT29 cell line with and without IFNy stimulation. CBX3 chromatin association was detected by Q-PCR using primers specific to the identified binding sites, as peaks 51235, 51236 for STAT1 and peak 91126 for CD274 (data not shown). Q-PCR with primer sets aside from indicated CBX3 binding domain (Non-specific primers) is performed.

In unstimulated HT29 cells, ChIP assays revealed a potent association of CBX3 to promoter regions for both genes. Interestingly, IFNY stimulation significantly decreased CBX3 chromatin association on these sites (data not shown). This decrease could not be attributed to a decrease of CBX3 level since 24h IFNy stimulation did not result in any significant change in the expression of CBX3 at both the mRNA (data not shown) and protein (data not shown) levels in HT29 cells.

We also noted an uncharacterized long non-coding RNA (LOC105373805), which located upstream to the STAT1 promoter in opposite orientation, most likely under the control of a bidirectional STAT1 promoter activity (data not shown). Q-PCR detection indicated that CBX3 KO significantly increased LOC105373805 transcript level, which could be further enhanced 2h after IFNy stimulation (data not shown). These results suggested that CBX3 depletion suppresses basal repression on STAT1 promoter, which liberates it for further activation upon IFNy stimulation.

Overall, these results are consistent with a model in which CBX3 binds at promoter regions to repress STAT1 and CD274 transcription. Upon IFNy stimulation, CBX3 decreased its binding to the promoter which favorite gene expression.

CBX3 expression correlates negatively to STAT1 or CD274 expression in colorectal cancer

We then examined clinical data from colorectal cancer (CRC) patients to investigate the potential antagonist effect of CBX3 and IFNy on the expression of STAT1/PD-L1 in CRC. Clinical co-expression analysis based on colorectal adenocarcinoma was performed with the source data from TCGA (Cerami et al, 2012) (Gao et al, 2013). The correlation between different gene expressions, such as CBX3 VS. STAT1, CBX3 VS. CD274, as well as IFNy VS. STAT1 or IFNy VS. CD274, was based on mRNA expression z-scores relative to diploid samples (RNA-Seq V2 RSEM, 592 samples). The patients were classified into two categories defined by the median of IFNy or CBX3 mRNA level. Interestingly, high IFNy expression correlated with high STAT1 or CD274 expression in colorectal cancer, but high CBX3 expression was significantly associated with low expression levels of STAT1 or CD274 (Figure 4A).

Furthermore, in contrast to IFNy which correlated positively with most members of JAK/STAT signaling, CBX3 mRNA expression correlated negatively with the majority of JAK/STAT signaling genes’ mRNA level, such as JAK1, JAK2, STAT2, and STAT3, etc. (Figure 4B-C). These findings suggest that the antagonist effect between CBX3 and IFNy may not be limited to STAT1/PD-L1, but rather to the entire JAK/STAT signaling pathway in colorectal cancer.

Colorectal cancer patients with low CBX3 expression exhibit better overall survival in considering both CBX3 and CD274 expressions

We then further investigated RNA-sequencing data from the TCGA colorectal cohort to identify the prognosis relation among CBX3, STAT1, and CD274 expressions. Optimal cut points of expression with overall survival as outcome were determined for each investigated marker (Figure 4D). Kaplan Meier and log rank analysis stratified on CBX3, STAT1, and CD274 expression.

First, combined stratification based on CBX3 and STAT1 expression revealed a worse prognosis for patients with CBX3-high ST AT I -low level expression as compared to all other CRC samples (Figure 4E).

The situation for CD274 is more complex. Combined stratification with CBX3 and CD274 expression revealed a significant relation of overall survival among four groups of samples: CBX3-high/CD274-low, CBX3-low/CD274-low, CBX3-high/CD274-high, and CBX3 low/CD274-high. First, the majority of the CRC patients presented a low CD274 expression (86.3% of total patients), CBX3-high/CD2744ow group exhibited a longer overall survival compared to CBX3-low/CD274-low group (Figure 4E). However, the CD274-high group (only 13.7% of total patients) showed a lower overall survival notwithstanding the high or low expression level of CBX3.

Taken together, these results suggest that expression levels of CBX3, STAT1, and CD274 have opposite prognostic significance for CRC patients. High expression of CBX3 is generally associated with a worse prognosis, particularly when it is associated with a low expression of STAT1. Moreover, low expression of CBX3 is indicative of a better prognosis for the majority of CRC patients who exhibit a low expression of CD274.

CBX3 KO sensitizes CRC cells to chemotherapy through regained IFNy sensitivity

Chemotherapy is one of the standard treatment strategies for CRC at various stages. Since CBX3 KO can render IFNy-insensitive CRC cells becoming dramatically sensitive to IFNy stimulation, we further investigated whether this restored sensitivity to IFNy could make CBX3 KO CRC cells more sensitive to chemotherapy.

Firstly, the effect of CBX3 deletion and IFNy stimulation on cell survival of HT29 and SW480 cells was examined. CBX3 KO led to higher cell proliferation in HT29 cells. Ki67 and Annexin labeling indicate CBX3 deletion led to higher cell proliferation in HT29 cells (Figure 5A) but makes HT29 more sensitive to IFNy induced apoptosis (Figure 5B). However, these effects are not observed in SW480 cells (data not shown).

Despite the different effects on the proliferation of different CRC cells, CBX3 KO significantly increased or tended to increase the chemosensitivity of both HT29 and SW480 cells to Irinotecan and Fluorouracil (5-FU), two of the first-line chemotherapy drugs in CRC treatment (Figure 6A-B). Furthermore, the sensitivity of CBX3 KO cells to Irinotecan or to 5- FU remarkably increased after IFNy stimulation (applied 24 hours before and 48 hours during the drug treatment). More specifically, for SW480 CBX3 KO cells, this effect was particularly pronounced in response to 5-FU treatment while for HT29 CBX3 KO cells, this effect was evident for both treatments.

Further analysis to compare the cell viability with or without additional IFNy stimulation revealed that IFNy stimulation increased only CBX3 KO cells’ chemosensitivity and had little effect on WT CRC cells (Figure 6C-D).

All together, these results show that IFNy stimulation can remarkably sensitize CBX3 KO CRC cells to chemotherapy, suggesting that the increased sensitivity to IFNy in CBX3 KO cells can deeply increase their chemosensitivity.

Cbx3KO increased the chemosensitivity of mice CRC tumors under IFNy stimulation

We finally confirmed that Cbx3 KO can increase CRC chemosensitivity under IFNy stimulation with MC38 syngeneic mouse tumor model.

We first generated CRISPR/Cas9 Cbx3 KO MC38 cell. Cbx3 KO increased the sensitivity of MC38 cells to IFNy, indicated by the increase of STAT1 expression under IFNy stimulation (data not shown). At the same time, Cbx3 KO increases also the chemosensitivity of MC38 cells to 5-FU combined to IFNy (Figure 6E).

We then grafted MC38 cells (WT or Cbx3 KO) to C57BL/6 mice. 7 days after implant, the mice with WT or Cbx3 KO tumors were then treated by 5-FU or 5-FU combined to IFNy until 22nd day (data not shown). Tumors’ weight at the end of treatment are presented in the Figure 6F. Tumors’ volumes were also quantified 3 times each week to compare the growth rate between WT and Cbx3 KO tumors under different treatments (Figure 6 G-H). The size of WT and Cbx3 KO tumors without treatment exhibited no significant difference, even the KO tumors seem showing a tendency to grow bigger than WT tumor around 21st and 22nd days (Figure 6G-H). Despite of this tendency, the deletion of Cbx3 significantly increased MC38 tumors’ chemosensitivity to 5-FU combined IFNy compared to WT MC38 tumors (Figure 6G-H)

In this study, we have identified a novel role of CBX3 in antagonizing the IFNy signaling cascade in the colon epithelium. We found that CBX3 represses the transcription of STAT1 and CD274 to antagonize IFNy stimulation. Likewise, upon IFNy stimulation, we observed a decreased CBX3 binding to the promoters of STAT1 and CD274, concomitant with their increased gene expression. Importantly, depletion of CBX3 led to a dramatic increase of STAT1 and PD-L1 expression under IFNy stimulation, strongly suggesting that CBX3 is an important checkpoint to control IFNy stimulated immune gene activation. Interestingly, deletion CBX3 in SW480 or HT29 cells is enough to induce dramatic STAT1 and CD274 expression under IFNy stimulation, even though there exists a compensatory increase of CBX5 (HPla) mRNA and CBX1 (HP1 ) mRNA is identified in SW480 cells. CBX3 antagonist effect to IFNy could not be compensated by other members of HP1 family.

Example 2:

Breast cancer patients with low CBX3, but high IFNg, STAT1 or CD274 expression exhibit better overall survival

As in colorectal cancer, the further investigation was done to identify the prognosis relation among CBX3, STAT1, CD274, IFNy expressions based on RNA-sequencing data from the TCGA breast cohort. Optimal cut points of expression with overall survival as outcome were determined for each investigated marker (Figure 7A-7B, Figure 9A). Kaplan Meier and log rank analysis stratified on CBX3, STAT1, and CD274 expression.

First, the patients with low CBX3 expression exhibit better overall survival. On the contrary, the patients with low STAT1, CD274, IFNy expression exhibit worse overall survival (Figure 7C-7D, Figure 9B).

The combined stratification based on CBX3 and STAT I , or CD274, IFNy expression revealed a worse prognosis for patients with CBX3-high STATI-low or with CBXJ-high CD277-1 ow or with CBA3-high TFAy-low level expression as compared to all other breast samples (Figure 8A-8B, Figure 9C)

Taken together, these results suggest that in breast cancer as in CRC cancer, low CBX3, but high STAT1 or CD274 or IFNy expression level are associated with a better overall survival. Example 3:

RSL3, ferroptosis inducer, (HY-100218A) was purchased from MedChemExpress (MCE, USA). For in vivo experiment, RSL3 was dissolve in buffer contain 10%DMSO 40%PEG4005% Tween-8045%Saline. C57BL/6 mice (7 weeks) were purchased from Janvier- Labs. After 1 week of settling down, a total of 0.5 x 106 MC38 WT and Cbx3 KO cells were injected subcutaneously into C57BL/6 mice at their fourth inguinal nipple. After 1 week, mice started treatment with control, 40 mg/kg (IS, 3R)-RSL3 (RSL3, ferroptosis inducer, HY- 100218A), 5000 units of recombinant marine IFN-y (315-05, Pepro Tech) and 40 mg/kg (IS, 3R)-RSL3 with 5000 units of recombinant marine IFN-y (315-05, Pepro Tech), respectively, every two days. Tumor size was measured three times per week in two dimensions, the longest tumor dimension (L, length) and the value at right angles to it (W, width), with an electronic caliper. Sacrifice mice after 15 days of treatment, separate tumor tissue, measure tumor weight, and undergo further analysis. Tumor volume was calculated as an ellipse (tumor volume = (width2 * length)/2).

To evaluate whether CBX3 depletion could sensitize CRC tumor to ferroptosis inducer under IFNy stimulation, we used the syngeneic MC38 tumor model by subcutaneously implanting wild-type (WT) or cbx3KO MC38 cells into the flank of C57BL/6 mice. The mice were then treated 7 days after implant with control solution (Ctrl group), ferroptosis inducer (RSL3 group), and RSL3 combined with IFNy (Combo group). Tumor volume was measured 3-4 times per week until 22^nd days and at the end of treatment, the tumors were isolated, weighted and harvested for further analysis.

In MC38 wild-type (WT) tumors, there was no significant difference in tumor growth/volume and final weight among the three treatment groups (Figure 10A). Similarly, in the MC38 Cbx3 knockout (CZ>x3KO) group, RSL3 treatment did not result in a significant decrease of tumor size compared to the control group (Figure 10B). However, when treated combined with RSL3 and IFNy, both KO tumor growth/volume and final tumor weight showed a significant decrease compared to other groups (Figure 10B-10C). These findings suggest that colorectal cancer cells lacking CBX3 are more susceptible to ferroptosis inducer under IFNy stimulation.

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Claims

CLAIMS;

1. A Chromobox protein homolog 3(CBX3) inhibitor for use in the treatment of cancer in a subject in need thereof.

2. The CBX3 inhibitor for use according to claim 1, wherein the cancer is: Colorectal cancer (CRC), breast cancer, osteosarcoma, melanoma, glioblastoma, glioma cancer (Brain lower grade glioma), neuroblastoma, lung cancer(Lung squamous cell carcinoma, Lung Adenocarcinoma), ovarian cancer, gastric cancer (Stomach adenocarcinoma), prostate cancer(Prostate adenocarcinoma), pancreatic cancer(Pancreatic adenocarcinoma), endometrial cancer, uterine cancer (Uterine corpus endometrial carcinoma), bladder cancer, cholangiocarcinoma, esophageal cancer, head and neck squamous cell cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors, kidney cancer (Kidney renal clear cell carcinoma, Kidney renal papillary cell carcinoma), liver cancer, thyroid cancer, Cervical squamous cell carcinoma and endocervical adenocarcinoma, myeloma cancer (Acute myeloid leukemia), B-cell lymphoma cancer (Lymphoid neoplasm diffuse large B-cell lymphoma), leukaemia (like acute myeloid leukaemia, acute lymphoid leukaemia, chronic myelomonocytic leukemia (CMML) . ), lymphoma and myelodysplastic syndrome (MDS), Hodgkin's disease, laryngeal and hypopharyngeal cancer, thymus cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, penile cancer, pituitary cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), vaginal cancer, vulvar cancer.

3. The CBX3 inhibitor for use according to claim 1, wherein the CBX3 inhibitor is selected from the group consisting of but not limited to: small organic molecule, peptide, peptidomimetic, antibody, aptamers, siRNA or antisense oligonucleotide.

4. The CBX3 inhibitor for use according to claims 1 to 3 wherein, the CBX3 inhibitor is a compound having the formula I.

5. A method for enhancing the efficacy of classical treatment by increasing the sensitivity of cancer cells to IFNy/STATl/PD-Ll axis comprising a step of administrating an inhibitor of CBX3 in a subject in need thereof.

6. i) A Chromobox protein homolog 3(CBX3) inhibitor and ii) a classical treatment, as a combined preparation for use in the treatment of a cancer in a subject in need thereof.

7. i) A Chromobox protein homolog 3 (CBX3) inhibitor, ii) an activator of IFNy/STATl/PD-Ll axis and iii) a classical treatment, as a combined preparation for use in the treatment of a cancer in a subject in need thereof.

8. The combined preparation for use according to claims 6 to 7, wherein the classical treatment is an activator of IFNy/STAT I/PD-L I axis, targeted therapy, radiation therapy, immunotherapy, chemotherapy or ferroptosis therapy.

9. The combined preparation for use according to claim 8, wherein the classical treatment is IFNy/STATl/PD-Ll axis.

10. The combined preparation for use according to claim 8, wherein the classical treatment is immunotherapy.

11. The combined preparation for use according to claim 10, wherein the immunotherapy is an immune-check point inhibitor.

12. The combined preparation for use according to claim 8, wherein the classical treatment is ferroptosis therapy.

13. A pharmaceutical composition comprising CBX3 inhibitor and pharmaceutically acceptable excipients.

14. A pharmaceutical composition comprising i) CBX3 inhibitor, ii) an activator of IFNy/STATl/PD-Ll axis and/or iii) a classical treatment, as a combined preparation.

15. The pharmaceutical composition according to claims 13 to 14 for use in the treatment of cancer in a subject in need thereof.