Combination of a NRAS inhibitor and an Anti-fibrotic agent for use in the treatment of NRAS mutated melanoma

Abstract

Inventors have examined the effect of RMC-6236 and Nintedanib as a combination on NRAS mutant melanoma cell proliferation and survival. Crystal violet assay performed on SKMEL2 cells demonstrated that RMC-6236 acts synergistically with Nintedanib to reduce cell proliferation. Using the Bliss prediction method, they further demonstrated a strong synergistic action of RMC-6236 / Nintedanib combination on the proliferation of NRAS mutated melanoma cells. Finally, western blot analysis showed that Nindedanib cooperated with RMC- 6236 to reduce the levels of cell survival markers, such as phosphorylated AKT and Survivin and to synergistically promoted the activation of caspase 3, an apoptosis executioner. The present invention relates to a method for treating NRAS mutated melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of: i) a NRAS inhibitor and ii) an anti-fibrotic agent as a combined preparation.

Description

[0001] METHODS AND COMPOSITION FOR TREATING NRAS MUTATED MELANOMA

[0002] FIELD OF THE INVENTION:

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

[0004] BACKGROUND OF THE INVENTION:

[0005] Activating mutations in NRAS are found in 15 to 30% of cutaneous melanomas. Patients with NRAS mutated melanomas have a worse prognosis due to the aggressiveness of the tumor, the lack of effective therapies, or the emergence of treatment resistance (1). In melanoma, the most common NRAS mutations occur at codon 61 (Q61R, Q61L, and Q61K). Less frequently, other mutations occur at codons 12 or 13. In addition, activating NRAS mutations arise in BRAFV600-mutated melanoma patients progressing on BRAF inhibitor therapy (ie vemurafenib or dabrafenib). Whereas NRAS G12X and NRAS G13X mutations decrease sensitivity to GTPase-accelerating proteins, NRAS Q61X mutations disrupt the GTPase activity of RAS, leading to a constitutively active conformation. Oncogenic NRAS then triggers constitutive activation of the MAPK and PI3K / AKT pathways.

[0006] Locally advanced, metastatic or non-resectable NRAS-mutant melanoma has proven challenging to treat, especially for patients unresponsive to immune checkpoint inhibitors. As a second-line treatment after anti-CTLA4 and / or anti-PDl immunotherapies, MEK inhibitors (MEKi) (i.e. Trametinib) are not sufficient to provide a benefit in terms of overall survival. Thus, there is an unmet need for new strategies aiming at improving current therapies and the management of NRAS-mutated melanomas.

[0007] SUMMARY OF THE INVENTION:

[0008] The invention relates to a method for treating NRAS mutated melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of i) a NRAS inhibitor and ii) an anti-fibrotic agent as a combined preparation.

[0009] In particular, the invention is defined by claims.

[0010] DETAILED DESCRIPTION OF THE INVENTION:

[0011] The objective of the inventors is to study the adaptive response of NRAS-mutated melanomas to novel treatments targeting oncogenic NRAS and to develop original combination therapies targeting non-genetic resistance mechanisms associated with mesenchymal plasticity and pro-fibrotic reprogramming to improve outcomes in RAS-driven melanoma. Here, theyhave investigated the impact of RMC-6236 also known as Daraxonrasib, a RAS multi-selective noncovalent inhibitor of the active, GTP -bound state of mutant and wild-type variants of RAS isoforms on NRAS mutant melanoma cells. Their results reveal that treatment of NRASQ61-mutated cells with RMC-6236 reduces cell proliferation and ERK and AKT signaling pathways, while promoting FAK signaling and a mesenchymal-like phenotypic reprogramming.

[0012] Importantly, the EMA / FDA approved anti-fibrotic drug Nintedanib (Ofev, Vargatef) prevents the adaptive mesenchymal like response and FAK activation induced by oncogenic NRAS inhibition and acts synergistically with RMC-6236 to reduce the viability of NRAS mutated melanoma cells. Their findings provide the first evidence that anti-fibrotic drugs enhance the efficacy of RAS inhibitors in NRAS mutant melanoma cells and provide the rationale of using RMC-6236 in combination with anti -fibrotic drugs to treat patients with NRAS-mutated melanomas.

[0013] Method for treating NRAS mutated melanoma

[0014] In a first aspect, the invention relates to a method for treating NRAS mutated melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of: i) a NRAS inhibitor and ii) an anti -fibrotic agent as a combined preparation.

[0015] 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 amaintenance 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.]).

[0016] 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.

[0017] In a particular embodiment, the melanoma is cutaneous melanoma.

[0018] In another embodiment, the melanoma is resistant melanoma.

[0019] As used herein, the term “resistant melanoma” refers to melanoma, which does not respond to a classical 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. The resistance of cancer for the medication is caused by mutations in the gene, which are involved in the proliferation, divisions or differentiation of cells. In the context of the invention, the resistance of melanoma is caused by the mutations (single or double) in the following genes: BRAF, NRAS, NF1 or PTEN, or the lack of BRAF, RAS or NF1 mutations (triple wild-type subtype). Resistance may also be due to BRAF and NRAS double mutation in patients who progress on BRAF inhibitor therapy. 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.

[0020] In a particular embodiment, NRAS mutations occur at codon 61 (Q61R, Q61L, and Q61K).

[0021] In a particular embodiment, NRAS mutations occur at the G12 or G13 codon.

[0022] The inhibitors of BRAF mutation and MEK are used to treat the melanoma with BRAF mutations.In a particular embodiment, the melanoma is resistant in which double-negative BRAF and NRAS mutant melanoma. The term “PTEN” refers to Phosphatase and TENsin homolog, it is one of the most frequently inactivated tumor suppressor genes in sporadic cancers.

[0023] The resistance can be also caused by a triple-negative NRAS, BRAF and NF 1 mutations as described above.

[0024] In a particular embodiment, the melanoma has NRAS mutation.

[0025] As used herein, the term “NRAS” refers to neuroblastoma RAS viral oncogene homolog. It is a member of the Ras gene family. The naturally occurring human NRAS gene has a nucleotide sequence as shown in Genbank Accession number NM_002524 and the naturally occurring human NRAS protein has an aminoacid sequence as shown in Genbank Accession number NP 002515. The murine nucleotide and amino acid sequences have also been described (Genbank Accession numbers NM_010937, NM_001368638).

[0026] 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. The inhibitors of MEK are used to treat the melanoma with NRAS mutations.

[0027] As used herein, the term “NRAS inhibitor” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of NRAS.

[0028] More particularly, such compound by inhibiting NRAS activity reduces cell division and differentiation. In a particular embodiment, the inhibitor of NRAS is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.

[0029] The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide. In a particular embodiment, the inhibitor of NRAS 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.

[0030] In a particular embodiment, the inhibitor of NRAS is a small organic molecule.

[0031] 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. The inhibitors of BRAF mutations are well known in the art.

[0032] In a particular embodiment, the inhibitor of NRAS is RMC-6236. RMC-6236, also known as Daraxonrasib is well known in the art and is the first investigational orallybioavailable, RAS(ON) multi-selective tricomplex inhibitor. RMC-6236 has the following formula, structure and CAS number 2765081-21-6:

[0033]

[0034] In some embodiments, the inhibitor of NRAS expression is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide, which inhibits the expression of NRAS. In a particular embodiment, the inhibitor of NRAS expression is siRNA. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene. Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 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 inhibitinggene 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.

[0035] In some embodiments, the inhibitor of NRAS expression is an endonuclease.

[0036] 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).

[0037] 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 interspacedshort palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

[0038] 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.

[0039] 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).

[0040] In some embodiments, the inhibitor of NRAS is an antibody.

[0041] 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.

[0042] 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.

[0043] In a particular embodiment, the inhibitor of NRAS 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.

[0044] In a particular embodiment, the inhibitor of NRAS is Mb24, as described in Whaby et al, Oncogene 2024 ; https: / / doi.org / 10.1038 / s41388-024-03186-y.As used herein, the term “subject” 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.

[0045] 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 melanoma resistant. In a particular embodiment, the subject has or is susceptible to have NRAS mutation. As used herein, the term “anti-fibrotic agent” refers to an agent that inhibits or reduces fibrosis.

[0046] Examples of anti-fibrotic agents include, without limitation, TGF-beta neutralizing antibodies, angiotension II inhibitors, inhibitors of fibrogenic cytokine signaling (e.g., TNF-alpha neutralizing antibodies, and inhibitors or neutralizing antibodies against IL6 signaling), integrin inhibitors, and other small molecules or inhibitors or neutralizing antibodies that may directly target fibrogenic signaling, indirectly target fibrogenic signaling (e.g., upstream or downstream modulators), or suppress fibrogenic signaling. Specific examples of anti-fibrotic agents include, without limitation, SHP-627, hydronidone PXS-25, disitertide, fresolimumab, LY2382770, STX-100, CWHM-12, SB-431542, THR-184, PF-06473871, RXI-109, FG-3019, imatinib, BOT-191, nilotinib, dasatinib, nintedanib, sorafenib, thalidomide, pomalidomide, etanercept, belimumab, refanalin (BB-3), dectrekumab (Q AX-576), tralokinumab, anakinra, rilonacept, SARI 56597, carlumab, bindarit, maraviroc, RS- 504393, actimmune, IFN-a, batimastat (BB-49), marimastat, macitentan, bosentan, ambrisentan, sparsentan (RE-021), atrasentan, losartan, BMS-986020, SAR-100842, PARI antagonists, curcumin, silymarin, b-caryophyllene, beraprost, iloprost, treprostinil, aviptadil, sivelestat, UK-396082, serelaxin, PRM-151, dioscin, NTU281, rapamycin, pal omid-529 (RES-529), ruxolitinib, baricitinib, omipalisib (GSK2126458), PF-562271, tanzisertib (CC-930), MMI-0100, IMD-1041, bardoxolone methyl (CDDO-Me), antisense NF-KB, baicalein, sulfasalazine, Y- 27632, bortezomib, emricasan, VX-166, Z-VAD-fmk, CTP-499, VBY-376, CA- 074Me, paquinimod, HOE-077, rosiglitazone, elafibranor (GFT-505), saroglitazar, pioglitazone, docosahexaenoic acid, INT-767, PX-102, obeti cholic acid, turofexorate isopropyl (WAY-362450), GW4064, triamcinolone, genistein, pentoxifylline, SIS-3, glycyrrhizin, anti-miR-21, ademetionine (SAM), b- aminopropionitrile (BAPN), simtuzumab (GS-6624), GM-CT-01, GR-MD-02, GCS- 100, GKT137831, N-acetylcysteine, mitoquinone, salvianolic acid B, resveratrol, pyridoxamine, a-tocopherol, and IW001.

[0047] In a particular embodiment, the anti-fibrotic agent is nintedanib.In a particular embodiment, the method for treating NRAS mutated melanoma according to the invention wherein the inhibitor of NRAS is RMC-6236 and the anti-fibrotic agent is Nintedanib.

[0048] In a particular embodiment, the invention relates to a method for treating NRAS mutated melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of: i) a NRAS inhibitor, ii) an anti -fibrotic agent, and iii) a classical treatment as a combined preparation.

[0049] As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy. The combined therapy may be tri-therapy. Such combined treatment can be administered simultaneously, separately or sequentially.

[0050] 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.

[0051] As used herein, the term “classical treatment” refers to any compound, natural or synthetic, used for the treatment of a melanoma or resistant melanoma.

[0052] In a particular embodiment, the classical treatment refers to radiation therapy, chemotherapy, targeted therapy, immunotherapy or ferroptosis therapy.

[0053] In a particular embodiment, the classical treatment is chemotherapy.

[0054] As used herein, the term "chemotherapy" has its general meaning in the art and refers to chemical compounds that are effective in inhibiting tumor growth. Examples of chemotherapeutic agents include multikinase inhibitors such as sorafenib and sunitinib, 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, triethylenethiophosphaoramide and trimethylolomelamine; acetogenins (especially bullatacin and bullatacinone); a camptothecin (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 aschlorambucil, chlornaphazine, 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 morpholinodoxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idanrbicin, marcellomycin, mitomycins, mycophenolic acid, nogalamycin, 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; antiadrenals 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-11 ; 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.

[0055] In a particular embodiment, the classical treatment is radiation therapy.

[0056] As used herein, the term “radiation therapy” has its 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 givento a subject per day; and hypofractionated radiation therapy, in which larger doses of radiation therapy per fraction is given but fewer fractions.

[0057] In a particular embodiment, the classical treatment is targeted therapy.

[0058] 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 or MEK inhibitors as described below.

[0059] In a further embodiment, the targeted therapy is the use of inhibitors of MEK.

[0060] As used herein, the term “MEK” refers to Mitogen-activated protein kinase, also known as MAP2K, MEK, MAPKK. It is a kinase enzyme, which phosphorylates mitogen-activated protein kinase (MAPK). As used herein, the term “inhibitor of MEK” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of MEK. More particularly, such compound by inhibiting BRAF activity reduces phosphorylation of MAPK. In a particular embodiment, the inhibitor of MEK is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide.

[0061] In a particular embodiment, the inhibitor of MEK is a small organic molecule.

[0062] The inhibitors of MEK are well known in the art. In a particular embodiment, the inhibitor of MEK is Trametinib also known as mekinist, which is commercialized by GSK. In a particular embodiment, the inhibitor of MEK is Cobimetinib also known as cotellic commercialized by Genentech. In a particular embodiment, the inhibitor of MEK is Binimetinib also known as MEK162, ARRY-162 is developed by Array Biopharma.

[0063] In another embodiment, the classical treatment is a combined treatment.

[0064] As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy. The combined therapy may be tri-therapy.

[0065] In the context of the invention, the melanoma is resistant to a combined treatment characterized by using an inhibitor of NRAS, an anti -fibrotic agent and a classical treatment as described above.

[0066] In a further embodiment, the classical treatment refers to a treatment with an immunotherapy.

[0067] As used herein, the term “immunotherapy” refers to a treatment of disease by activating or suppressing the immune system. Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies, while immunotherapies that reduce or suppress are classified as suppression immunotherapies. Immunotherapy is wellknown in the art and is selected from the group consisting of but not limited to: Checkpoint Inhibitors, CAR-T Cell Therapy; monoclonal antibodies or cancer vaccines.

[0068] In a further embodiment, the classical treatment is immune checkpoint inhibitor treatment.

[0069] As used herein, the term "immune checkpoint inhibitor" refers to molecules that totally or partially reduce, inhibit, interfere with or modulate one or more immune checkpoint proteins. As used herein, the term "immune checkpoint protein" has its general meaning in the art and refers to a molecule that is expressed by T cells in that either turn up a signal (stimulatory checkpoint molecules) or turn down a signal (inhibitory checkpoint molecules). Immune checkpoint molecules are recognized in the art to constitute immune checkpoint pathways similar to the CTLA-4 and PD-1 dependent pathways (see e.g. Pardoll, 2012. Nature Rev Cancer 12:252-264; Mellman et al., 2011. Nature 480:480- 489). Examples of stimulatory checkpoint include CD27 CD28 CD40, CD122, CD137, 0X40, GITR, and ICOS. Examples of inhibitory checkpoint molecules include A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 and VISTA. The Adenosine A2A receptor (A2AR) is regarded as an important checkpoint in cancer therapy because adenosine in the immune microenvironment, leading to the activation of the A2a receptor, is negative immune feedback loop and the tumor microenvironment has relatively high concentrations of adenosine. B7-H3, also called CD276, was originally understood to be a co-stimulatory molecule but is now regarded as co-inhibitory. B7-H4, also called VTCN1, is expressed by tumor cells and tumor-associated macrophages and plays a role in tumour escape. B and T Lymphocyte Attenuator (BTLA) and also called CD272, has HVEM (Herpesvirus Entry Mediator) as its ligand. Surface expression of BTLA is gradually downregulated during differentiation of human CD8+ T cells from the naive to effector cell phenotype, however tumor-specific human CD8+ T cells express high levels of BTLA. CTLA-4, Cytotoxic T-Lymphocyte-Associated protein 4 and also called CD152. Expression of CTLA-4 on Treg cells serves to control T cell proliferation. IDO, Indoleamine 2,3-dioxygenase, is a tryptophan catabolic enzyme. 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 inSeptember 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.

[0070] 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.

[0071] In a particular embodiment, the immune checkpoint inhibitor is an antibody.

[0072] Typically, antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

[0073] 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).

[0074] 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).

[0075] In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154.

[0076] In the context of the invention, the immune checkpoint inhibitor inhibits Tim-3 or its ligand.In a particular embodiment, the immune checkpoint inhibitor is an anti-Tim-3 antibody such as described in WO03063792, WO2011155607, WO2015117002, WO2010117057 and W02013006490.

[0077] In some embodiments, the immune checkpoint inhibitor is a small organic molecule. The term "small organic molecule" as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (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.

[0078] 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.

[0079] 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.

[0080] 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.

[0081] 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 carb oxi mi dami de :h N S

[0082] — K'H

[0083]

[0084] 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:

[0085] N --N

[0086] N

[0087]

[0088] H

[0089] 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.

[0090] In some embodiments, the immune checkpoint inhibitor is an aptamer.

[0091] 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.

[0092] 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.

[0093] In a further embodiment, the classical treatment refers to a treatment with an immunotherapy.

[0094] As used herein, the term “ferroptosis inducer” denotes a compound able to increase ferroptosis occurrence. Ferroptosis inducers are well-known in the art. As example, the ferroptosis inducer may be APR-246, Ras Synthetic Lethal 3 (RSL3), ML 162, ML210, acrolein,erastin, Imidazole Ketone Erastin (IKE), Piperazine Erastin (PE), sulfasalazine, sorafenib, Ferroptosis Inducer 56 (FIN56), Ferroptosis inducer endoperoxide (FIN02), Caspase-Independent Lethal 56 (CIL56), mevalonate-derived coenzyme Q10, buthionine sulfoximine (BSO), amentoflavone, dihydroartemisinin (DHA), typhaneoside, artesunate, Withaferin A (WA), auranofin.

[0095] In a particular embodiment, the ferroptosis inducer is Ras Synthetic Lethal 3 (RSL3). As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g., inhibitors of NRAS and anti -fibrotic agent) into the subject, such as by intraparenchymal, intracerebroventricular, intrathecal, intranasal, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery, oral 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.

[0096] In a particular embodiment, the inhibitor of NRAS and anti-fibrotic agent are administered orally.

[0097] By a "therapeutically effective amount" is meant a sufficient amount of inhibitor of NRAS and an anti-fibrotic agent for use in a method for the treatment of melanoma at a reasonable benefit / risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions 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 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 coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient,typically 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.

[0098] Pharmaceutical composition

[0099] The NRAS inhibitor and the anti-fibrotic agent as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions.

[0100] A further objective the invention relates to a pharmaceutical composition comprising i) an inhibitor of NRAS and ii) an anti -fibrotic agent.

[0101] In another embodiment, the pharmaceutical composition comprising i) an inhibitor of NRAS, ii) an anti-fibrotic agent and iii) a classical treatment.

[0102] In another embodiment, the pharmaceutical composition according to the invention, for use in the therapy.

[0103] In another embodiment, the pharmaceutical composition according to the invention for use in the treatment of NRAS mutated melanoma.

[0104] In a particular embodiment, the pharmaceutical composition according to the invention comprising RMC-6236 and Nintedanib.

[0105] The inhibitors of NRAS and the anti -fibrotic agent as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capableof 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 filteredsterilization. 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] A further object of the present invention relates to a method of screening a drug suitable for the treatment of melanoma comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the activity of NRAS.

[0107] Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit the activity of NRAS. In some embodiments, the assay first comprises determining the ability of the test compound to bind to NRAS. In some embodiments, a population of cells is then contacted and activated so as to determine the ability of the test compound to inhibit the activity of NRAS. 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 NRAS as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo.Typically, the test compound is selected from the group consisting of peptides, peptidomimetics, small organic molecules, aptamers or nucleic acids. For example the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules.

[0108] 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.

[0109] FIGURES:

[0110] Figure 1. Nintedanib prevents the adaptive mesenchymal-like response and FAK activation induced by oncogenic NRAS inhibition. (A) Western blot analysis of markers of cell proliferation, FAK signalling and ECM on cell lysates from SKMEL2 cells treated by RMC-6236 or Trametinib, or co-treated with RMC-6236 and Nintedanib. HSP60, loading control. (B) RT-qPCR analysis of the expression of mesenchymal, ECM and YAP target genes on SKMEL2 cells treated with RMC-6236 (50nM) and / or Nintedanib (IpM) for 72h. Data is represented as mean ± sd from a triplicate representative of at least 2 independent experiments Figure 2. The combination of RMC-6236 with Nintedanib acts synergistically to reduce the viability of NRAS mutated melanoma cells. (A) Crystal violet cell proliferation assay performed on SKMEL2 cells treated with the indicated doses of RMC-6236 and Nintedanib. Cells were plated in triplicate on 12-well plates and treated with the indicated doses of Trametinib and RMC-6236 for 72h. (B) Western blot analysis of P-AKT, Survivin and cleaved caspase 3 levels on lysates from SKMEL2 cells treated with vehicle, RMC-6236 (50nM) or / and Nintedanib (IpM) for 72 h. P-ACTIN, loading control.

[0111] Figure 3. (A) Mouse Ma Nras 1014 melanoma cells were subcutaneously inoculated into C57BL / 6 syngeneic mice. When tumors reached 100 mm3, mice were treated every other day with vehicle (Control), Nintedanib, RMC-6236 (Daraxonrasib), or RMC-6236 plus Nintedanib (n= 6). (B) Representative median graphics showing tumor growth following treatment (n= 6). Two-way ANOVA was used for statistical analysis. (C) Kaplan-Meier survival curves of mice treated with the indicated therapies (n = 6). The Gehan-Breslow-Wilcoxon statistical test was used for RMC-6236 vs RMC-6236 / Nintedanib. **P <0.001. (D) Mouse body weight was measured at the indicated times. Data shown are mean SD (n = 6).EXAMPLE:

[0112] Material & Methods

[0113] Cell lines and reagents

[0114] SKMEL2 human NRAS-mutated malignant melanoma cell line was from ATCC. Melanoma cells were cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 7% FBS (Hyclone) and 1% penicillin / streptomycin. Culture reagents were purchased from Thermo Fisher Scientific. RASi (RMC-6236) was from Medkoo. MEKi (GSK1120212, Trametinib) and Nintedanib (BIBF1120) were from Selleckem.

[0115] Crystal violet cell proliferation assay

[0116] Cells (104) were plated in triplicate on 12-well plates for 24h and treated with the indicated inhibitor for 72h. Cells were then stained with 0.04% crystal violet, 20% ethanol in PBS for 30 min. Following extensive washing of the plate, representative photographs were taken. The crystal violet dye was solubilized by 10% acetic acid in PBS and measured by absorbance at 595 nm. RNA extraction and real-time quantitative PCR Total RNA was extracted from tumors and cell samples with the miRNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. 1 pg of extracted RNA from cells was reverse transcribed into cDNA using the Multiscribe reverse transcriptase kit provided by Applied Biosystems. Primers were designed using PrimerBank or adopted from published studies. Gene expression levels were measured using Platinum SYBR Green qPCR Supermix (Fisher Scientific) and Step One thermocycler (Applied Biosystem). Results from RT-qPCR were normalized using the reference gene RPL32 and relative gene expression was quantified with the AACt method.

[0117] Western blot analysis

[0118] After treatment, cells were lysed at 4°C in stop buffer (50mM TrisHCl, 150mM NaCl, 0.25% sodium deoxycholate, 1% NP-40, ImM EDTA) supplemented with protease and phosphatase inhibitors (Pierce). Lysates were immersed in liquid nitrogen and centrifuged for 15 min at 13,300 rpm at 4°C. After colorimetric assay (DC protein assay-Biorad), 30 pg of total proteins are taken up in buffer (25mM Tris (pH 6.8), 2% SDS, 5% glycerol, 1% P-mercaptoethanol, 0.01% bromophenol blue), heated to 95°C 5min, plated on electrophoresis gels, separated, and transferred to nitrocellulose membrane (Millipore). Membranes were saturated Ih at RT in saturation buffer (5% BSA, lOmM Tris-HCl (pH7.5), 500mM NaCl) and incubated with primary antibodies at 4°C ON. The next day, membranes were rinsed in wash buffer (lOmM Tris-HCl (pH7.5), 500mM NaCl, 0.1% Tween 20), incubated Ih at RT with theperoxidase-coupled secondary antibodies. After washing, membranes were revealed by chemiluminescence (ECL, Millipore).

[0119] Syngeneic mice model

[0120] 6-week-old female C57BL / 6 mice were injected subcutaneously in the right flank with 1 x io6mouse NRASQ61Kmelanoma cells (Ma Nras 1014). Tumors were measured with caliper, and treatments were started when the tumors reached a volume of 0.1 cm3, after randomization of mice into control and test groups. RMC-6236 (25 mg / kg), Nintedanib (50 mg / kg), or the combination were administered by oral gavage every other day during up to 4 weeks. Control mice were treated with vehicle only. Animals were sacrificed when the tumors reached a volume of 1 cm3.

[0121] Statistical analysis

[0122] Unless otherwise stated, all experiments were repeated at least three times and representative data / images are shown. Statistical data analysis was performed using GraphPad Prism software. Unpaired two-tailed Mann-Whitney test was used for statistical comparisons between two groups and multiple Mann-Whitney test to compare three or more groups. Histogram plots and curves represent mean ± sd. P values < 0.05 were considered statistically significant.

[0123] Results

[0124] To find new therapeutic approach to target Y MS'-mut at ed melanomas, we first compare the action of Trametinib, a clinically approved MEKi and RMC-6236 (Daraxonrasib), a novel inhibitor of wild-type and mutated forms of KRAS, HRAS and NRAS (2) on the NRAS Q61R mutant human melanoma cell line SKMEL2. As with Trametinib, treatment with RMC-6236 led to a dose-dependent reduction in SKMEL2 cell proliferation (data not shown). Consistent with their inhibitory effect on cell proliferation, western blot analysis of SKMEL2 cell lysates showed that both Trametinib and RMC-6236 decreased the levels of markers of cell cycle progression such as phosphorylated form of Rb and cyclin dependent kinase CDK2 expression (Figure 1A). We recently reported that in response to BRAF oncogenic pathway inhibition, BRAF mutated melanoma cells induce a fibrotic-like mesenchymal program that include the expression of extracellular matrix (ECM) proteins, YAP target genes and features typical of myofibroblasts including collagen-remodeling activities and PDGFRP expression (3,4). Here, we show that when exposed to Trametinib or RMC-6236, NRAS mutant SKMEL2 cells up-regulated the expression of mesenchymal and ECM (PDGFR FN1, COL1A1, LOXL2) genes and YAP target genes (AMOTL2, THBS1) (Figure IB and data not shown). The increased expression at protein levels of PDGFRP, FN1, aSMA (ACTA2 gene product) upon Trametiniband RMC-6236 treatment and MAPK pathway inhibition (as shown to a decreased levels of phosphorylated ERK) was confirmed by western blot analysis (Figure 1A and data not shown). We further show that whereas RMC-6236 inhibited the constitutive activation of ERK, RSK and AKT, it increased the activation of FAK signaling pathway (as indicated by increased levels of phosphorylated FAK and its substrate Paxillin) and the expression of mesenchymal FN1 marker, and of the YAP target TSP1 (THBS1 gene product) (Figure 1A and data not shown).

[0125] Gene expression analysis on SKMEL2 cells shows that the anti-fibrotic Nintedanib strongly attenuated the fibrotic-like mesenchymal signature induced by RMC-6236, including a decrease in the expression of ECM markers (FNJ, LOXL2, COL1A7), PDGFRfi and YAP target genes (AMOTL2, THBS1) (Figure IB). In addition, western blot showed that Nintedanib prevents the activation of FAK signaling and FN1 expression induced by oncogenic NRAS inhibition (Figure 1A). Note that while Nintedanib prevented the adaptive reprogramming promoted by NRAS inhibition, it cooperated with RMC-6236 to reduce the levels of phosphorylated Rb and CDK2, and to increase the expression of the cell cycle arrest marker p27KIPl (Figure 1A).

[0126] Next, we examined the effect of RMC-6236 and Nintedanib combination on NRAS mutant human melanoma cell proliferation and survival. Crystal violet assay performed on SKMEL2 cells demonstrated that RMC-6236 acts synergistically with Nintedanib to reduce cell proliferation (Figure 2A). Using the Bliss prediction method, we further demonstrated a strong synergistic action of RMC-6236 / Nintedanib combination on the proliferation and survival of NRAS mutated melanoma cells (Bliss mean 35.77, p=2.58 10-5) (Figure 2B and data not shown). Western blot analysis showed that Nindedanib cooperated with RMC-6236 to reduce the levels of cell survival markers, such as phosphorylated AKT and Survivin and to synergistically promoted the activation of caspase 3, an apoptosis executioner (Figure 2B).

[0127] These data indicate that the anti-fibrotic drug Nintedanib enhances the efficacy of the RAS inhibitor RMC-6236 in melanoma cells.

[0128] Finally, we showed, in vivo in a syngeneic model of NRAS-mutant murine melanoma with a Q61K mutation, the benefic effect of the combined action of the RAS inhibitor RMC-6236 and the antifibrotic Nintedanib on tumor growth and animal survival (Figures 3A to 3C).

[0129] The combined treatment significantly improved mouse survival without body weight loss or sign of toxicity throughout the study (Figure 3D).In conclusion, our findings provide in vitro and in vivo pre-clinical evidence of using the novel multi-selective RAS inhibitor RMC-6236, alone or in combination with anti -fibrotic drugs such as Nintedanib, to treat patients with AT Ymutated melanomas.

[0130] REFERENCES:

[0131] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

[0132] 1. Randic T, Kozar I, Margue C, Utikal J, Kreis S. NRAS mutant melanoma: Towards better therapies. Cancer Treat Rev 2021;99: 102238

[0133] 2. Jiang J, Jiang L, Maldonato BJ, Wang Y, Holderfield M, Aronchik I, et al. Translational and Therapeutic Evaluation of RAS-GTP Inhibition by RMC-6236 in RAS-Driven Cancers. Cancer Discov 2024;14:994-1017

[0134] 3. Girard CA, Lecacheur M, Ben Jouira R, Berestjuk I, Diazzi S, Prod'homme V, et al. AFeed-Forward Mechanosignaling Loop Confers Resistance to Therapies Targeting the MAPK Pathway in BRAF-Mutant Melanoma. Cancer Res 2020;80: 1927-41

[0135] 4. Diazzi S, Baeri A, Fassy J, Lecacheur M, Marin-Bejar O, Girard CA, et al. Blockade of the pro-fibrotic reaction mediated by the miR-143 / -145 cluster enhances the responses to targeted therapy in melanoma. EMBO Mol Med 2022:el5295

[0136] 5. Hilberg F, Roth GJ, Krssak M, Kautschitsch S, Sommergruber W, Tontsch-Grunt U, et al. BIBF 1120: triple angiokinase inhibitor with sustained receptor blockade and good antitumor efficacy. Cancer Res 2008;68:4774-82

[0137] 6. Rivera-Ortega P, Hayton C, Blaikley J, Leonard C, Chaudhuri N. Nintedanib in the management of idiopathic pulmonary fibrosis: clinical trial evidence and real -world experience. Ther Adv Respir Dis 2018;12:1753466618800618 7. Lamb YN. Nintedanib: A Review in Fibrotic Interstitial Lung Diseases. Drugs 2021;81:575-86

Claims

1. - 27 -CLAIMS:

1. A method for treating NRAS mutated melanoma in a subj ect in need thereof comprising a step of administering said subject with a therapeutically effective amount of: i) a NRAS inhibitor and ii) an anti-fibrotic agent as a combined preparation.

2. The method according to claim 1, wherein the NRAS mutated melanoma is a resistant melanoma.

3. The method according to claim 2, wherein, the NRAS mutated melanoma is resistant to a classical treatment.

4. The method according to claim 3, wherein, the classical treatment is selected from the group consisting of but not limited to: radiation therapy, targeted therapy, immunotherapy, ferroptosis therapy or chemotherapy.

5. The method according to claim 1, wherein the inhibitor of NRAS is RMC-6236.

6. The method according to claim 1, wherein the anti -fibrotic agent is selected from the group consisting of but not limited to: TGF-beta neutralizing antibodies, angiotension II inhibitors, inhibitors of fibrogenic cytokine signaling (e.g., TNF- alpha neutralizing antibodies, and inhibitors or neutralizing antibodies against IL6 signaling), integrin inhibitors, and other small molecules or inhibitors or neutralizing antibodies that may directly target fibrogenic signaling, indirectly target fibrogenic signaling (e.g., upstream or downstream modulators), or suppress fibrogenic signaling.

7. The method according to claim 1, wherein the anti -fibrotic agent is selected from the group consisting of but not limited to: SHP-627, hydronidone PXS-25, disitertide, fresolimumab, LY2382770, STX-100, CWHM-12, SB-431542, THR-184, PF- 06473871, RXI-109, FG-3019, imatinib, BOT-191, nilotinib, dasatinib, nintedanib, sorafenib, thalidomide, pomalidomide, etanercept, belimumab, refanalin (BB-3), dectrekumab (Q AX-576), tralokinumab, anakinra, rilonacept, SARI 56597, carlumab, bindarit, maraviroc, RS- 504393, actimmune, IFN-a, batimastat (BB-49), marimastat, macitentan, bosentan, ambrisentan, sparsentan (RE-021), atrasentan, losartan, BMS- 986020, SAR-100842, PARI antagonists, curcumin, silymarin, b-caryophyllene, beraprost, iloprost, treprostinil, aviptadil, sivelestat, UK-396082, serelaxin, PRM-151,dioscin, NTU281, rapamycin, pal omid-529 (RES-529), ruxolitinib, baricitinib, omipalisib (GSK2126458), PF-562271, tanzisertib (CC-930), MMI-0100, IMD-1041, bardoxolone methyl (CDDO-Me), antisense NF-KB, baicalein, sulfasalazine, Y- 27632, bortezomib, emricasan, VX-166, Z-VAD-fmk, CTP-499, VBY-376, CA- 074Me, paquinimod, HOE-077, rosiglitazone, elafibranor (GFT-505), saroglitazar, pioglitazone, docosahexaenoic acid, INT-767, PX-102, obeti cholic acid, turofexorate isopropyl (WAY-362450), GW4064, triamcinolone, genistein, pentoxifylline, SIS-3, glycyrrhizin, anti-miR-21, ademetionine (SAM), b- aminopropionitrile (BAPN), simtuzumab (GS-6624), GM-CT-01, GR-MD-02, GCS- 100, GKT137831, N- acetylcysteine, mitoquinone, salvianolic acid B, resveratrol, pyridoxamine, a- tocopherol, and IW001.

8. The method according to claim 1, wherein the anti -fibrotic agent is Nintedanib.

9. A pharmaceutical composition comprising i) an inhibitor of NRAS and ii) an anti- fibrotic agent.

10. The pharmaceutical composition according to claim 8, for use in the therapy.

11. The pharmaceutical composition according to claim 9, for use in the treatment of NRAS mutated melanoma.

Images