Setdb1 inhibitor for use in the treatment of uveal melanoma

Abstract

Metastatic uveal melanomas are highly resistant to all existing treatments. To identify actionable vulnerabilities, the inventors conducted a CRISPR-Cas9 knockout screen using a library composed of chromatin remodelers. They revealed that the histone H3 methyltransferase SETDB1 plays a critical role in metastatic uveal melanoma cell proliferation and survival. Functionally, SETDB1 knockdown triggers decreased expression of genes related to replication and cell cycle and promotes growth arrest associated with increased markers for DNA damage and senescence entry. Using pre-clinical model, they further demonstrated that anti-SETDB1 therapy tumor growth in vivo. The inventors identify SETDB1 as a new relevant therapeutic target for the treatment of metastatic uveal melanomas. The present invention relates to a method for treating uveal melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of SETDB1 inhibitor.

Description

[0001] SETDB1 INHIBITOR FOR USE IN THE TREATMENT OF UVEAL MELANOMA

[0002] FIELD OF THE INVENTION:

[0003] The invention is in the field of oncology, in particular in the field of melanoma. More particularly, the invention is in the field of metastatic uveal melanomas.

[0004] BACKGROUND OF THE INVENTION:

[0005] Uveal melanoma (UM) is the most common primary intraocular malignancy in adult and a deadly neoplasm. Despite successful treatment of the primary lesion by proton therapy or enucleation, up to 50% of UM patients develop metastases (mUM), predominantly in the liver (reviewed in '). Metastatic uveal melanomas are highly refractory to existing treatments. Recently, tebentafusp, a bispecific protein immunotherapy targeting CD3 and the melanoma antigen GP100 has been shown to improve the overall survival of small subset of HLA-A2 haplotype patients2. Hence, another ninety percent of patients will die within 6 months after the diagnosis of their metastases, highlighting an unmet clinical need. The characterization of novel oncogenic molecular mechanisms driving uveal melanoma progression and treatment resistance is essential to improve patients’ survival.

[0006] The main oncogenic drivers in uveal melanomas are mutations in the heterotrimeric G- protein alpha subunit GNAQ or its paralog GNA11 (GNAQ / 11). Eighty percent of uveal melanomas harbor a mutation in one of these two genes3. The most frequent GNAQ and GNA11 mutation is the substitution of glutamate at position 209 by proline or leucine (GNAQ / l lQ209P / L) that results in loss of GTPase activity, producing constitutive activation of GNAQ / GNA11. Rare mutations in CYSLTR2 and PLCB4, which function upstream and downstream of GNAQ / GNA11 respectively, have also been identified3.

[0007] Complementary to these driver mutations, uveal melanoma is characterized by later alterations, the most frequent is the loss of the tumor suppressor BRCA-1 associated protein-1 (BAPP) gene. BAP1 loss is associated with a high metastatic risk and a poor prognosis3,4BAP1 is a deubiquitinase with a substrate preference for histone H2A lysine 119 (H2AK119), meaning that BAP1 loss triggers H2AK119 mono-ubiquitination which in turn promotes transcriptional repression5. Accumulating evidence indicate that epigenetic changes play important role in cancer progression but also therapy resistance6. In line with that, HDAC inhibitors (HDACi) are reported to decrease histone H2A ubiquitination to induce uveal melanoma cell growth arrest and death7. Hence, they emerged as promising drugs in uveal melanoma treatment, yet none of the pan-HDACi (vorinostat, entinostat, belinostat) tested so far showed clinical efficacy.

[0008] Collectively, the understanding of the molecular mechanisms of uveal melanoma has increased over the past decade, yet it has not translated into a survival benefit for the majority of patients. Hence, it remains to identify pivotal players in metastatic uveal melanoma proliferation and survival that would be amenable to therapeutic intervention.

[0009] In order to probe the role of epigenetic-related mechanisms involved in uveal melanoma proliferation and survival, here the inventors performed a CRISPR-Cas9 screen in GNAQQ209Lhuman melanoma cells targeting chromatin modifiers with enzymatic activities. They identified the histone methyltransferase SETDB1, thereby providing the first evidence of SETDB1 implication in uveal melanoma cell growth. They then comprehensively evaluated the role and function of SETDB1 in uveal melanoma cell proliferation and survival.

[0010] SUMMARY OF THE INVENTION:

[0011] The present invention relates to a method for treating uveal melanoma in a subject in need thereof comprising a step of administering said subject with a SETDB1 inhibitor. In particular, the invention is defined by the claims.

[0012] DETAILED DESCRIPTION OF THE INVENTION:

[0013] Metastatic uveal melanomas are highly resistant to all existing treatments. To identify actionable vulnerabilities, the inventors conducted a CRISPR-Cas9 knockout screen using a library composed of chromatin remodelers. They revealed that the histone H3 methyltransferase SETDB1 plays a critical role in metastatic uveal melanoma cell proliferation and survival. Functionally, SETDB1 knockdown triggers decreased expression of genes related to replication and cell cycle and promotes growth arrest associated with increased markers for DNA damage and senescence entry. Moreover, deficiency in CDC6, an essential regulator of DNA replication in eukaryotic cells, phenocopies SETDB1 loss. Using pre-clinical model, they further demonstrated that anti-SETDBl therapy tumor growth in vivo. Not only they provide for the first time evidence that SETDB1 plays a critical role in metastatic uveal melanoma cell progression, but they also identify SETDB1 as a new relevant therapeutic target for the treatment of metastatic uveal melanomas.

[0014] Method for treatins uveal melanoma Accordingly, in a first aspect, the present invention relates to a method for treating uveal melanoma in a subject in need thereof comprising a step of administering said subject with a SETDB1 inhibitor.

[0015] More particularly, the present invention relates to a SETDB 1 inhibitor for use in the treatment of uveal melanoma in a subject in need thereof.

[0016] As used herein, the term “subject” or “patient” denotes a mammal, such as a rodent, a feline, a canine, and a primate. Particularly, the subject according to the invention is a human. More particularly, the subject according to the invention has or is susceptible to have melanoma. In particular embodiment, the subject has or is susceptible to have cutaneous melanoma. In a particular embodiment, the subject has or is susceptible to have metastatic melanoma. In a particular embodiment, the subject has or is susceptible to have uveal melanoma. In a particular embodiment, the subject has or is susceptible to have metastatic uveal melanoma. In a particular embodiment, the subject has or is susceptible to have uveal melanoma resistant.

[0017] As used herein, the term “melanoma” also known as malignant melanoma, refers to a type of cancer that develops from the pigment-containing cells, called melanocytes. There are three general categories of melanoma: 1) cutaneous melanoma which corresponds to melanoma of the skin; it is the most common type of melanoma; 2) mucosal melanoma which can occur in any mucous membrane of the body, including the nasal passages, the throat, the vagina, the anus, or in the mouth; and 3) ocular melanoma also known as uveal melanoma or choroidal melanoma, is a rare form of melanoma that occurs in the eye.

[0018] In a particular embodiment, the melanoma is uveal melanoma.

[0019] As used herein, the term “uveal melanoma” refers to a disease in which malignant (cancer) cells form in the tissues of the eye. It is an aggressive and deadly neoplasm, which develops from melanocytes in the choroid. At diagnosis, only 1-3% of the patients have detectable metastases.

[0020] As used herein, the terms “treating” or “treatment” refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subject at risk of contracting the disease or suspected to have contracted the disease as well as subject who are ill or have been diagnosed as suffering from a disease or medical condition, and includes suppression of clinical relapse. The treatment may be administered to a subject having a medical disorder or who ultimately may acquire the disorder, in order to prevent, cure, delay the onset of, reduce the severity of, or ameliorate one or more symptoms of a disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. By "therapeutic regimen" is meant the pattern of treatment of an illness, e.g., the pattern of dosing used during therapy. A therapeutic regimen may include an induction regimen and a maintenance regimen. The phrase "induction regimen" or "induction period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the initial treatment of a disease. The general goal of an induction regimen is to provide a high level of drug to a subject during the initial period of a treatment regimen. An induction regimen may employ (in part or in whole) a "loading regimen", which may include administering a greater dose of the drug than a physician would employ during a maintenance regimen, administering a drug more frequently than a physician would administer the drug during a maintenance regimen, or both. The phrase "maintenance regimen" or "maintenance period" refers to a therapeutic regimen (or the portion of a therapeutic regimen) that is used for the maintenance of a subject during treatment of an illness, e.g., to keep the subject in remission for long periods of time (months or years). A maintenance regimen may employ continuous therapy (e.g., administering a drug at a regular intervals, e.g., weekly, monthly, yearly, etc.) or intermittent therapy (e.g., interrupted treatment, intermittent treatment, treatment at relapse, or treatment upon achievement of a particular predetermined criteria [e.g., pain, disease manifestation, etc.]).

[0021] As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g. SETDB1 inhibitor) into the subject, such as by topical, intravitreal, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and / or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

[0022] In a particular embodiment, the administration is a intravitreal administration. In another particular embodiment, the administration is a topical administration.

[0023] A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient (e.g. SETDB1 inhibitor) for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg / kg to about 20 mg / kg of body weight per day, especially from about 0.001 mg / kg to 7 mg / kg of body weight per day.

[0024] As used herein, the term “histones” are highly basic proteins abundant in lysine and arginine residues that are found in eukaryotic cell nuclei and in most Archaeal phyla. They act as spools around which DNA winds to create structural units called nucleosomes. Nucleosomes in turn are wrapped into 30-nanometer fibers that form tightly packed chromatin. Histones prevent DNA from becoming tangled and protect it from DNA damage. In addition, histones play important roles in gene regulation and DNA replication. Without histones, unwound DNA in chromosomes would be very long. For example, each human cell has about 1.8 meters of DNA if completely stretched out; however, when wound about histones, this length is reduced to about 90 micrometers (0.09 mm) of 30 nm diameter chromatin fibers. There are five families of histones which are designated H1 / H5 (linker histones), H2, H3, and H4 (core histones). The nucleosome core is formed of two H2A-H2B dimers and a H3-H4 tetramer. The tight wrapping of DNA around histones is to a large degree a result of electrostatic attraction between the positively charged histones and negatively charged phosphate backbone of DNA. Histones may be chemically modified through the action of enzymes to regulate gene transcription. The most common modification are the methylation of arginine or lysine residues or the acetylation of lysine. Methylation can affect how other protein such as transcription factors interact with the nucleosomes. Lysine acetylation eliminates a positive charge on lysine thereby weakening the electrostatic attraction between histone and DNA resulting in partial unwinding of the DNA making it more accessible for gene expression.

[0025] As used herein, the term “histone methyltransferases” (HMT) are hi stone-modifying enzymes (e.g., histone-lysine N-methyltransferases and histone-arginine N- methyltransferases), that catalyze the transfer of one, two, or three methyl groups to lysine and arginine residues of histone proteins. The attachment of methyl groups occurs predominantly at specific lysine or arginine residues on histones H3 and H4. Two major types of histone methyltranferases exist, lysine-specific (which can be SET (Su(var)3-9, Enhancer of Zeste, Trithorax) domain containing or non-SET domain containing) and arginine-specific. The class of lysine-specific histone methyltransferases is subdivided into SET domain-containing and non-SET domain-containing. As indicated by their monikers, these differ in the presence of a SET domain, which is a type of protein domain. Human genes encoding proteins with histone methyltransferase activity include: ASH IL, DOT IL, EHMT1, EHMT2, EZH1, EZH2, MLL, MLL2, MLL3, MLL4, MLL5, NSD1, PRDM2, SET, SETBP1, SETD1A, SETD1B, SETD2, SETD3, SETD4, SETD5, SETD6, SETD7, SETD8, SETD9, SETDB1, SETDB2, SETMAR, SMYD1, SMYD2, SMYD3, SMYD4, SMYD5, SUV39H1, SUV39H2, KMT5B, SUV420H2.

[0026] As used herein, the term “SET domain bifurcated methyltransferase 1” (SETDB1) refers to a mRNA or protein product of the SETDB1 gene found on chromosome 1, locus 1 q21 (Gene ID: 9869 (human). The SETDB1 protein is a histone lysine methyltransferase, catalyzing the di- and tri -methylation of histone H3K9. SETDB1 referred to in the literature as ESET; KG1T; KMT1E; KIAA0067; H3-K9-HMTase4; and SETDB1 SET.

[0027] As used herein, the term “inhibitor of SETDB1” refers to an agent that inhibits the histone methyltransferase activity of SETDB1 by at least 10% in a subject compared to the SETDB1 activity prior to, or in the absence of, administration of the agent. The agent can be any organic or inorganic molecule, including modified and unmodified nucleic acids such as antisense nucleic acids, RNA interference agents such as siRNA, shRNA, or miRNA; peptides, peptidomimetics, receptors, ligands, and antibodies.

[0028] In some embodiments, a SETDB 1 inhibitor reduces the activity of SETDB 1 by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99%, or even 100% (i.e., no detectable activity of SETDB 1) in the presence of the inhibitor compared to the activity of SETDB 1 in the absence of the inhibitor. In a particular embodiment, the SETDB1 inhibitor is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide. The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide.

[0029] In a particular embodiment, SETDB1 inhibitor is a small organic molecule. The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

[0030] In some embodiment, the SETDB1 inhibitor include but are not limited to (R,R)-59 (also known as SETDBli or SETDB1-TTD-IN-1), VH01, VH06, 5-allyloxy-2-(pyrrolidin-l- yl)quinoline (APQ), 3-Deazaneplanocin a (DZNep), Mithramycin A (Mit-A), EC-8042, Nogalamycin, Adavosertib (AZD1775), Arsenic trioxide (AS2O3), Paclitaxel, Piperlongumine, UNC0642, SETDBli.

[0031] In particular, the SETDB1 inhibitor is (R,R)-59 also known as SETDBli or SETDB1- TTD-IN-1 is a potent, selective and endogenous binder competitive ligand of SET domain bifurcated protein 1 tandem tudor domain (SETDB1-TTD) that binds to TTD, with a Kd of 88 nM. SETDBli is having the following CAS number : 2755823-12-0 and the following chemical structure :

[0032] In particular, the SETDB1 inhibitor is mithramycin A (also referred to as plicamycin, MIT) or a derivative thereof. Mithramycin A is an antineoplastic antibiotic produced by Streptomyces plicatus. Mithramycin A is having the following CAS number : 18378-89-7 and the following chemical structure :

[0033] In a particular embodiment, the SETDB1 inhibitor is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

[0034] In some embodiments, the SETDB1 inhibitor is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of metabolites involved in SETDB1 metabolism.

[0035] In a particular embodiment, the SETDB1 inhibitor 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 doublestranded 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.

[0036] In a particular embodiment, the SETDB1 inhibitor 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.

[0037] In some embodiments, the SETDB1 inhibitor 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).

[0038] 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.

[0039] 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 Marraffini, 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:e2671.), 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.l 13.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038 / cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR / Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

[0040] 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).

[0041] In some embodiments, the SETDB1 inhibitor 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 0 368 684, WO 06 / 030220 and WO 06 / 003388.

[0042] In a particular embodiment, the SETDB1 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.

[0043] In a particular embodiment, the SETDB1 inhibitor is an intrabody. 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.

[0044] Method for treating resistant uveal melanoma

[0045] In a second aspect, the invention relates to a method for treating resistant uveal melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of SETDB1 inhibitor.

[0046] As used herein, the term “resistant melanoma” refers to melanoma 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 of melanoma.

[0047] As used herein, the term “resistant melanoma cell” refers to cell which does not respond to a treatment. As used herein, the term “sensitive melanoma cell” refers to cell which does respond to a treatment.

[0048] In some embodiments, the melanoma is resistant to BRAF inhibitors. BRAF is a member of the Raf kinase family of serine / threonine-specific protein kinases. This protein plays a role in regulating the MAP kinase / ERKs signaling pathway, which affects cell division, differentiation, and secretion. A number of mutations in BRAF are known. In particular, the V600E mutation is prominent. Other mutations which have been found are R461I, I462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, K600E, A727V, and most of these mutations are clustered to two regions: the glycine-rich P loop of the N lobe and the activation segment and flanking regions. In a particular embodiment, the BRAF mutation is V600E. The inhibitors of BRAF mutations are well known in the art.

[0049] In some embodiments, the melanoma is resistant to MEK inhibitors. MEK refers to Mitogen-activated protein kinase kinase, also known as MAP2K, MEK, MAPKK. It is a kinase enzyme which phosphorylates mitogen-activated protein kinase (MAPK). MEK is activated in melanoma. In some embodiments, the melanoma is resistant to NRAS inhibitors. The NRAS gene is in the Ras family of oncogene and involved in regulating cell division. NRAS mutations in codons 12, 13, and 61 arise in 15-20 % of all melanomas.

[0050] In some embodiments, the melanoma is resistant to immune checkpoint inhibitors.

[0051] 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.

[0052] 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. A related immune-inhibitory enzymes. 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, Killer-cell 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 -turn or T-cell response.

[0053] 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.

[0054] In a particular embodiment, the immune checkpoint inhibitor is an antibody.

[0055] Typically, antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

[0056] 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).

[0057] 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).

[0058] 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.

[0059] In a particular embodiment, the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, WO2011155607, WO2015117002, WO2010117057 and W02013006490.

[0060] In some embodiments, the immune checkpoint inhibitor is a small organic molecule.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] In a particular embodiment, the small organic molecules interfere with Indoleamine- pyrrole 2,3-dioxygenase (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.

[0065] 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 :

[0066] 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:

[0067] 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.

[0068] In some embodiments, the immune checkpoint inhibitor is an aptamer.

[0069] 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.

[0070] 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.

[0071] In a particular embodiment, the melanoma is a metastatic uveal melanoma.

[0072] As used herein, the term “metastasis” refers to the spread of cancer cells from a primary site and the formation of new tumors in another region of the body. Metastasis is responsible for as much as 90% of cancer-associated mortality. The liver is often the first metastatic site in patients with uveal melanoma. Accordingly, metastatic uveal melanoma refers migration of ciliary or choroid cells to the liver and induces liver metastasis.

[0073] In a particular embodiment, the resistant melanoma is a uveal resistant melanoma.

[0074] As used herein, the term “uveal melanoma resistant” refers to uveal melanoma 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 of uveal melanoma.

[0075] 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.

[0076] In a particular embodiment, the uveal melanoma resistant has at least one mutation in the five following genes: BAP1, EIF1AX, GNA11, GNAQ, and / or SF3B1.

[0077] In a particular embodiment, the resistant melanoma is resistant to to a treatment with an immune check point inhibitor as described above.

[0078] Combined preparation

[0079] In a third aspect, the present invention relates to i) SETDB 1 inhibitor, and ii) a classical treatment as a combined preparation for use in the treatment of uveal melanoma and / or uveal resistant uveal melanoma.

[0080] In a particular embodiment, the invention i) SETDB 1 inhibitor, and ii) a classical treatment as a combined preparation for simultaneous, separate or sequential use in the treatment of uveal melanoma and / or uveal resistant melanoma.

[0081] As used herein, the term “administration simultaneously” refers to administration of 2 active ingredients by the same route and at the same time or at substantially the same time. The term “administration separately” refers to an administration of 2 active ingredients at the same time or at substantially the same time by different routes. The term “administration sequentially” refers to an administration of 2 active ingredients at different times, the administration route being identical or different.

[0082] As used herein, the term “classical treatment” refers to treatments well known in the art and used to treat melanoma. In the context of the invention, the classical treatment refers to targeted therapy, radiation therapy, chemotherapy immunotherapy, HD AC inhibitor or calcium channel blocker CCB.

[0083] As used herein, the term “targeted therapy” refers to drugs which attack specific genetic mutations within cancer cells, such as melanoma while minimising harm to healthy cells. Typically, the targeted therapy for melanoma refers to use of BRAF, MEK or NBAS inhibitors as described above.

[0084] As used herein, the term “immunotherapy” has its general meaning in the art and refers to the treatment that consists in administering an immunogenic agent i.e. an agent capable of inducing, enhancing, suppressing or otherwise modifying an immune response. In a particular embodiment, the immunotherapy consists of use of an immune check point inhibitor as described above.

[0085] 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.

[0086] 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; pancrati statin; 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.

[0087] 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.

[0088] In a particular embodiment, the present invention relates to i) SETDB1 inhibitor, and ii) an histone deacetylase inhibitor as a combined preparation for use in the treatment of uveal melanoma and / or uveal resistant uveal melanoma.

[0089] In a particular embodiment, the invention i) SETDB1 inhibitor, and ii) an histone deacetylase inhibitor as a combined preparation for simultaneous, separate or sequential use in the treatment of uveal melanoma and / or uveal resistant melanoma.

[0090] As used herein, the term histone “histone deacetylase inhibitor” called also HDACi, refers to a class of compounds that interfere with the function of histone deacetylase. Histone deacetylases (HDACs) play important roles in transcriptional regulation and pathogenesis of cancer. Typically, inhibitors of HDACs modulate transcription and induce cell growth arrest, differentiation and apoptosis. HDACis also enhance the cytotoxic effects of therapeutic agents used in cancer treatment, including radiation and chemotherapeutic drugs. In a particular embodiment, the histone deacetylase inhibitor is valproic acid (VP A). The term "valproic acid" refers to acid-2- propylpentanoic (CsHieCE), 5 which has the following CAS number and formula 99-66-1 in the art:

[0091] In a particular embodiment, the HDAC inhibitor is suberoylanilide hydroxamic acid, also called Vorinostat (N-Hydroxy-N'-phenyloctanediamide) was the first histone deacetylase inhibitor approved by the U.S. Food and Drug Administration (FDA) on 2006 (Marchion DC et al 2004; Valente et al 2014).

[0092] In a particular embodiment the HDAC inhibitor is Panobinostat (LBH-589) has received the FDA approval on 2015 and has the structure as described in Valente et al 2014.

[0093] In a particular embodiment the HDAC inhibitor is Givinostat (ITF2357) has been granted as an orphan drug in the European Union (Leoni et al 2005; Valente et al 2014).

[0094] In a particular embodiment the HDAC inhibitor is Belinostat also called Beleodaq (PXD-101) has received the FDA approval on 2014 (Ja et al 2003; Valente et al 2014).

[0095] In a particular embodiment the HDAC inhibitor is Entinostat (as SNDX-275 or MS- 275). This molecule has the following chemical formula (C21H20N4O3) and has structure as described in Valente et al 2014.

[0096] In a particular embodiment the HDAC inhibitor is Mocetinostat (MGCD01030) having the following chemical formula (C23H20N6O) (Valente et al 2014).

[0097] In a particular embodiment the HDAC inhibitor is Practinostat (SB939) having the following chemical formula (C20H30N4O2) and the structure as described in Diermayr et al 2012.

[0098] In a particular embodiment the HDAC inhibitor is Chidamide (CS055 / HBI-8000) having the following chemical formula (C22H19FN4O2).

[0099] In a particular embodiment the HDAC inhibitor is Quisinostat (JNJ-26481585) having the following chemical formula (C21H26N6O2).

[0100] In a particular embodiment the HDAC inhibitor is Abexinostat (PCI24781) having the following chemical formula (C21H23N3O5) (Valente et al 2014).

[0101] In a particular embodiment the HDAC inhibitor is CHR-3996 having the following chemical formula (C20H19FN6O2) (Moffat D et al 2010; Banerji et al 2012). In a particular embodiment the HD AC inhibitor is AR-42 having the following chemical formula (C18H20N2O3) (Lin et al 2012).

[0102] Pharmaceutical composition

[0103] The SETDB1 inhibitor and the classical treatment for use according to the invention as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

[0104] Accordingly, in a fourth aspect, the invention relates to a pharmaceutical composition comprising a combination of SETDB1 inhibitor and a classical treatment for use in the treatment of uveal melanoma and / or uveal resistant melanoma.

[0105] The SETDB1 inhibitor and the combined preparation as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intravitreal administration, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are 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.

[0106] Method of screening

[0107] In a fifth aspect, the present invention relates to a method of screening a drug suitable for the treatment of uveal melanoma or uveal resistant melanoma comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression and / or activity of SETDB 1.

[0108] Typically, such test compound is able to inhibit the expression and / or activity of inhibitor of SETDB 1.

[0109] Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit SETDB 1. In some embodiments, the assay first comprises determining the ability of the test compound to bind to SETDB 1 metabolism . 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 SIK2 metabolism, 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, petptidomimetics, 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.

[0110] 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.

[0111] FIGURES:

[0112] Figure 1: Anti-SETDBl therapy reduces metastatic uveal melanoma cell proliferation and survival. (A-C) Quantification of colony formation assay of human 0MM1.3, OMM2.5, 0MM1 metastatic uveal melanoma cells grown for 10 days in absence or presence of increasing concentration of Mithramycin A.

[0113] Figure 2: Anti-SETDBl therapy reduces cell proliferation and viability of uveal melanoma cells. (A-C) Quantification of colony formation assay of human 0MM1.3, OMM2.5, 0MM1 metastatic uveal melanoma cells grown for 10 days in absence or presence of increasing concentration of SETDDB 1 inhibitor (SETDBi).

[0114] Figure 3: SETDBI inhibition has anti-tumoral effect in vivo. Survival curves for mice bearing the 0MM1.3 cell xenografts showing time to 1 cm3 in vehicle (red) or 1 mg / kg Mithramycin A (blue) treated mice. Log-rank (Mantel-Cox) test was performed for comparison between groups. **p=0.0027.

[0115] Figure 4: Mithramycin A effect on normal choroidal melanocytes and fibroblasts.

[0116] (A) Growth inhibition curve is shown for Mithramycin A at 96 h of treatment of normal choroidal melanocytes (n = 3). Data are mean ± SEM. (B) Growth inhibition curve is shown for Mithramycin A at 96 h of treatment of fibroblasts (n = 3). Data are mean ± SEM.

[0117] EXAMPLE:

[0118] Material & Methods

[0119] Cell cultures

[0120] Human uveal melanoma cell lines 0MM1.3 (GNAQQ209P)39, 0MM1 (GNA11Q2O9L) and OMM2.5 (GNAQQ209P)39were grown in RPMI supplemented with 10% FBS at 37°C in a humidified atmosphere containing 5%CO240. Cell lines are regularly tested for mycoplasma and are mycoplasma-free. They were authenticated through short tandem repeat (STR) profiling.

[0121] Biochemicals

[0122] Mithramycin A was from SantaCruz (sc-200909) and SETDBli from was from medchem express (HY-141539)

[0123] Pooled CRISPR library details

[0124] In total, 3,052 unique sgRNAs targeting -140 human chromatin remodeler genes for 4 guides per target were used for pooled CRISPR screens8. Libraries were amplified following the Broad Institute protocol. To ensure library diversity, colonies were collected from 15 bacterial plates after transformation of STBL4 electrocompetent cells (New England Biolabs). The pool of plasmids was prepared for infection using an endotoxin-free Maxi prep kit (Qiagen).

[0125] CRISPR-Cas9 screen

[0126] Human 0MM1.3 uveal melanoma cells were first infected with the lentiCas9-Hygro (LCH) (Addgene # 104995) and selected with hygromycin (10 pg / mL). Cells were then infected with the sgRNA library at a low MOI (<1) to ensure a single sgRNA vector per cell. After 4 days of infection, cells were analyzed by flow cytometry and <20% of cells were EGFP- positive, corresponding to single vector copy. EGFP-positive cells were expanded for 10 days. A fraction of cells was collected at day 0 to ensure a proper coverage of sgRNAs. Medium was changed every 3 days. At day 35, cells from all conditions were collected and genomic DNA was extracted. Since melanin pigment may interfere with DNA-and / or RNA-based molecular profiling41, we purified the samples using the OneStepTM PCR inhibitor Removal Kit (Zymo Research). The integrated sgRNAs were then amplified by PCR with primers containing multiplexing barcodes and adaptors and sequenced on the Illumina NextSeq500. Hits were selected based on the log2 fold change of sgRNA reads at day 35. Analyses and plots of the sequencing data were conducted using Prism 6 software (GraphPad Software) and Rank Products Analysis to determine P values. Data were analysed using the software Mageck which calculates a score based on a fold change where either sgRNA is depleted or enriched compared to the control condition. mRNA preparation and real-time / quantitative PCR The mRNAs were prepared using TRIzol (Fisher Scientific, 15596026T) according to a standard procedure. QRT-PCR was performed using SYBR® Green I (Fisher Scientific, 4368708) and Multiscribe Reverse Transcriptase (Applied Biosystems) and subsequently monitored using the ABI Prism 7900 Sequence Detection System (Applied Biosystems, Foster City, CA) as previously done42. The detection of the RPLP0 gene was used to normalize the results. Primer sequences for each cDNA were designed using either Primer bank (https: / / pga.mgh.harvard.edu / primerbank / ). Sequences are available upon request.

[0127] RNA-sequencing

[0128] Reads were preprocessed to remove adapter and low-quality sequences (Phred quality score below 20). After this preprocessing, reads shorter than 40 bases were discarded for further analysis. These preprocessing steps were performed using cutadapt version 1.10. Reads were mapped to rRNA sequences using bowtie version 2.2.8 and reads mapping to rRNA sequences were removed for further analysis. Reads were mapped onto the hg38 assembly of Homo sapiens genome using STAR version 2.5.3a. Gene expression quantification was performed from uniquely aligned reads using htseq-count version 0.6. Ipl, with annotations from Ensembl version 99 and “union” mode. Only non-ambiguously assigned reads have been retained for further analyses. Read counts have been normalized across samples with the median-of-ratios method43. Differential gene expression analysis was performed using the methodology implemented in the Bioconductor package DESeq2 version 1.16.144. P-values were adjusted for multiple testing by the method proposed by Benjamini and Hochberg45. Deregulated genes were defined as genes with log2(foldchange) > 1 or < -1 and adjusted P-value < 0.05.

[0129] Transient transfection of siRNA and infection of shRNA

[0130] Briefly, a single pulse of 50 nM of control siRNA, siRNA to SETDB1 (Sigma SASI HsOl OO 150485 and SASI_Hs02_00344324) or siRNA to CDC6 (Company) was administered to the cells at 50% confluency through transfection with 5 Dl of Lipofectamine™ RNAiMAX in Opti-MEM medium (Invitrogen, San Diego, CA, USA) as described46.

[0131] Cell cycle analysis

[0132] The Click-iT Plus EdU Alexa Fluor 647 Flow Cytometry Assay Kit (Invitrogen C10634) was used for detection of replicating 0MM1.3 cells based on incorporation of 2 pM EdU (5-ethynyl 2’ -deoxyuridine) into newly synthesized DNA for 2h followed by its recognition with azide dyes via copper mediated “click” reaction, according to the manufacturer’s protocol. DAPI and EdU (C10634, Invitrogen) double staining was used to measure DNA content in live cells by flow cytometry.

[0133] Western blot assays

[0134] Briefly, cell lysates (30 pg) were separated using SDS-PAGE, transferred onto a PVDF membrane as previously described47and subsequently exposed to the appropriate antibodies, anti-SETDBl (VMA00243; 1 / 1,000) from Biorad, anti-p21 (2947; 1 / 1,000), anti-CDC6 (3387; 1 / 1,000), anti-phospho CHEK2 (2197; 1 / 1,000), anti-CHEK2 (6334; 1 / 1,000), and anti-PARP (9542, 1 / 1,000), from Ozyme, anti MCM6 (ab201683; 1 / 1,000) from abeam, anti-ACTIN (sc- 47778; 1 / 1,000), anti-GAPDH (sc-47724; 1 / 1,000) and anti-HSP90 (sc-13119; 1 / 1,000) from Santa Cruz Biotechnology. The proteins were visualized using the ECL system (Amersham). The western blots shown are representative of at least 3 independent experiments.

[0135] Colony formation assay

[0136] Human uveal melanoma cells were seeded onto six-well plates at low density, allowed to adhere overnight and cultured as indicated. Then, the colonies were stained with 0.04% crystal violet / 2% ethanol in PBS for 30 min. Photographs of the stained colonies were captured. Crystal violet was then solubilized and growth was monitored by measuring the absorbance at 561nm as previously reported48. Photographs of the stained colonies were captured. The colony formation assay was performed in triplicate.

[0137] Immunofluorescence staining

[0138] Immunofluorescence experiments were carried out as previously described49. Briefly, cells grown on glass coverslips were fixed in 4% formaldehyde supplemented with 0.1% Triton X-100 for 15 min at room temperature prior to permeabilisation in 0.5% Triton X-100 for 5 min. After blocking with 3% BSA in PBS containing 0.05% Tween 20, the cells were stained for 1 h in blocking solution with antibodies to g-H2AX (1 / 500, Abeam abl 1174), 53BP1 (1 / 50, Bethyl, IHC-00001). Primary antibody detection was achieved via incubation with anti-rabbit or anti-mouse Alexa Fluor 594- or 488-conjugated secondary antibodies (Invitrogen) for 45 min at room temperature. The slides were mounted in DAKO mounting medium supplemented with Hoechst (1 / 1,000, Invitrogen, #H3570) and examined oil immersion with a NIKON ARI confocal microscope. Representative experiments are shown.

[0139] Animal experimentation Animal experiments were performed in accordance with French law and approved by a local institutional ethical committee. The animals were maintained on a 12-h light / dark cycle in a temperature-controlled facility at 22°C and provided free access to food (standard laboratory chow diet). Human 0MM1.3 melanoma cells (4xl06cells) were subcutaneously inoculated into 8-wk-old male and female immune-deficient Nod scid gamma (NSG) mice (Janvier Laboratory). When the tumors became palpable, mice received a daily intratumoral injection for 7 days of Mithramycin (1 mg / kg) 3 times per week dissolved in phosphate buffered saline (PBS). Control mice were injected with PBS alone. The growth tumor curves were determined after measuring the tumor volume using the equation V = (L x W2) / 2 as previously reported50.

[0140] Statistics

[0141] Statistical significance between groups was determined as indicated in the legends. *p- value <0.05; **p-value<0.01; ***p-value<0.001; ****pvalue< 0.0001.

[0142] Results

[0143] A CRISPR-Cas9 screen identify SETDB1 as a key driver of metastatic uveal melanoma cell growth

[0144] To identify actionable vulnerabilities in metastatic uveal melanoma cells, we performed a CRISPR-Cas9 knockout screen targeting -140 chromatin remodelers containing enzymatic activity in GNAQQ209Puveal human melanoma cells8. Briefly, representative 0MM1.3 uveal melanoma cells, originally derived from liver metastasis and harboring a GNAQQ209Pmutation, were engineered to stably express Cas9, transduced with GFP -tagged single-guide RNA (sgRNA) library (3-4 sgRNAs per gene encoded in pLKO.1) and GFP-positive cells were sorted for expansion.

[0145] Next, genomic DNA was isolated from cells at day 0, which represents the library distribution prior to the screening selection process, and at day 35, and the abundance of each sgRNA was determined using next-generation sequencing. Analysis of the CRISPR-Cas9 screen dataset with MaGeck software, which calculates a score based on a fold change, revealed depleted (left part of the volcano plot) or enriched (right part of the volcano plot) sgRNA compared to the control condition (Data not shown).

[0146] To identify genes involved in cellular proliferation and survival, we screened for genes whose loss conferred a reduced proliferative advantage on cells. The screen yielded several valuable candidates, among which the histone methyltransferase SETDB1, that catalyzes the addition of methyl groups to histone H3 at lysine 9 (H3K9) (Data not shown). Given that SETDB1 was identified as one of the top critical genes for metastatic uveal melanoma cell proliferation, its role in uveal melanoma biology remained unknown and treatment resistant cancer cells displayed high level of H3K9me39, we thought that SETDB 1 was a good candidate to pursue.

[0147] The functional impact of SETDB 1 knockdown (KD) on proliferation was validated by introducing individual sgRNAs. SETDB 1 inhibition in pool 0MM1.3 mUM cells was confirmed by immunoblot (Data not shown) and resulted in a substantially decreased colonyforming capacity (Data not shown). We also used another genetic approach with siRNA to inhibit SETDB 1 (Data not shown). Our data showed that two different siRNA, that efficiently inhibited SETDB 1 at both the mRNA and protein level, also translated into reduced cell number and colony -formation ability (Data not shown). Next, we showed that adding back SETDB 1 rescued the reduced colony formation ability mediated by SETDB 1 knockdown (Data not shown). We also confirmed these findings in two other metastatic uveal melanoma cell lines and showed that SETDB 1 knockdown also reduced the colony formation ability of BAP 1 negative MP46 primary uveal melanoma cells (Data not shown). Then, we assessed the expression of SETDB 1 in a panel of metastatic uveal melanoma cell lines, and normal choroidal melanocytes isolated from patient biopsies. All three metastatic uveal melanoma cells expressed about 15-fold higher SETDB 1 mRNA level compared to normal human choroidal melanocytes (Data not shown). This observation was confirmed at the protein level showing higher SETDB 1 expression in metastatic uveal melanoma cells compared to normal choroidal melanocytes (Data not shown).

[0148] Collectively, these data indicate that SETDB 1 plays a critical role in metastatic uveal melanoma cell growth.

[0149] SETDB1 regulates replication and genomic integrity

[0150] SETDB 1 mechanisms of action in uveal melanoma remains to be elucidated. To delineate the mechanisms by which SETDB 1 regulates metastatic uveal melanoma cell growth, we profiled the transcriptome of five different uveal melanoma cell lines, 3 metastatic BAP1 positive cell lines and 2 primary BAP1 negative cell lines that were either treated with a control siRNA or with a siRNA to SETDB 1 (Data not shown). We identified genes common to the 5 SETDB 1-KD cell lines that were significantly upregulated (n=1784) and downregulated n=1562) compared to control cells (adjp-value<0,05) (Data not shown). Gene Set Enrichment Analysis (GSEA) uncovered 4 gene sets (out of 10 statistically significant) related to replication, and others related to telomere and cell cycle in SETDB1-KD cells compared to control cells (Data not shown). Supporting this, the heatmap revealed several genes implicated in DNA replication pre-initiation complex (ORC1, CDC6, MCM6, MCM7) and cell cycle (E2F2) (Data not shown). It is worth noting that CDC6 is regulated by E2F proteins10. RNA- seq datasets also revealed reduced mRNA level of other components of the pre-initiation complex (CDT1, CDC45 and SLD3 / TICRR) in SETDB1-KD cells compared to control cells. Inhibition of CDC6 and MCM6 in SETDB1 KD cells was confirmed at the protein level (Data not shown). These data suggest that SETDB1 plays a critical role in metastatic uveal melanoma cell proliferation through regulation of DNA replication.

[0151] SETDB1 inhibition triggers DNA damage and senescence-like phenotypes

[0152] Initiation of DNA replication takes place in the S phase of the cell cycle. To get insights into the kinetics of cell cycle, 0MM1.3 proliferating cells treated with control siRNA or siRNA to SETDB1 were stained for incorporated EdU against total DNA content using Hoechst 33342. cells. SETDB1-KD displayed reduced percent of cells in late S phase and increased percent of cells in G0 / G1 phase.

[0153] Given that accurate DNA replication is critical for achieving genome integrity and cell survival, and that Given that eukaryotic cells dampen their progression through S phase upon DNA damage, we hypothesized that SETDB1 down-regulation would promote DNA damage. To address this point, we analyzed phosphorylated CHEK2 and H2AX (y-H2AX), canonical markers of DNA breaks signing replication stress. We also performed staining of 53BP1 that rapidly localizes to DNA double-strand breaks following DNA replication stress and is a marker of unrepaired DNA damage and an important component of the DNA damage responsen. Immunoblot of 0MM1.3 cells treated with a siRNA to SETDB1 showed enhanced phosphorylation of CHEK2 (Data not shown). Moreover, as shown by immunofluorescence analyses, SETDB1-KD enhanced y-H2AX and 53BP1 stainings compared to the control cells (Data not shown). Enhanced y-H2AX and 53BP1 stainings were also observed when expression of SETDB1 was reduced using the CRISPR-Cas9 approach (Data not shown), indicating that SETDB 1 KD triggers DNA damage. Time course analysis showed that SETDB 1 KD caused enhanced p21 expression (Data not shown). Persistent DNA damage is well known to promote a senescent phenotype. Thus, we then conducted P-Galactosidase (SA-PGal) staining at pH6 to measure senescence entry. Our data demonstrated SA-PGal staining in 0MM1.3 treated with a SETDB 1 siRNA compared to control cells (Data not shown). SA-PGal staining following SETDB1 KD by siRNA was repeated in 0MM1 cells (Data not shown). Collectively, our findings indicate that SETDB1 overcomes the process of senescence to favor metastatic uveal melanoma cell proliferation.

[0154] CDC6 knockdown phenocopies SETDB1 phenotypes

[0155] Cell division cycle 6 (CDC6) and Minichromosome Maintenance Complex Component 6 (MCM6) are essential regulator of DNA replication in eukaryotic cells and they play critical roles in the activation and maintenance of the checkpoint mechanisms in the cell cycle. However, only CDC6 inhibition, using two different siRNA, translated into reduced 0MM1.3 colony formation capacity (Data not shown). OMM2.5 cells treated with the CDC6 siRNA also exhibited reduced colony formation capacity (Data not shown). These data prompted us to focus our attention on CDC6. We observed that CDC6 KD triggered an increase in DNA damage as illustrated by enhanced y-H2AX and 53BP1 stainings in immunofluorescence compared to control cells (Data not shown). SA-PGal activity was also detected in cells treated with CDC6 siRNA (Data not shown). Altogether, our findings demonstrate the critical role of CDC6 in uveal melanoma cell proliferation and suggest that SETDB1 exerts its effect through CDC6.

[0156] Anti-SETDBl therapy reduces metastatic uveal melanoma cell growth

[0157] Our data point out to SETDB1 as a potential relevant therapeutic target in metastatic uveal melanomas. Mithramycin A, an antitumor antibiotic used in phase II clinical trials for the treatment of patients with a broad range of malignancies (ClinicalTrials.gov: NCTO 1624090) has been demonstrated to inhibit SETDB1 expression12. Mithramycin A is reported to impair SETDB1 expression by blocking binding of the SP-1 transcription factor at the SETDB1 promoter13. We tested the effect of Mithramycin A on the different GNAQ / 11 -mutated human metastatic uveal melanoma cells in vitro. 0MM1.3 cells exposed to increasing concentration of Mithramycin A showed reduced colony formation ability (Figures 1A to 1C). Similar observations were performed in 2 other human metastatic uveal cell lines, thereby demonstrating that the growth defects are not restrained to a unique cell line. Interestingly, a more direct SETDB1 inhibitor (SETDBli), which prevents the interaction of SETDB1 with histones, has been recently reported14In line with what we observed with Mithramycin A, increasing concentration of SETDBli also caused reduced colony formation ability of the different human metastatic uveal melanoma cell lines (Figures 2A to 2C). Therefore, our data show that human metastatic uveal melanoma cells are sensitive to two different distinct SETDB1 inhibitors. To know how Mithramycin A and SETDBli were promoting reduced colony numbers, we performed SA-bGal staining and determined apoptotic cell death induction by FACS analysis of AnnexinV / PI. Although Mithramycin A induces p21, which plays a key role in senescence induction, both inhibitors didn’t appear to be associated with SA-bGal staining. In contrast, they both promoted apoptosis that was most visible at the highest concentration tested (30 nM) as illustrated by annexin V / PI labelling (Data not shown) and the detection of cleaved PARP, a well-studied caspase 3 substrate (Data not shown). Moreover, Mithramycin A treated cells triggered decreased SETDB1 expression as well as that of CDC6 (Data not shown).

[0158] These data indicate that SETDB1 represents a promising anti -metastatic uveal melanoma therapy. Given that Mithramycin A works at nanomolar concentration compared to SETDBi and has been assessed in clinical trials, we decided to pursue with Mithramycin A. To address this point, we next investigated the therapeutic relevance of inhibiting SETDBI on tumor growth in vivo. Both male and female mice were used in these experiments since uveal melanoma incidence is similar in men and women. 0MM1.3 cells were subcutaneously injected into NSG immunodeficient mice and when the tumors were palpable (approximately 100 mm3), mice were injected intraperitoneally every 3 days with Mithramycin A (1 mg / kg) (Data not shown). When the tumor reached ~1 cm3, the mouse was sacrificed. Our data show that Mithramycin A strongly impaired uveal melanoma growth as illustrated by reduced tumor volume compared to the vehicle control group (Data not shown). Interestingly while we observed no difference in tumor growth between male and female, the anti-uveal melanoma effect of Mithramycin A appeared more robust in female than in male mice. Moreover, Mithramycin A effect translated into a survival advantage as measured by time to a tumor size of 1 cm3(Figure 3).

[0159] Overall, Mithramycin A was well tolerated, with some transient weight loss in some mice that resolved after treatment discontinuation for one round. Section of Mithramycin A- treated tumors revealed reduced SETDBI expression compared to control tumor sections (Data not shown). Noteworthy, no toxicity was observed in normal choroidal melanocytes or fibroblasts (Figures 4A and 4B). Altogether, our findings demonstrate that SETDBI critically supports metastatic uveal melanoma progression in vivo, and establish SETDBI as a promising effective therapeutic strategy in these often untreatable tumours.

[0160] Discussion

[0161] We hereby present a chromatin-focused CRISPR-Cas9 screen to identify factors that play a critical role in proliferation and survival of metastatic uveal melanoma cells, and identified the histone H3K9 methyltransferase SETDB1. Accumulating evidence indicate that epigenetic regulators play important role for cancer initiation and progression but also for therapy resistance6. Little is known about the mechanisms of epigenetic regulation in uveal melanoma cell biology.

[0162] Our data show that SETDB1 level is higher in metastatic uveal melanoma cells compared to control normal choroidal melanocytes. In line with that, SETDB1 is amplified in different types of cancers, including breast cancers, ovarian cancers, bladder cancers and cutaneous melanomas in which it can also be found mutated (Cancer Genome Atlas Research Network, 2014;15,16. Our analysis of public datasets revealed no alteration in copy number or mutations in SETDB1. However, increased SETDB1 expression could occur through other mechanisms, including chromosomal translocation, single nucleotide polymorphism in regulatory regions and mutation or activation of upstream signaling pathways. How SETDB1 expression is regulated in uveal melanoma remains to be determined.

[0163] SETDB1 has been reported to enhance the proliferation of different tumor cells in vitro and in vivo16 20. In human cutaneous melanoma cells, overexpression of SETDB1 stimulates their tumorigenic properties and favors skin melanoma development in cooperation with BRAFV600Emutation in a zebrafish model in vivo15. In uveal melanoma cells, we found that SETDB1 knockdown by genetic approaches induces growth arrest and senescence-like phenotypes. These data reveal how SETDB1 by overcoming the process of senescence, a major barrier against tumor progression, controls metastatic uveal melanoma cell proliferation. A metastasis promoting role for SETDB1 has also been reported in different cancer types such as in colorectal cancer, and in cutaneous melanomas18,21in which high SETDB1 expression was detected at the invasive front17Supporting this, in cutaneous malignant melanomas, SETDB1 regulates expression of thrombospondin- 1, known to stimulate metastasis formation17Our data did not show any change in the motile ability of metastatic uveal melanoma cells after SETDB1 knockdown nor in THBS1 expression. In contrast, our transcriptomic analysis highlighted depletion in factors involved in the formation of the pre-repli cation complex in SETDB1 knocked-down cells. Among them, reduced CDC6 and MCM6 expression, was validated at the protein level but, only CDC6 knockdown impaired metastatic uveal melanoma cell colony formation ability. The effect of MCM6 reduction is likely to be offset by other MCM members in contrast to CDC6 which role is essential and non-compensable. High CDC6 expression is associated with enhanced malignant behavior22,23and drug resistance24 26. Moreover, upon CDC6 knockdown, uveal melanoma cells exhibited signs of DNA damage and senescent phenotypes, thereby mimicking the effect of SETDB1 inhibition. Altogether, these observations suggest that SETDB1 can mediate its effect in part through CDC6. Hence, both SETDB1 and CDC6 might represent new prognosis biomarkers and new potential therapeutic targets in uveal melanomas.

[0164] How SETDB 1 inhibits the expression of these factors remains to be elucidated. Lysine methylation of histones is recognized as important regulators of gene expression27,28. For instance, di- and tri -methylation of lysine 9 or 27 of histone H3 (H3K9me2-3 and H3K27me2- 3) are generally linked to transcriptional repression, while their monomethylation (H3K9me and H3K27me) is found in transcriptional regulatory elements controlling active areas29. In mammalian cells, H3K27me3 is achieved by EZH2. Abnormal EZH2 expression has been associated with various cancers30including uveal melanomas31,32. Likewise, H3K27me3 is associated with a poor prognosis in uveal melanomas31. Pharmacologic inhibition of EZH2 triggers uveal melanoma cell death in vitro31,32, suggesting that EZH2 is a valid therapeutic targets. However, clinical trials with different EZH2 inhibitors have failed to demonstrate efficacy in uveal melanoma patients (ref).

[0165] SETDB 1 is known to deposit the repressive H3K9me3 mark and mediate gene repression. However, we observed virtually no difference in H3K9me3 level after SETDB 1 knockdown. This finding suggests the existence of compensation mechanisms by other H3K9 methyltransferases for the regulation of overall H3K9me3 metabolism in metastatic uveal melanoma cells. Indeed, histone H3 lysine 9 tri-methylation (H3K9me3) is catalyzed by two enzymatic systems, SUV39h and SETDB1ZESET1 (SET domain bifurcated)33. Another possibility is that SETDB 1 regulates the transcription program independently of its H3K9me3 activity. This is reminiscent of other publications demonstrating that SETDB 1 functions through methylation of non-histone protein such as p53 and AKT34,35. SETDB 1 knockdown might enhance the expression of a transcriptional repressor which in turn reduce expression of pre-replication factors.

[0166] Whatever the mechanisms of SETDB 1 action, our data point out to SETDB 1 as a relevant therapeutic target for the treatment of metastatic uveal melanoma cell. This is evidenced by both in vitro and in vivo effect of Mithramycin A and SETDB li, two reported SETDB 1 inhibitors. In vitro, both molecules reduced metastatic uveal melanoma cell proliferation and / or survival. Importantly, in a pre-clinical model, Mithramycin A also strongly reduced metastatic uveal melanoma cell growth and robustly extended mouse lifespan.

[0167] In our experiment, Mithramycin A appeared to display a more efficient effect in female mice compared to male in our experiments. Although this remains to be firmly demonstrated, this response could be ascribed to hormone regulation, which in combination with Mithramycin A improve its therapeutic effect. Thus, our data highlight for the first time possible genderspecific disparities in response to anti-uveal melanoma therapeutics.

[0168] In sum, our findings strongly demonstrate that SETDB1 represent a valid therapeutic option for the treatment of metastatic uveal melanomas.

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Claims

CLAIMS:

1. A method for treating uveal melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of SETDB 1 inhibitor.

2. The method according to claim 1 wherein the melanoma is uveal melanoma resistant.

3. The method according to claims 1 and 2 wherein the SETDB 1 inhibitor is Mithramycin A.

4. The method according to claims 1 and 2 wherein the SETDB 1 inhibitor is SETDB li.

5. The method according to claims 1 to 4 wherein the SETDB 1 inhibitor is administered by topical or intravitreal administration.

6. i) SETDB 1 inhibitor, and ii) a classical treatment, as a combined preparation for use in the treatment of uveal melanoma.

7. The combined preparation for use according to claim 6 wherein the SETDB 1 inhibitor is Mithramycin A.

8. The combined preparation for use according to claim 6 wherein the SETDB 1 inhibitor is SETDB li.

9. A pharmaceutical composition comprising a combination of SETDB 1 inhibitor and a classical treatment for use in the treatment of uveal resistant melanoma.

10. The pharmaceutical composition for use according to claim 9 wherein the SETDB 1 inhibitor is Mithramycin A.

11. The pharmaceutical composition for use according to claim 9 wherein the SETDB 1 inhibitor is SETDB li.

12. A method of screening a drug suitable for the treatment of uveal melanoma or uveal resistant melanoma comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression and / or activity of SETDB 1.

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