WO2025073765A1 - Methods of prognosis and treatment of patients suffering from melanoma - Google Patents

Abstract

Melanoma cell plasticity plays a key role in the acquisition of resistance to therapy. TRIM24/TIF1α, which encodes for the tripartite RING/B-Box/Coil-coiled transcriptional coactivator, is frequently amplified in human melanomas. The inventors observed a strong correlation between elevated TRIM24 expression and metastatic disease, adverse outcome to immune checkpoint inhibitor therapy and a worse relapse-free survival. shRNA-mediated knock-down of TRIM24 decreased the migratory capacities and increased the sensitivity to BRAF inhibitors in two cellular melanoma models. RNA-sequencing analyses revealed that TRIM24 knock-down significantly represses undifferentiated/invasive transcriptional programs. A protac-based approach for degradation of Trim24 was shown to resensitize resistant melanoma cells to BRAF inhibitors. Thus, the present invention relates to a method for predicting the survival time of a patient suffering from melanoma comprising the step of measuring the level of TRIM24, and the use of TRIM24 inhibitor for treating melanoma, in particular resistant melanoma.

Description

METHODS OF PROGNOSIS AND TREATMENT OF PATIENTS SUFFERING FROM MELANOMA

FIELD OF THE INVENTION:

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

BACKGROUND OF THE INVENTION:

Metastatic melanoma is the most aggressive form of skin cancer, with nearly 15,000 new cases per year in France. Patient treatment has recently been revolutionized by the advent of targeted therapies inhibiting BRAF/MEK (BRAFi, MEKi) and immunotherapies targeting immune checkpoint proteins (anti-PD-1, anti-CTLA4). However, nearly one in two patients still develops resistance.

Acquired resistance to BRAF/MEK-targeted therapies is not solely dependent on genetic alterations (Villanueva et al., 2013) but also involves non-genetic mechanisms, driven by transcriptional, epigenetic, or translational phenotypic changes (Rambow et al., 2019). The phenotypic plasticity of melanoma cells allows rapid adaptation of tumors in response to signals from the microenvironment, including anti-cancer therapies, and contributes to intra-tumoral heterogeneity. Furthermore, the plasticity of tumor cells may also facilitate evasion of the immune system and resistance to immunotherapies in melanoma (Benboubker et al., 2022). Additionally, the epigenetic reprogramming occurring in melanoma cells allows reversible modulation of gene expression, and could explain their rapid adaptation in response to treatments. While epigenetic regulators such as EZH2 have been linked to tumor plasticity and immune evasion in melanoma (Zingg et al., 2015, 2017), it remains important to gain a better understanding of the epigenetic mechanisms that underlie phenotypic plasticity to comprehensively grasp therapy resistance in melanoma. Thus, the aim of the study was to identify a novel epigenetic regulator involved in melanoma cell plasticity.

SUMMARY OF THE INVENTION:

The present invention is defined by the claims. The present invention relates to methods of prognosis and treatment of patients suffering from melanoma. The present invention also relates to use of TRIM24 inhibitors for overcoming resistance to BRAF inhibitors, MEK inhibitor or immune checkpoint inhibitor in melanoma. DETAILED DESCRIPTION OF THE INVENTION:

The inventors show that TRIM24 orchestrates the plasticity of melanoma cells and governs an undifferentiated/neural-crest-like phenotype. An elevated expression of TRIM24 is associated with increased invasiveness and resistance to targeted therapies in melanoma. Their study reveals a greater TRIM24 expression in the tumor compared to surrounding tissues, and high expression of TRIM24 is associated with unfavorable prognoses and a negative response to therapy in melanoma patients. Their results firmly establish TRIM24 as a robust marker of aggressiveness in melanoma. Furthermore, they successfully and specifically degraded TRIM24 in melanoma cells which increases the sensitivity to BRAFi and restore responsiveness in resistant cell line, positioning TRIM24 as a promising targetable protein for melanoma treatment, and in particular in combination with the current therapies available.

Methods for predicting the survival time

Thus, in a first aspect, the invention relates to an in vitro method for predicting the survival time of a patient suffering from melanoma comprising i) determining the level of Tripartite motif-containing 24 (TRIM24) from a sample obtained from the patient, ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the patient will have a short survival time when the level determined at step i) is higher than the predetermined reference value or concluding that the patient will have a long survival time when the level determined at step i) is lower than the predetermined reference value.

As used herein, the term “subject” or “patient” refers to any mammal, such as a rodent, a feline, a canine, and a primate. Notably, in the present invention, the subject is a human. More particularly, the subject according to the invention has or is susceptible to have melanoma.

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. As used herein, the term “sample” refers to any sample obtained from the subject for the purpose of performing the method of the present invention. In some embodiments, the sample is a bodily fluid (e.g. a blood sample) or a tissue. In some embodiments, the sample is a tissue sample. The term “tissue sample” includes sections of tissues such as biopsy or autopsy samples, fixed or and frozen sections taken for histological purposes. As used herein, the term “blood sample” means any blood sample derived from the subject. Collections of blood samples can be performed by methods well known to those skilled in the art. In some embodiments, the blood sample is a serum or plasma sample.

In a particular embodiment, the sample has been previously obtained from the subject.

As used herein, the term “Tripartite motif-containing 24” or “TRIM24”, also known as transcriptional intermediary factor la (TIFla) refers to a protein which mediates transcriptional control by interaction with the activation function 2 (AF2) region of several nuclear receptors, including the estrogen, retinoic acid, and vitamin D3 receptors. Its Entrez reference is 8805 and its UniProt reference is 015164.

The detection and quantification of TRIM24 in the sample can be detected by any method known in the art.

As used herein, the term "expression level" refers, e.g., to a determined level of expression of gene of interest or protein of interest (i.e TRIM24). The expression level of expression indicates the amount of expression product in a sample. The expression product of a gene of interest can be the nucleic acid of interest itself, a nucleic acid transcribed or derived therefrom, or the a polypeptide or protein derived therefrom.

Measuring the expression level of TRIM24 can be done by measuring the gene expression level of these genes and can be performed by a variety of techniques well known in the art.

Typically, the expression level of a gene may be determined by determining the quantity of mRNA. Methods for determining the quantity of mRNA are well known in the art. For example, the nucleic acid contained in the samples (e.g., cell or tissue prepared from the patient) is first extracted according to standard methods, for example using lytic enzymes or chemical solutions or extracted by nucleic-acid-binding resins following the manufacturer's instructions. The extracted mRNA is then detected by hybridization (e. g., Northern blot analysis, in situ hybridization) and/or amplification (e.g., RT-PCR). Other methods of Amplification include ligase chain reaction (LCR), transcription- mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence-based amplification (NASBA).

Nucleic acids having at least 10 nucleotides and exhibiting sequence complementarity or homology to the mRNA of interest herein find utility as hybridization probes or amplification primers. It is understood that such nucleic acids need not be identical, but are typically at least about 80% identical to the homologous region of comparable size, more preferably 85% identical and even more preferably 90-95% identical. In certain embodiments, it will be advantageous to use nucleic acids in combination with appropriate means, such as a detectable label, for detecting hybridization.

Typically, the nucleic acid probes include one or more labels, for example to permit detection of a target nucleic acid molecule using the disclosed probes. In various applications, such as in situ hybridization procedures, a nucleic acid probe includes a label (e.g., a detectable label). A “detectable label” is a molecule or material that can be used to produce a detectable signal that indicates the presence or concentration of the probe (particularly the bound or hybridized probe) in a sample. Thus, a labelled nucleic acid molecule provides an indicator of the presence or concentration of a target nucleic acid sequence (e.g., genomic target nucleic acid sequence) (to which the labelled uniquely specific nucleic acid molecule is bound or hybridized) in a sample. A label associated with one or more nucleic acid molecules (such as a probe generated by the disclosed methods) can be detected either directly or indirectly. A label can be detected by any known or yet to be discovered mechanism including absorption, emission and/ or scattering of a photon (including radio frequency, microwave frequency, infrared frequency, visible frequency and ultra-violet frequency photons). Detectable labels include colored, fluorescent, phosphorescent and luminescent molecules and materials, catalysts (such as enzymes) that convert one substance into another substance to provide a detectable difference (such as by converting a colourless substance into a coloured substance or vice versa, or by producing a precipitate or increasing sample turbidity), haptens that can be detected by antibody binding interactions, and paramagnetic and magnetic molecules or materials.

Particular examples of detectable labels include fluorescent molecules (or fluorochromes). Numerous fluorochromes are known to those of skill in the art, and can be selected, for example from Life Technologies (formerly Invitrogen), e.g., see, The Handbook — A Guide to Fluorescent Probes and Labeling Technologies). Examples of particular fluorophores that can be attached (for example, chemically conjugated) to a nucleic acid molecule (such as a uniquely specific binding region) are provided in U.S. Pat. No. 5,866, 366 to Nazarenko et al., such as 4-acetamido-4'-isothiocyanatostilbene-2,2' disulfonic acid, acridine and derivatives such as acridine and acridine isothiocyanate, 5-(2'-aminoethyl) aminonaphthalene- 1 -sulfonic acid (EDANS), 4-amino -N- [3 vinylsulfonyl)phenyl]naphthalimide-3,5 disulfonate (Lucifer Yellow VS), N-(4-anilino-l- naphthyl)maleimide, antllranilamide, Brilliant Yellow, coumarin and derivatives such as coumarin, 7-amino-4-methylcoumarin (AMC, Coumarin 120), 7-amino-4- trifluoromethylcouluarin (Coumarin 151); cyanosine; 4',6-diarninidino-2-phenylindole (DAPI); 5',5"dibromopyrogallol-sulfonephthalein (Bromopyrogallol Red); 7 -diethylamino -3 (4'-isothiocyanatophenyl)-4-methylcoumarin; diethylenetriamine pentaacetate; 4,4'- diisothiocyanatodihydro-stilbene-2,2'-disulfonic acid; 4,4'-diisothiocyanatostilbene-2,2'- disulforlic acid; 5-[dimethylamino] naphthalene- 1 -sulfonyl chloride (DNS, dansyl chloride); 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL); 4-dimethylaminophenylazophenyl- 4'-isothiocyanate (DABITC); eosin and derivatives such as eosin and eosin isothiocyanate; erythrosin and derivatives such as erythrosin B and erythrosin isothiocyanate; ethidium; fluorescein and derivatives such as 5-carboxyfluorescein (FAM), 5-(4,6diclllorotriazin-2- yDaminofluorescein (DTAF), 2'7'dimethoxy-4'5'-dichloro-6-carboxyfluorescein (JOE), fluorescein, fluorescein isothiocyanate (FITC), and QFITC Q(RITC); 2',7'-difluorofluorescein (OREGON GREEN®); fluorescamine; IR144; IR1446; Malachite Green isothiocyanate; 4- methylumbelliferone; ortho cresolphthalein; nitrotyrosine; pararosaniline; Phenol Red; B- phycoerythrin; o-phthaldialdehyde; pyrene and derivatives such as pyrene, pyrene butyrate and succinimidyl 1 -pyrene butyrate; Reactive Red 4 (Cibacron Brilliant Red 3B-A); rhodamine and derivatives such as 6-carboxy-X-rhodamine (ROX), 6-carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride, rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, rhodamine green, sulforhodamine B, sulforhodamine 101 and sulfonyl chloride derivative of sulforhodamine 101 (Texas Red); N,N,N',N'-tetramethyl-6-carboxyrhodamine (TAMRA); tetramethyl rhodamine; tetramethyl rhodamine isothiocyanate (TRITC); riboflavin; rosolic acid and terbium chelate derivatives. Other suitable fluorophores include thiol-reactive europium chelates which emit at approximately 617 mn (Heyduk and Heyduk, Analyt. Biochem. 248:216-27, 1997; J. Biol. Chem. 274:3315-22, 1999), as well as GFP, LissamineTM, diethylaminocoumarin, fluorescein chlorotriazinyl, naphthofluorescein, 4,7-dichlororhodamine and xanthene (as described in U.S. Pat. No. 5,800,996 to Lee et al.) and derivatives thereof. Other fluorophores known to those skilled in the art can also be used, for example those available from Life Technologies (Invitrogen; Molecular Probes (Eugene, Oreg.)) and including the ALEXA FLUOR® series of dyes (for example, as described in U.S. Pat. Nos. 5,696,157, 6, 130, 101 and 6,716,979), the BODIPY series of dyes (dipyrrometheneboron difluoride dyes, for example as described in U.S. Pat. Nos. 4,774,339, 5,187,288, 5,248,782, 5,274,113, 5,338,854, 5,451,663 and 5,433,896), Cascade Blue (an amine reactive derivative of the sulfonated pyrene described in U.S. Pat. No. 5,132,432) and Marina Blue (U.S. Pat. No. 5,830,912).

In addition to the fluorochromes described above, a fluorescent label can be a fluorescent nanoparticle, such as a semiconductor nanocrystal, e.g., a QUANTUM DOTTM (obtained, for example, from Life Technologies (QuantumDot Corp, Invitrogen Nanocrystal Technologies, Eugene, Oreg.); see also, U.S. Pat. Nos. 6,815,064; 6,682,596; and 6,649, 138). Semiconductor nanocrystals are microscopic particles having size-dependent optical and/or electrical properties. When semiconductor nanocrystals are illuminated with a primary energy source, a secondary emission of energy occurs of a frequency that corresponds to the handgap of the semiconductor material used in the semiconductor nanocrystal. This emission can he detected as colored light of a specific wavelength or fluorescence. Semiconductor nanocrystals with different spectral characteristics are described in e.g., U.S. Pat. No. 6,602,671. Semiconductor nanocrystals that can he coupled to a variety of biological molecules (including dNTPs and/or nucleic acids) or substrates by techniques described in, for example, Bruchez et al., Science 281 :20132016, 1998; Chan et al., Science 281 :2016-2018, 1998; and U.S. Pat. No. 6,274,323. Formation of semiconductor nanocrystals of various compositions are disclosed in, e.g., U.S. Pat. Nos. 6,927, 069; 6,914,256; 6,855,202; 6,709,929; 6,689,338; 6,500,622; 6,306,736; 6,225,198; 6,207,392; 6,114,038; 6,048,616; 5,990,479; 5,690,807; 5,571,018; 5,505,928; 5,262,357 and in U.S. Patent Publication No. 2003/0165951 as well as PCT Publication No. 99/26299 (published May 27, 1999). Separate populations of semiconductor nanocrystals can he produced that are identifiable based on their different spectral characteristics. For example, semiconductor nanocrystals can he produced that emit light of different colors based on their composition, size or size and composition. For example, quantum dots that emit light at different wavelengths based on size (565 mn, 655 mn, 705 mn, or 800 mn emission wavelengths), which are suitable as fluorescent labels in the probes disclosed herein are available from Life Technologies (Carlshad, Calif.).

Additional labels include, for example, radioisotopes (such as 3 H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes. Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, beta-galactosidase, beta-glucuronidase, or beta-lactamase.

Alternatively, an enzyme can he used in a metallographic detection scheme. For example, silver in situ hybridization (SISH) procedures involve metallographic detection schemes for identification and localization of a hybridized genomic target nucleic acid sequence. Metallographic detection methods include using an enzyme, such as alkaline phosphatase, in combination with a water-soluble metal ion and a redox-inactive substrate of the enzyme. The substrate is converted to a redox-active agent by the enzyme, and the redoxactive agent reduces the metal ion, causing it to form a detectable precipitate. (See, for example, U.S. Patent Application Publication No. 2005/0100976, PCT Publication No. 2005/ 003777 and U.S. Patent Application Publication No. 2004/ 0265922). Metallographic detection methods also include using an oxido-reductase enzyme (such as horseradish peroxidase) along with a water soluble metal ion, an oxidizing agent and a reducing agent, again to form a detectable precipitate. (See, for example, U.S. Pat. No. 6,670,113).

Probes made using the disclosed methods can be used for nucleic acid detection, such as ISH procedures (for example, fluorescence in situ hybridization (FISH), chromogenic in situ hybridization (CISH) and silver in situ hybridization (SISH)) or comparative genomic hybridization (CGH).

In situ hybridization (ISH) involves contacting a sample containing target nucleic acid sequence (e.g., genomic target nucleic acid sequence) in the context of a metaphase or interphase chromosome preparation (such as a cell or tissue sample mounted on a slide) with a labeled probe specifically hybridizable or specific for the target nucleic acid sequence (e.g., genomic target nucleic acid sequence). The slides are optionally pretreated, e.g., to remove paraffin or other materials that can interfere with uniform hybridization. The sample and the probe are both treated, for example by heating to denature the double stranded nucleic acids. The probe (formulated in a suitable hybridization buffer) and the sample are combined, under conditions and for sufficient time to permit hybridization to occur (typically to reach equilibrium). The chromosome preparation is washed to remove excess probe, and detection of specific labeling of the chromosome target is performed using standard techniques.

For example, a biotinylated probe can be detected using fluorescein-labeled avidin or avidin-alkaline phosphatase. For fluorochrome detection, the fluorochrome can be detected directly, or the samples can be incubated, for example, with fluorescein isothiocyanate (FITC)- conjugated avidin. Amplification of the FITC signal can be effected, if necessary, by incubation with biotin-conjugated goat antiavidin antibodies, washing and a second incubation with FITC- conjugated avidin. For detection by enzyme activity, samples can be incubated, for example, with streptavidin, washed, incubated with biotin-conjugated alkaline phosphatase, washed again and pre-equilibrated (e.g., in alkaline phosphatase (AP) buffer). For a general description of in situ hybridization procedures, see, e.g., U.S. Pat. No. 4,888,278.

Numerous procedures for FISH, CISH, and SISH are known in the art. For example, procedures for performing FISH are described in U.S. Pat. Nos. 5,447,841; 5,472,842; and 5,427,932; and for example, in Pirlkel et al., Proc. Natl. Acad. Sci. 83:2934-2938, 1986; Pinkel et al., Proc. Natl. Acad. Sci. 85:9138-9142, 1988; and Lichter et al., Proc. Natl. Acad. Sci. 85:9664-9668, 1988. CISH is described in, e.g., Tanner et al., Am. .1. Pathol. 157: 1467-1472, 2000 and U.S. Pat. No. 6,942,970. Additional detection methods are provided in U.S. Pat. No. 6,280,929.

Numerous reagents and detection schemes can be employed in conjunction with FISH, CISH, and SISH procedures to improve sensitivity, resolution, or other desirable properties. As discussed above probes labeled with fluorophores (including fluorescent dyes and QUANTUM DOTS®) can be directly optically detected when performing FISH. Alternatively, the probe can be labeled with a nonfluorescent molecule, such as a hapten (such as the following nonlimiting examples: biotin, digoxigenin, DNP, and various oxazoles, pyrrazoles, thiazoles, nitroaryls, benzofurazans, triterpenes, ureas, thioureas, rotenones, coumarin, courmarin-based compounds, Podophyllotoxin, Podophyllotoxin-based compounds, and combinations thereof), ligand or other indirectly detectable moiety. Probes labeled with such non-fluorescent molecules (and the target nucleic acid sequences to which they bind) can then be detected by contacting the sample (e.g., the cell or tissue sample to which the probe is bound) with a labeled detection reagent, such as an antibody (or receptor, or other specific binding partner) specific for the chosen hapten or ligand. The detection reagent can be labeled with a fluorophore (e.g., QUANTUM DOT®) or with another indirectly detectable moiety, or can be contacted with one or more additional specific binding agents (e.g., secondary or specific antibodies), which can be labeled with a fluorophore.

In other examples, the probe, or specific binding agent (such as an antibody, e.g., a primary antibody, receptor or other binding agent) is labeled with an enzyme that is capable of converting a fluorogenic or chromogenic composition into a detectable fluorescent, colored or otherwise detectable signal (e.g., as in deposition of detectable metal particles in SISH). As indicated above, the enzyme can be attached directly or indirectly via a linker to the relevant probe or detection reagent. Examples of suitable reagents (e.g., binding reagents) and chemistries (e.g., linker and attachment chemistries) are described in U.S. Patent Application Publication Nos. 2006/0246524; 2006/0246523, and 2007/ 01 17153.

It will be appreciated by those of skill in the art that by appropriately selecting labelled probe-specific binding agent pairs, multiplex detection schemes can he produced to facilitate detection of multiple target nucleic acid sequences (e.g., genomic target nucleic acid sequences) in a single assay (e.g., on a single cell or tissue sample or on more than one cell or tissue sample). For example, a first probe that corresponds to a first target sequence can he labelled with a first hapten, such as biotin, while a second probe that corresponds to a second target sequence can be labelled with a second hapten, such as DNP. Following exposure of the sample to the probes, the bound probes can he detected by contacting the sample with a first specific binding agent (in this case avidin labelled with a first fluorophore, for example, a first spectrally distinct QUANTUM DOT®, e.g., that emits at 585 mn) and a second specific binding agent (in this case an anti-DNP antibody, or antibody fragment, labelled with a second fluorophore (for example, a second spectrally distinct QUANTUM DOT®, e.g., that emits at 705 mn). Additional probes/binding agent pairs can he added to the multiplex detection scheme using other spectrally distinct fluorophores. Numerous variations of direct, and indirect (one step, two step or more) can he envisioned, all of which are suitable in the context of the disclosed probes and assays.

Probes typically comprise single-stranded nucleic acids of between 10 to 1000 nucleotides in length, for instance of between 10 and 800, more preferably of between 15 and 700, typically of between 20 and 500. Primers typically are shorter single-stranded nucleic acids, of between 10 to 25 nucleotides in length, designed to perfectly or almost perfectly match a nucleic acid of interest, to be amplified. The probes and primers are “specific” to the nucleic acids they hybridize to, i.e. they preferably hybridize under high stringency hybridization conditions (corresponding to the highest melting temperature Tm, e.g., 50 % formamide, 5x or 6x SCC. SCC is a 0.15 M NaCl, 0.015 M Na-citrate).

The nucleic acid primers or probes used in the above amplification and detection method may be assembled as a kit. Such a kit includes consensus primers and molecular probes. A preferred kit also includes the components necessary to determine if amplification has occurred. The kit may also include, for example, PCR buffers and enzymes; positive control sequences, reaction control primers; and instructions for amplifying and detecting the specific sequences.

In a particular embodiment, the methods of the invention comprise the steps of providing total RNAs extracted from cumulus cells and subjecting the RNAs to amplification and hybridization to specific probes, more particularly by means of a quantitative or semi- quantitative RT-PCR.

In another preferred embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a sample from a test subject, optionally first subjected to a reverse transcription, is labelled and contacted with the microarray in hybridization conditions, leading to the formation of complexes between target nucleic acids that are complementary to probe sequences attached to the microarray surface. The labelled hybridized complexes are then detected and can be quantified or semi-quantified. Labelling may be achieved by various methods, e.g. by using radioactive or fluorescent labelling. Many variants of the microarray hybridization technology are available to the man skilled in the art (see e.g. the review by Hoheisel, Nature Reviews, Genetics, 2006, 7:200-210).

In another embodiment, the expression level is determined by metabolic imaging (see for example Yamashita T et al., Hepatology 2014, 60: 1674-1685 or Ueno A et al., Journal of hepatology 2014, 61 : 1080-1087).

Expression level of a gene may be expressed as absolute expression level or normalized expression level. Typically, expression levels are normalized by correcting the absolute expression level of a gene by comparing its expression to the expression of a gene that is not a relevant for determining the response of antipsychotic treatment, e.g., a housekeeping gene that is constitutively expressed. Suitable genes for normalization include housekeeping genes such as the actin gene ACTB, ribosomal 18S gene, GUSB, PGK1, TFRC, GAPDH, TBP and ABLP This normalization allows the comparison of the expression level in one sample, e.g., a patient sample, to another sample, or between samples from different sources.

Measuring the expression level of the proteins TRIM24 can be performed by a variety of techniques well known in the art. Typically protein expression level may be measured for example by capillary electrophoresis-mass spectroscopy technique (CE-MS), flow cytometry, mass cytometry or ELISA performed on the sample.

In the present application, the “level of protein” or the “protein level expression” means the quantity or concentration of said protein. In still another embodiment, the “level of protein” means the quantitative measurement of the protein expression relative to a negative control. Such methods comprise contacting a sample with a binding partner capable of selectively interacting with proteins present in the sample. The binding partner is generally an antibody that may be polyclonal or monoclonal, preferably monoclonal.

The presence of the protein can be detected using standard electrophoretic and immunodiagnostic techniques, including immunoassays such as competition, direct reaction, or sandwich type assays. Such assays include, but are not limited to, Western blots; agglutination tests; enzyme-labeled and mediated immunoassays, such as ELISAs; biotin/avidin type assays; radioimmunoassays; immunoelectrophoresis; immunoprecipitation, capillary electrophoresismass spectroscopy technique (CE-MS).etc. The reactions generally include revealing labels such as fluorescent, chemioluminescent, radioactive, enzymatic labels or dye molecules, or other methods for detecting the formation of a complex between the antigen and the antibody or antibodies reacted therewith.

The aforementioned assays generally involve separation of unbound protein in a liquid phase from a solid phase support to which antigen-antibody complexes are bound. Solid supports which can be used in the practice of the invention include substrates such as nitrocellulose (e. g., in membrane or microtiter well form); polyvinylchloride (e. g., sheets or microtiter wells); polystyrene latex (e.g., beads or microtiter plates); polyvinylidine fluoride; diazotized paper; nylon membranes; activated beads, magnetically responsive beads, and the like.

More particularly, an ELISA method can be used, wherein the wells of a microtiter plate are coated with a set of antibodies against the proteins to be tested. A sample containing or suspected of containing the marker protein is then added to the coated wells. After a period of incubation sufficient to allow the formation of antibody-antigen complexes, the plate(s) can be washed to remove unbound moieties and a detectably labeled secondary binding molecule is added. The secondary binding molecule is allowed to react with any captured sample marker protein, the plate is washed and the presence of the secondary binding molecule is detected using methods well known in the art.

Particularly, a mass spectrometry-based quantification methods may be used. Mass spectrometry-based quantification methods may be performed using either labelled or unlabelled approaches [DeSouza and Siu, 2012], Mass spectrometry-based quantification methods may be performed using chemical labeling, metabolic labeling or proteolytic labeling. Mass spectrometry-based quantification methods may be performed using mass spectrometry label free quantification, a quantification based on extracted ion chromatogram (EIC) and then profile alignment to determine differential level of polypeptides. Particularly, a mass spectrometry-based quantification method particularly useful can be the use of targeted mass spectrometry methods as selected reaction monitoring (SRM), multiple reaction monitoring (MRM), parallel reaction monitoring (PRM), data independent acquisition (DIA) and sequential window acquisition of all theoretical mass spectra (SWATH) [Moving target Zeliadt N 2014 The Scientist;Liebler Zimmerman Biochemistry 2013 targeted quantitation pf proteins by mass spectrometry; Gallien Domon 2015 Detection and quantification of proteins in clinical samples using high resolution mass spectrometry. Methods v81 pl5-23 ; Sajic, Liu, Aebersold, 2015 Using data-independent, high-resolution mass spectrometry in protein biomarker research: perspectives and clinical applications. Proteomics Clin Appl v9 p 307-21],

Particularly, the mass spectrometry-based quantification method can be the mass cytometry also known as cytometry by time of flight (CYTOF) (Bandura DR, Analytical chemistry, 2009).

Particularly, the mass spectrometry-based quantification is used to do peptide and/or protein profiling can be use with matrix-assisted laser desorption/ionisation time of flight (MALDI-TOF), surface-enhanced laser desorption/ionization time of flight (SELDI-TOF; CLINPROT) and MALDI Biotyper apparatus [Solassol, Jacot, Lhermitte, Boulle, Maudelonde, Mange 2006 Clinical proteomics and mass spectrometry profiling for cancer detection. Journal: Expert Review of Proteomics V3, 13, p311-320 ; FDA K130831],

Methods of the invention may comprise a step consisting of comparing the proteins and fragments concentration in circulating cells with a control value. As used herein, "concentration of protein" refers to an amount or a concentration of a transcription product, for instance the proteins of the invention. Typically, a level of a protein can be expressed as nanograms per microgram of tissue or nanograms per milliliter of a culture medium, for example. Alternatively, relative units can be employed to describe a concentration. In a particular embodiment, "concentration of proteins" may refer to fragments of the proteins of the invention.

The predictive method of the present invention is particularly suitable for predicting the duration of the overall survival (OS), progression-free survival (PFS) and/or the disease-free survival (DFS) of the cancer patient. Those of skill in the art will recognize that OS survival time is generally based on and expressed as the percentage of people who survive a certain type of cancer for a specific amount of time. Cancer statistics often use an overall five-year survival rate. In general, OS rates do not specify whether cancer survivors are still undergoing treatment at five years or if they've become cancer-free (achieved remission). DSF gives more specific information and is the number of people with a particular cancer who achieve remission. Also, progression-free survival (PFS) rates (the number of people who still have cancer, but their disease does not progress) includes people who may have had some success with treatment, but the cancer has not disappeared completely.

Typically, the expression “short survival time” indicates that the patient will have a survival time that will be lower than the median (or mean) observed in the general population of patients suffering from said cancer. When the patient will have a short survival time, it is meant that the patient will have a “poor prognosis”. Inversely, the expression “long survival time” indicates that the patient will have a survival time that will be higher than the median (or mean) observed in the general population of patients suffering from said cancer. When the patient will have a long survival time, it is meant that the patient will have a “good prognosis”.

Accordingly, in other words, the invention relates to an in vitro method for assessing a melanoma patient’s risk of having a poor prognostic of survival comprising the steps of: i) determining in a sample obtained from the patient the level of TRIM24, ii) comparing the level of TRIM24 determined at step i) with a predetermined reference, and iii) concluding that the patient is at high risk of having a bad prognostic of survival when the level of TRIM24 determined at step i) is significantly higher than the predetermined reference value or concluding that the patient is at high risk of having a good prognostic of survival when the level of TRIM24 determined at step i) is significantly higher than the predetermined reference value.

As used herein, the term "risk", in the context of the present invention, relates to the probability that an event will occur over a specific period and can mean a subject's "absolute" risk or "relative" risk. Absolute risk can be measured with reference to either actual observation post-measurement for the relevant time cohort or with reference to index values developed from statistically valid historical cohorts that have been followed for the relevant time period. Relative risk refers to the ratio of a subject's absolute risks compared to the absolute risks of low-risk cohorts or an average population risk, which can vary by how clinical risk factors are assessed. Odds ratios, the proportion of positive events to negative events for a given test result, are also commonly used (odds are according to the formula p/(l-p) where p is the probability of an event and (1- p) is the probability of no event) to no- conversion. "Risk evaluation," or "evaluation of risk" in the context of the present invention, encompasses predicting the probability, odds, or likelihood that an event or disease state may occur, the rate of occurrence of the event or conversion from one disease state to another. Risk evaluation can also comprise the prediction of future clinical parameters, traditional laboratory risk factor values, or other indices of relapse, either in absolute or relative terms in reference to a previously measured population. The methods of the present invention may be used to make continuous or categorical measurements of the risk of conversion, thus diagnosing and defining the risk spectrum of a category of subjects defined as being at risk of conversion. In the categorical scenario, the invention can be used to discriminate between normal and other subject cohorts at higher risk. In some embodiments, the present invention may be used so as to discriminate those at risk from normal.

In some embodiments, the “predetermined reference value” is relative to a number or value derived from population studies, including without limitation, patients of the same or similar age range, patients in the same or similar ethnic group, and patients having the same severity of cancer. Such predetermined reference values can be derived from statistical analyses and/or risk prediction data of populations obtained from mathematical algorithms and computed indices of the disease. Typically, the predetermined reference value is a threshold or cutoff value. Typically, a "threshold value" or "cutoff value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based on the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skill in the art. For example, retrospective measurement in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after determining the level of anti-VASN autoantibodies in a group of reference, one can use algorithmic analysis to statistically treat the levels determined in samples to be tested and thus obtain a classification standard having significance for sample classification. The full name of the ROC curve is the receiver operator characteristic curve, also known as the receiver operation characteristic curve. It is mainly used for clinical and biochemical diagnostic tests. The ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cutoff values (thresholds or critical values, boundary values between normal and abnormal diagnostic test results) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate, and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point with high sensitivity and specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result improves as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used to draw the ROC curve, such as MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER. SAS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

In some embodiments, the predetermined reference value is typically determined by carrying out a method comprising the steps of: a) providing a collection of samples from patients suffering from melanoma; b) providing, for each sample provided at step a), information relating to the actual clinical outcome for the corresponding patient (i.e. the duration of the disease-free survival (DFS) and/or the overall survival (OS)); c) providing a serial of arbitrary quantification values; d) determining the level of the marker of interest (i.e. TRIM24) for each sample contained in the collection provided at step a); e) classifying said samples in two groups for one specific arbitrary quantification value provided at step c), respectively: (i) a first group comprising samples that exhibit a quantification value for level that is lower than the said arbitrary quantification value contained in the said serial of quantification values; (ii) a second group comprising samples that exhibit a quantification value for said level that is higher than the said arbitrary quantification value contained in the said serial of quantification values; whereby two groups of samples are obtained for the said specific quantification value, wherein the samples of each group are separately enumerated; f) calculating the statistical significance between (i) the quantification value obtained at step e) and (ii) the actual clinical outcome of the patients from which samples contained in the first and second groups defined at step f) derive; g) reiterating steps f) and g) until every arbitrary quantification value provided at step d) is tested; h) setting the said predetermined reference value as consisting of the arbitrary quantification value for which the highest statistical significance (most significant) has been calculated at step g)-

For example the level of the marker of interest (i.e. TRIM24 ) has been assessed for 100 samples of 100 patients. The 100 samples are ranked according to the level of the marker of interest (i.e. TRIM24). Sample 1 has the highest level and sample 100 has the lowest level. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding cancer patient, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated. The predetermined reference value is then selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the level of the marker of interest (i.e. TRIM24) corresponding to the boundary between both subsets for which the p value is minimum is considered as the predetermined reference value. It should be noted that the predetermined reference value is not necessarily the median value of levels of the marker of interest (i.e. TRIM24). Thus in some embodiments, the predetermined reference value thus allows discrimination between a poor and a good prognosis with respect to DFS and OS for a patient. Practically, high statistical significance values (e.g. low P values) are generally obtained for a range of successive arbitrary quantification values, and not only for a single arbitrary quantification value. Thus, in one alternative embodiment of the invention, instead of using a definite predetermined reference value, a range of values is provided. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g. lower P value) are retained, so that a range of quantification values is provided. This range of quantification values includes a "cut-off value as described above. For example, according to this specific embodiment of a "cut-off value, the outcome can be determined by comparing the level of the marker of interest (i.e. TRIM24) with the range of values which are identified. In certain embodiments, a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum p value which is found). For example, on a hypothetical scale of 1 to 10, if the ideal cut-off value (the value with the highest statistical significance) is 5, a suitable (exemplary) range may be from 4-6. Therefore, a patient may be assessed by comparing values obtained by measuring the level of the marker of interest (i.e. TRIM24), where values greater than 5 reveal an increased risk of having a poor prognosis and values less than 5 reveal a decreased risk of a poor prognosis. In some embodiments, a patient may be assessed by comparing values obtained by measuring the level of the marker of interest (i.e. TRIM24) and comparing the values on a scale, where values above the range of 4-6 indicate an increased risk having a poor prognosis and values below the range of 4-6 indicate a decreased risk of having a poor prognosis, with values falling within the range of 4-6 indicating an intermediate prognosis.

A further object of the present invention relates to an in vitro method of predicting the risk of relapse in a subject suffering from melanoma i) comprising determining the level of TRIM24 in a sample obtained from the subject ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the subject is at high risk of relapse when the level of TRIM24 determined at step i) is higher than the predetermined reference value.

Typically, high levels of TRIM24 indicate that the subject is at high risk of relapse, whereas low levels of TRIM24 indicate that the subject is at low risk of relapse.

As used herein, the term "relapse" refers to the return of signs and symptoms of a disease after a subject has enjoyed a remission after treatment. Thus, if initially, the target disease is alleviated or healed, or the progression of the disease is halted or slowed down and subsequently, when the disease or one or more characteristics of the disease resume, the subject is referred to as being "relapsed." Typically, the treatment is surgery, immunotherapy, chemotherapy, radiation therapy, MAPK inhibitors (such as MEK inhibitors and B-RAF inhibitors).

In some embodiments, the predictive method of the present invention is performed during the course of treatment, where the quantification of TRIM24 is carried out before, during and as follow-up to a course of therapy. Desirably, therapy targeted to melanoma results in a decrease in the TRIM24 level in the patient’s sample. A further object of the present invention relates to an in vitro method for predicting the response to treatment in a patient suffering from melanoma, comprising the step of determining in a sample obtained from said patient the level of TRIM24, wherein a low level of TRIM24 indicate that the subject is at high risk of achieving a response.

Typically, high levels of TRIM24 indicate that the subject is at low risk of achieving a response, whereas low levels of TRIM24 indicate that the subject is at high risk of achieving a response.

As used herein, the term “high” refers to a measure that is significantly greater than normal, greater than a standard, such as a predetermined reference value or a subgroup measure, or that is relatively greater than another subgroup measure. As used herein, the term “low” refers to a level that is less than normal or less than a standard, such as a predetermined reference value or a subgroup measure that is relatively less than another subgroup level.

In other word, the inventions refers to an in vitro method of predicting whether a subject suffering from melanoma will achieves a response with treatment comprising i) determining the level of TRIM24 in a sample obtained from the subject, ii) comparing the level determined at step i) with the predetermined reference value and iii) concluding that the subject is at high risk of achieving a response when the level determined at step ii) is lower than the level determined at step i) or concluding that the subject is at low risk of achieving a response when the level determined at step ii) is higher than the level determined at step i).

In another words, the invention refers to an in vitro method for predicting the risk of having a resistant melanoma in a subject suffering from melanoma comprising i) determining the level of TRIM24 in a sample obtained from the subject ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the subject is at high risk of having a resistant melanoma when the level of TRIM24 determined at step i) is higher than the predetermined reference value and concluding that the subject is at low risk of having a resistant melanoma when the level of TRIM24 determined at step i) is lower than the predetermined reference value.

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. 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, MEK, NRAS or PTEN. The resistance can be also caused by a double-negative BRAF and NRAS mutation. 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 BRAF mutation or MEK are used to treat the melanoma with NRAS 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. Inactivating mutations and deletions of the PTEN gene are found in many types of cancers, including melanoma. The resistance can be also caused by a triple-negative NRAS, BRAF and MEK mutations as described above. Accordingly, such resistance is against the treatments using an inhibitor of BRAF mutation or an inhibitor of MEK, as described below. In a particular embodiment, the melanoma is resistant to a combined treatment characterized by using an inhibitor of BRAF mutation and an inhibitor of MEK as described below.

In particular embodiment, the treatment is targeted therapy or immune checkpoint therapy.

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.

Thus, in particular embodiment, the invention refers to an in vitro method for predicting the response treatment to BRAF or MEK inhibitors in a patient suffering from melanoma, comprising the step of determining in a sample obtained from said patient the level of TRIM24, wherein a high levels of TRIM24 indicates that the subject is at low risk of achieving a response to BRAF or MEK inhibitors and a low level of TRIM24 indicates that the subj ect is at high risk of achieving a response to BRAF or MEK inhibitors.

As used herein, the term “BRAF” or “B-RAF” 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 survival. 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/K in the context of the invention.

As used herein, the term “inhibitor of BRAF” refers to a natural or synthetic compound that has a biological effect to inhibit the activity or the expression of BRAF. More particularly, such compound by inhibiting BRAF activity reduces cell division, differentiation, and secretion. In a particular embodiment, the inhibitor of BRAF 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. In a particular embodiment, the inhibitor of BRAF is an aptamer. “Aptamers” are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. In a particular embodiment, the inhibitor of BRAF 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. The inhibitors of BRAF mutations are well known in the art. For instance, reviews are published disclosing such BRaf kinase inhibitors (Tsai et al, PNAS February 26, 2008 105 (8) 3041-3046, Garnett et Marais, 2004 Cancer cell, Volume 6, Issue 4, Pages 313-319; Wilmott et al 2012, Cancer Therapy: Clinical, Volume 18, Issue 5; Fujimura et al, Expert Opin Investig Drugs. 2019 Feb;28(2): 143-148, Trojaniello et al, Expert Rev Clin Pharmacol. 2019 Mar;12(3):259-266; Kakadia et al, Onco Targets Ther. 2018 Oct 17; 11:7095- 7107; Roskoski, Pharmacol Res. 2018 Sep;135:239-258; Eroglu et al, Ther Adv Med Oncol. 2016 Jan;8(l):48-56), the disclosure of which being incorporated herein by reference. Patent applications also disclose B-Raf kinase inhibitors, for instance and non-exhaustively WO14164648, WO14164648, WO14206343, W013040515, WO11147764, WO11047238, WO1 1025968, WO11025951, WO11025938, WO11025965, WO11090738, WO09143389, WO091 11280, WO09111279, WO09111278, WO09111277, W008068507, W008020203, WO071 19055, WO07113558, W007071963, WO07113557, W006079791, WO06067446, W006040568, WO06024836, WO06024834, W006003378, WO05123696, the disclosure of which being incorporated herein by reference. In a particular embodiment, the inhibitor of BRAF is Vemurafenib. Vemurafenib also known as PLX4032, RG7204 or RO5185426 and commercialized by Roche as zelboraf. In a particular embodiment, the inhibitor of BRAF is Dabrafenib also known as tafinlar, which is commercialized by Novartis. In a particular embodiment, the melanoma is resistant to a treatment with dacarbazine. Dacarbazine also known as imidazole carboxamide is commercialized as DTIC-Dome by Bayer.

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

The inhibitors of MEK are well known in the art. For instance, reviews are published disclosing such MEK kinase inhibitors (Kakadia et al, Onco Targets Ther. 2018 Oct 17; 11 :7095-7107; Steeb et al, Eur J Cancer. 2018 Nov; 103:41-51; Sarkisian and Davar, Drug Des Devel Ther. 2018 Aug 20;12:2553-2565; Roskoski, Pharmacol Res. 2018 Sep;135:239-258; Eroglu et al, Ther Adv Med Oncol. 2016 Jan;8(l):48-56), the disclosure of which being incorporated herein by reference. Patent applications also disclose MEK kinase inhibitors, for instance and non- exhaustively WO15022662, WO15058589, W014009319, WO14204263, WO13107283, WO13 136249, WO13136254, W012095505, W012059041, WO11047238, WO11047055, WO1 1054828, W010017051, W010108652, WO10121646, WO10145197, WO09129246, W009018238, WO09153554, W009018233, W009013462, W009093008, WO08089459, W007014011, W007044515, W007071951, WO07022529, W007044084, WO07088345, WO07121481, WO07123936, W006011466, W006011466, WO06056427, WO06058752, WO06133417, W005023251, WO05028426, W005051906, W005051300, W005051301, W005051302, WO05023759, W004005284, WO03077855, W003077914, W002069960, WO0168619, W00176570, W00041994, W00042022, W00042003, W00042002, W00056706, W00068201, WO9901426, the disclosure of which being incorporated herein by reference. 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.

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 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, an immune checkpoint inhibitor refers to any compound inhibiting the function of an immune checkpoint protein. The inhibition includes reduction of function and full blockade.

In a particular embodiment, the invention refers to an in vitro method for predicting the response treatment to immune checkpoint inhibitors in a patient suffering from melanoma, comprising the step of determining in a sample obtained from said patient the level of TRIM24, wherein a high levels of TRIM24 indicates that the subject is at low risk of achieving a response to immune checkpoint inhibitors and a low level of TRIM24 indicates that the subject is at high risk of achieving a response to immune checkpoint inhibitors.

In some embodiments, the immune checkpoint inhibitor could be an antibody, synthetic or native sequence peptides, small molecules or aptamers which bind to the immune checkpoint proteins and their ligands. In a particular embodiment, the immune checkpoint inhibitors is an PD1 inhibitor.

In a particular embodiment, the immune checkpoint inhibitor is an antibody. Typically, antibodies are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

In a particular embodiment, the immune checkpoint inhibitor is an anti -PD-1 antibody such as described in WO2011082400, W02006121168, W02015035606, W02004056875, W02010036959, W02009114335, W02010089411, WO2008156712, WO2011110621, WO2014055648 and WO2014194302. Examples of anti-PD-1 antibodies which are commercialized: Nivolumab (Opdivo®, BMS), Pembrolizumab (also called Lambrolizumab, KEYTRUDA® or MK-3475, MERCK).

In some embodiments, the immune checkpoint inhibitor is an anti-PD-Ll antibody such as described in WO2013079174, W02010077634, W02004004771, WO2014195852, W02010036959, WO2011066389, W02007005874, W02015048520, US8617546 and WO2014055897. Examples of anti-PD-Ll antibodies which are on clinical trial: Atezolizumab (MPDL3280A, Genentech/Roche), Durvalumab (AZD9291, AstraZeneca), Avelumab (also known as MSB0010718C, Merck) and BMS-936559 (BMS). In some embodiments, the immune checkpoint inhibitor is an anti-PD-L2 antibody such as described in US7709214, US7432059 and US8552154. In some embodiments, 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. In some embodiments, the immune checkpoint inhibitor is a small organic molecule. Typically, the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA. In a particular embodiment, small organic molecules interfere with transduction pathway of PD-1 and Tim-3. For example, they can interfere with molecules, receptors or enzymes involved in PD-1 and Tim-3 pathway. In a particular embodiment, the small organic molecules interfere with Indoleamine-pyrrole 2,3 -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-CLindoxyl 1,3- diacetate, 9- vinylcarbazole, acemetacin, 5 -bromo-tryptophan, 5 -bromoindoxyl diacetate, 3- Amino-naphtoic acid, pyrrolidine dithiocarbamate, 4-phenylimidazole a brassinin derivative, a thiohydantoin derivative, a P-carboline derivative or a brassilexin derivative. In a particular embodiment, the IDO inhibitor is selected from 1-methyl-tryptophan, P-(3- benzofuranyl)- alanine, 6-nitro-L-tryptophan, 3-Amino-naphtoic acid and P-[3- benzo(b)thienyl] -alanine or a derivative or prodrug thereof. In a particular embodiment, the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to -N-(3- bromo-4-fluorophenyl)-N'-hydroxy-4-{[2-(sulfamoylamino)-ethyl]amino}-l,2,5-oxadiazole-3 carb oximi dami de. 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], In a particular embodiment, the inhibitor is CA-170 (or AUPM- 170): an oral, small molecule immune checkpoint antagonist targeting programmed death ligand-1 (PD-L1) and V-domain Ig suppressor of T cell activation (VISTA) (Liu et al 2015). Preclinical data of CA-170 are presented by Curis Collaborator and Aurigene on November at ACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics. In some embodiments, the immune checkpoint inhibitor is an aptamer. Typically, the aptamers are directed against A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG- 3, TIM-3 or VISTA. In a particular embodiment, aptamers are DNA aptamers such as described in Prodeus et al 2015. A major disadvantage of aptamers as therapeutic entities is their poor pharmacokinetic profiles, as these short DNA strands are rapidly removed from circulation due to renal filtration. Thus, aptamers according to the invention are conjugated to with high molecular weight polymers such as polyethylene glycol (PEG). In a particular embodiment, the aptamer is an anti-PD-1 aptamer. Particularly, the anti-PD-1 aptamer is MP7 pegylated as described in Prodeus et al 2015.

Subject identified as responder to targeted therapy or immune checkpoint inhibitors according to the method of the invention can be treated with a compound selected from the group consisting of BRAF inhibitor, MEK inhibitor or immune checkpoint inhibitor.

Thus, in particular embodiment, the invention refers to a method for treating melanoma in a subject in need thereof comprising i) determining the level of TRIM24 from a sample obtained from the patient, ii) comparing the level determined at step i) with a predetermined reference value and iii) administering to said subject a therapeutically effective amount of a compound selected from the group consisting of BRAF inhibitor, MEK inhibitor or immune checkpoint inhibitor when the level determined at step i) is higher than the predetermined reference value.

In particular embodiment, the compound is BRAF inhibitor.

Method for treating melanoma

The inventors demonstrate that the inhibition of TRIM24 decreased the migratory capacities and increased the sensitivity to BRAF inhibitors in two human melanoma cellular models.

In another aspect, the invention refers to a method for treating melanoma in a subject in need thereof comprising administering a therapeutically effective amount of TRIM24 inhibitors.

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

As used herein, the term “TRIM24 inhibitor” refers to any compound natural or not which is capable of neutralizing, blocking, inhibiting, abrogating, reducing, degrading or interfering with the activities of TRIM24. TRIM24 inhibitors are well known in the art. The term encompasses any TRIM24 inhibitor that is currently known in the art or that will be identified in the future. The term also encompasses inhibitor of expression. The TRIM24 inhibition of the compounds may be determined using various methods well known in the art.

By "biological activity" of TRIM24 is meant regulating a transcriptional signature associated with reprogramming towards a stem-like, invasive state, resistant to treatment, or inducing tumor cell migration and decreasing their sensitivity to targeted therapy and immune checkpoint inhibitor therapy.

Tests for determining the capacity of a compound to be TRIM24 inhibitor are well known to the person skilled in the art. In a preferred embodiment, the inhibitor specifically binds to TRIM24 in a sufficient manner to inhibit the biological activity of TRIM24. Binding to TRIM24 and inhibition of the biological activity of TRIM24 may be determined by any competing assays well known in the art. For example, the assay may consist in determining the ability of the agent to be tested as TRIM24 inhibitor to bind to TRIM24. The binding ability is reflected by the Kd measurement. The term "KD", as used herein, is intended to refer to the dissociation constant, which is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M). KD values for binding biomolecules can be determined using methods well established in the art. In specific embodiments, an antagonist that "specifically binds to TRIM24 is intended to refer to an inhibitor that binds to human TRIM24 polypeptide with a KD of IpM or less, lOOnM or less, lOnM or less, or 3nM or less. Then a competitive assay may be settled to determine the ability of the agent to inhibit biological activity of TRIM24. The functional assays may be envisaged such evaluating the ability to inhibit a) induction of tumor cell migration and/or b) promoting their sensitivity to targeted therapy (i.e BRAF or MEK inhibitors) (see example with anti-TRIM24 shRNA and protac anti-TRIM24).

In some embodiments, the TRIM24 inhibitor is an inhibitor of TRIM24 activity.

Accordingly, the TRIM24 inhibitor may be a molecule that binds to TRIM24 selected from the group consisting of antibodies, aptamers, proteolyse targeting chimeric molecules, molecular glue degraders and polypeptides.

In one embodiment, the TRIM24 activity inhibitor of the invention is an aptamer.

“Aptamers” are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996). Then after raising aptamers directed against TRIM24 of the invention as above described, the skilled man in the art can easily select those inhibiting TRIM24.

In another embodiment, the TRIM24 activity inhibitor of the invention is an antibody (the term including “antibody portion”).

In one embodiment of the antibodies or portions thereof described herein, the antibody is a monoclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a polyclonal antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a humanized antibody. In one embodiment of the antibodies or portions thereof described herein, the antibody is a chimeric antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a light chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a heavy chain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fab portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a F(ab')2 portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fc portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a Fv portion of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises a variable domain of the antibody. In one embodiment of the antibodies or portions thereof described herein, the portion of the antibody comprises one or more CDR domains of the antibody.

As used herein, "antibody" includes both naturally occurring and non-naturally occurring antibodies. Specifically, "antibody" includes polyclonal and monoclonal antibodies, and monovalent and divalent fragments thereof. Furthermore, "antibody" includes chimeric antibodies, wholly synthetic antibodies, single chain antibodies, and fragments thereof. The antibody may be a human or nonhuman antibody. A nonhuman antibody may be humanized by recombinant methods to reduce its immunogenicity in man.

Antibodies are prepared according to conventional methodology. Monoclonal antibodies may be generated using the method of Kohler and Milstein (Nature, 256:495, 1975). To prepare monoclonal antibodies useful in the invention, a mouse or other appropriate host animal is immunized at suitable intervals (e.g., twice-weekly, weekly, twice-monthly or monthly) with antigenic forms of TRIM24. The animal may be administered a final "boost" of antigen within one week of sacrifice. It is often desirable to use an immunologic adjuvant during immunization. Suitable immunologic adjuvants include Freund's complete adjuvant, Freund's incomplete adjuvant, alum, Ribi adjuvant, Hunter's Titermax, saponin adjuvants such as QS21 or Quil A, or CpG-containing immunostimulatory oligonucleotides. Other suitable adjuvants are well-known in the field. The animals may be immunized by subcutaneous, intraperitoneal, intramuscular, intravenous, intranasal or other routes. A given animal may be immunized with multiple forms of the antigen by multiple routes. Briefly, the antigen may be provided as synthetic peptides corresponding to antigenic regions of interest in TRIM24. Following the immunization regimen, lymphocytes are isolated from the spleen, lymph node or other organ of the animal and fused with a suitable myeloma cell line using an agent such as polyethylene glycol to form a hydridoma. Following fusion, cells are placed in media permissive for growth of hybridomas but not the fusion partners using standard methods, as described (Coding, Monoclonal Antibodies: Principles and Practice: Production and Application of Monoclonal Antibodies in Cell Biology, Biochemistry and Immunology, 3rd edition, Academic Press, New York, 1996). Following culture of the hybridomas, cell supernatants are analyzed for the presence of antibodies of the desired specificity, i.e., that selectively bind the antigen. Suitable analytical techniques include ELISA, flow cytometry, immunoprecipitation, and western blotting. Other screening techniques are well-known in the field. Preferred techniques are those that confirm binding of antibodies to conformationally intact, natively folded antigen, such as non-denaturing ELISA, flow cytometry, and immunoprecipitation.

Significantly, as is well-known in the art, only a small portion of an antibody molecule, the paratope, is involved in the binding of the antibody to its epitope (see, in general, Clark, W. R. (1986) The Experimental Foundations of Modern Immunology Wiley & Sons, Inc., New York; Roitt, I. (1991) Essential Immunology, 7th Ed., Blackwell Scientific Publications, Oxford). The Fc' and Fc regions, for example, are effectors of the complement cascade but are not involved in antigen binding. An antibody from which the pFc' region has been enzymatically cleaved, or which has been produced without the pFc' region, designated an F(ab')2 fragment, retains both of the antigen binding sites of an intact antibody. Similarly, an antibody from which the Fc region has been enzymatically cleaved, or which has been produced without the Fc region, designated an Fab fragment, retains one of the antigen binding sites of an intact antibody molecule. Proceeding further, Fab fragments consist of a covalently bound antibody light chain and a portion of the antibody heavy chain denoted Fd. The Fd fragments are the major determinant of antibody specificity (a single Fd fragment may be associated with up to ten different light chains without altering antibody specificity) and Fd fragments retain epitope-binding ability in isolation. Within the antigen-binding portion of an antibody, as is well-known in the art, there are complementarity determining regions (CDRs), which directly interact with the epitope of the antigen, and framework regions (FRs), which maintain the tertiary structure of the paratope (see, in general, Clark, 1986; Roitt, 1991). In both the heavy chain Fd fragment and the light chain of IgG immunoglobulins, there are four framework regions (FR1 through FR4) separated respectively by three complementarity determining regions (CDR1 through CDRS). The CDRs, and in particular the CDRS regions, and more particularly the heavy chain CDRS, are largely responsible for antibody specificity.

It is now well-established in the art that the non CDR regions of a mammalian antibody may be replaced with similar regions of conspecific or heterospecific antibodies while retaining the epitopic specificity of the original antibody. This is most clearly manifested in the development and use of "humanized" antibodies in which non-human CDRs are covalently joined to human FR and/or Fc/pFc' regions to produce a functional antibody. This invention provides in certain embodiments compositions and methods that include humanized forms of antibodies. As used herein, "humanized" describes antibodies wherein some, most or all of the amino acids outside the CDR regions are replaced with corresponding amino acids derived from human immunoglobulin molecules. Methods of humanization include, but are not limited to, those described in U.S. Pat. Nos. 4,816,567, 5,225,539, 5,585,089, 5,693,761, 5,693,762 and 5,859,205, which are hereby incorporated by reference. The above U.S. Pat. Nos. 5,585,089 and 5,693,761, and WO 90/07861 also propose four possible criteria which may used in designing the humanized antibodies. The first proposal was that for an acceptor, use a framework from a particular human immunoglobulin that is unusually homologous to the donor immunoglobulin to be humanized, or use a consensus framework from many human antibodies. The second proposal was that if an amino acid in the framework of the human immunoglobulin is unusual and the donor amino acid at that position is typical for human sequences, then the donor amino acid rather than the acceptor may be selected. The third proposal was that in the positions immediately adjacent to the 3 CDRs in the humanized immunoglobulin chain, the donor amino acid rather than the acceptor amino acid may be selected. The fourth proposal was to use the donor amino acid reside at the framework positions at which the amino acid is predicted to have a side chain atom within 3 A of the CDRs in a three dimensional model of the antibody and is predicted to be capable of interacting with the CDRs. The above methods are merely illustrative of some of the methods that one skilled in the art could employ to make humanized antibodies. One of ordinary skill in the art will be familiar with other methods for antibody humanization. In one embodiment of the humanized forms of the antibodies, some, most or all of the amino acids outside the CDR regions have been replaced with amino acids from human immunoglobulin molecules but where some, most or all amino acids within one or more CDR regions are unchanged. Small additions, deletions, insertions, substitutions or modifications of amino acids are permissible as long as they would not abrogate the ability of the antibody to bind a given antigen. Suitable human immunoglobulin molecules would include IgGl, IgG2, IgG3, IgG4, IgA and IgM molecules. A "humanized" antibody retains a similar antigenic specificity as the original antibody. However, using certain methods of humanization, the affinity and/or specificity of binding of the antibody may be increased using methods of "directed evolution", as described by Wu et al., I. Mol. Biol. 294: 151, 1999, the contents of which are incorporated herein by reference. Fully human monoclonal antibodies also can be prepared by immunizing mice transgenic for large portions of human immunoglobulin heavy and light chain loci. See, e.g., U.S. Pat. Nos. 5,591,669, 5,598,369, 5,545,806, 5,545,807, 6,150,584, and references cited therein, the contents of which are incorporated herein by reference. These animals have been genetically modified such that there is a functional deletion in the production of endogenous (e.g., murine) antibodies. The animals are further modified to contain all or a portion of the human germ-line immunoglobulin gene locus such that immunization of these animals will result in the production of fully human antibodies to the antigen of interest. Following immunization of these mice (e.g., XenoMouse (Abgenix), HuMAb mice (Medarex/GenPharm)), monoclonal antibodies can be prepared according to standard hybridoma technology. These monoclonal antibodies will have human immunoglobulin amino acid sequences and therefore will not provoke human anti-mouse antibody (KAMA) responses when administered to humans. In vitro methods also exist for producing human antibodies. These include phage display technology (U.S. Pat. Nos. 5,565,332 and 5,573,905) and in vitro stimulation of human B cells (U.S. Pat. Nos. 5,229,275 and 5,567,610). The contents of these patents are incorporated herein by reference. Thus, as will be apparent to one of ordinary skill in the art, the present invention also provides for F(ab') 2 Fab, Fv and Fd fragments; chimeric antibodies in which the Fc and/or FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric F(ab')2 fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; chimeric Fab fragment antibodies in which the FR and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences; and chimeric Fd fragment antibodies in which the FR and/or CDR1 and/or CDR2 regions have been replaced by homologous human or non-human sequences. The present invention also includes so-called single chain antibodies. The various antibody molecules and fragments may derive from any of the commonly known immunoglobulin classes, including but not limited to IgA, secretory IgA, IgE, IgG and IgM. IgG subclasses are also well known to those in the art and include but are not limited to human IgGl, IgG2, IgG3 and IgG4. 1

In another embodiment, 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. The term “VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH. The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation. VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pi chia) (see for example US 6,838,254).

In one embodiment, the TRIM24 activity inhibitor of the invention is a proteolyse targeting chimeric molecule or a molecular glue degrader. As used herein, the term “proteolyse targeting chimeric molecules”, also known as “PROTAC”, has its general meaning in the art and refers to a heterobifunctional molecule composed of two active domains and a linker, capable of removing specific unwanted proteins. PROTAC works by bringing together the E3 ligase with the target protein thus allowing its ubiquitination and degradation by the proteasome. PROTAC as well known in the art as described Gechijian et al, 2018. As used herein, the term “molecular glue degraders” has its general meaning in the art and refers to monovalent compounds that orchestrate interactions between a target protein and an E3 ubiquitin ligase, prompting the proteasomal degradation of the former.

In one embodiment, the TRIM24 activity inhibitor is IACS-9571 (CAS No. : 1800477- 30-8)

In one embodiment, the PROTAC is dTRIM24 (CAS No. : 2170695-14-2)

In one embodiment, the inhibitor of the invention is a TRIM24 expression inhibitor.

TRIM24 expression inhibitor for use in the present invention may be based on antisense oligonucleotide constructs. Anti-sense oligonucleotides, including anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of TRIM24 mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of TRIM24 proteins, 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 encoding TRIM24 can be synthesized, e.g., by conventional phosphodiester techniques and administered by e.g., intravenous injection or infusion. Methods for using antisense techniques for specifically alleviating 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).

Small inhibitory RNAs (siRNAs) or short hairpin RNAs (shRNAs) can also function as TRIM24 expression inhibitor for use in the present invention. TRIM24 gene expression can be reduced by contacting the subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that TRIM24 expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01/36646, WO 99/32619, and WO 01/68836). Examples of said siRNA against TRIM24 include, but are not limited to, those described in Wang P, Shen N, Liu D, Ning X, Wu D, Huang X. TRIM24 siRNA induced cell apoptosis and reduced cell viability in human nasopharyngeal carcinoma cells. Mol Med Rep. 2018.

Examples of said shRNA against TRIM24 include, but are not limited to, shRNA supplied by Abbexa® under reference abx955805, shRNA supplied by Origene® under reference tl308650

Inhibitors of TRIM24 gene expression according to the present invention may be based nuclease therapy (like Talen or Crispr).

The term “nuclease” or “endonuclease” means synthetic nucleases consisting of a DNA binding site, a linker, and a cleavage module derived from a restriction endonuclease which are used for gene targeting efforts. The synthetic nucleases according to the invention exhibit increased preference and specificity to bipartite or tripartite DNA target sites comprising DNA binding (i.e. TALEN or CRISPR recognition site(s)) and restriction endonuclease target site while cleaving at off-target sites comprising only the restriction endonuclease target site is prevented. The guide RNA (gRNA) sequences direct the nuclease (i.e. Cas9 protein) to induce a site-specific double strand break (DSB) in the genomic DNA in the target sequence. Restriction endonucleases (also called restriction enzymes) as referred to herein in accordance with the present invention are capable of recognizing and cleaving a DNA molecule at a specific DNA cleavage site between predefined nucleotides. In contrast, some endonucleases such as for example Fokl comprise a cleavage domain that cleaves the DNA unspecifically at a certain position regardless of the nucleotides present at this position. Therefore, preferably the specific DNA cleavage site and the DNA recognition site of the restriction endonuclease are identical. Moreover, also preferably the cleavage domain of the chimeric nuclease is derived from a restriction endonuclease with reduced DNA binding and/or reduced catalytic activity when compared to the wildtype restriction endonuclease. According to the knowledge that restriction endonucleases, particularly type II restriction endonucleases, bind as a homodimer to DNA regularly, the chimeric nucleases as referred to herein may be related to homodimerization of two restriction endonuclease subunits. Preferably, in accordance with the present invention the cleavage modules referred to herein have a reduced capability of forming homodimers in the absence of the DNA recognition site, thereby preventing unspecific DNA binding. Therefore, a functional homodimer is only formed upon recruitment of chimeric nucleases monomers to the specific DNA recognition sites. Preferably, the restriction endonuclease from which the cleavage module of the chimeric nuclease is derived is a type IIP restriction endonuclease. The preferably palindromic DNA recognition sites of these restriction endonucleases consist of at least four or up to eight contiguous nucleotides. Preferably, the type IIP restriction endonucleases cleave the DNA within the recognition site which occurs rather frequently in the genome, or immediately adjacent thereto, and have no or a reduced star activity. The type IIP restriction endonucleases as referred to herein are preferably selected from the group consisting of: Pvull, EcoRV, BamHl, Bcnl, BfaSORF1835P, Bfil, Bgll, Bglll, BpuJl, Bse6341, BsoBl, BspD6I, BstYl, CfrlOl, Ecll8kl, EcoO1091, EcoRl, EcoRll, EcoRV, EcoR1241, EcoR12411, HinPl l, Hindi, Hindlll, Hpy991, Hpyl881, Mspl, Muni, Mval, Nael, NgoMIV, Notl, OkrAl, Pabl, Pad, PspGl, Sau3 Al, Sdal, Sfil, SgrAl, Thai, VvuYORF266P, Ddel, Eco571, Haelll, Hhall, Hindll, and Ndel.

Examples of said CRISPR guide RNA against PDGF-A include, but are not limited to, CRISPR Kit supplied by Adgene® under reference BRDN0001146855, and CRISPR Kit supplied by Origene® under reference KN405332.

Ribozymes can also function as TRIM24 expression inhibitor for use in the present invention. “Ribozymes” are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of TRIM24 mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

Both antisense oligonucleotides and ribozymes useful as TRIM24 expression inhibitor can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

Antisense oligonucleotides siRNAs, shRNA and ribozymes 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 or ribozyme nucleic acid to the cells and preferably cells expressing TRIM24. Preferably, 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 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 rouse 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.

Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in KRIEGLER (A Laboratory Manual," W.H. Freeman C.O., New York, 1990) and in MURRY ("Methods in Molecular Biology," vol.7, Humana Press, Inc., Cliffton, N.J., 1991). Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wildtype adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno-associated virus can also function in an extrachromosomal fashion.

Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g., SANBROOK et al., "Molecular Cloning: A Laboratory Manual," Second Edition, Cold Spring Harbor Laboratory Press, 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigenencoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC/CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intravenous, intramuscular, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencap sul ati on .

As used herein, the term "therapeutically effective amount" of the TRIM24 inhibitor as above described is meant a sufficient amount to provide a therapeutic effect. It will be understood, however, 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 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 coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

Subject identified with a poor prognosis or resistant melanoma according to the invention can be treated with TRIM24 inhibitor.

Thus, in a particular embodiment, the subject has been identified with a poor prognosis or resistant melanoma according to the invention,

Thus, in particular embodiment, the invention refers to a method for treating melanoma in a subject in need thereof comprising i) determining the level of TRIM24 from a sample obtained from the patient, ii) comparing the level determined at step i) with a predetermined reference value and iii) administering to said subject a therapeutically effective amount of TRIM24 inhibitor when the level determined at step i) is higher than the predetermined reference value.

In a particular embodiment, the TRIM24 inhibitors restores the response to a compound selected from the group consisting of BRAF inhibitor, MEK inhibitor or immune checkpoint inhibitor. In a particular embodiment, the melanoma is a resistant melanoma, and more particularly resistant to a treatment with a compound selected from the group consisting of BRAF inhibitor, MEK inhibitors or an immune checkpoint inhibitor.

Thus, in a particular embodiment, the TRIM24 inhibitor is administered in combination with a compound selected in the group consisting from BRAF inhibitor, MEK inhibitor and immune checkpoint inhibitor.

Accordingly, the invention refers to a method of treating melanoma in a subject in need thereof comprising administering to the subject a therapeutically effective combination comprising a TRIM24 inhibitor and a compound selected in the group consisting from BRAF inhibitor, MEK inhibitor and immune checkpoint inhibitor.

In other words, the inventions refers to a method delaying and/or preventing development of a melanoma resistant in a subject comprising administering to the subject a therapeutically effective amount of a compound selected in the group consisting from BRAF inhibitor, MEK inhibitor and immune checkpoint inhibitor in combination with a TRIM24 inhibitor.

In particular embodiment, the melanoma is resistant to a treatment with BRAF inhibitor, MEK inhibitors or an immune checkpoint inhibitor.

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. In the context of the invention, the melanoma is resistant to a combined treatment characterized by using an inhibitor of BRAF mutation and an inhibitor of MEK as described above. For example, the combined treatment may be a combination of Vemurafenib and Cotellic.

Typically, the TRIM24 inhibitor of the present invention is combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are 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.

Thus the present invention also relates to a pharmaceutical composition comprising a TRIM24 inhibitor according to the invention and a pharmaceutically acceptable carrier for use in the treatment of melanoma.

In a particular embodiment, the melanoma is a resistant melanoma. In a particular embodiment, the resistant melanoma is resistant to a BRAF inhibitor, MEK inhibitor or immune checkpoint inhibitor.

In a particular embodiment, the pharmaceutical composition further comprises a compound selected in the group consisting from BRAF inhibitor, MEK inhibitor or immune checkpoint inhibitor.

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.

FIGURE:

Figure 1. TRIM24 knock-down induces differentiation of melanoma cells. A. Frequency of genetic alterations on the TRIM24 locus in the TCGA Firehose Legacy skin cutaneous melanoma cohort. B. Relative frequency of primary tumors and metastasis according to TRIM24 levels. High and low TRIM24 levels were defined relatively to the median RNAseq expression in the TCGA Firehose Legacy skin cutaneous melanoma cohort (Chi-2 test was performed). C and D. Validation of TRIM24 exctinction upon shRNA-mediated knock-down in the A375 and the C.09-10 cells by qPCR. (n=3, Mean +/- SEM are plotted on the graphs. Student’s t-test was performed). E and F. Volcano plot representation of gene expression changes in RNAseq comparing TRIM24 knock-down to control cells in C.09-10 (E.) or A375 (F.). Genes with p. adj-value < 0.05 were considered differentially expressed. Genes with p. adjvalue < 0.001 and |log(fold change)| > 0.58 were considered for signature analyses. 3 independent samples were sequenced for each condition. G and H. GeneSet Enrichment Analyses (GSEA) of melanoma-specific gene signatures applied to differentially expressed genes upon TRIM24 knock-down in C.09-10 (F.) or A375 (G.). All listed pathways have p. adjvalue < 0.05. Articles of origin for each signature are specified between parentheses. I. qPCR analyses of relative gene expression changes upon TRIM24 knock-down in A375 cells. Gene expression was normalized to GAPDH expression. (n=4, Mean +/- SEM are represented on the graphs. Student’s t-test was performed for each gene.). J. Western Blot analyses of gene expression changes upon TRIM24 knock-down in A375 cells. (n=3)

Figure 2. TRIM24 knock-down inhibits functional properties associated with invasive melanoma cell states. A. Wound healing assays were performed to measure the migration of TRIM24 knock-down cells in A375. Cell division was impaired by mitomycin C treatment prior to experiment. Live-imaging of the cells was performed with an Incucyte device. (n=3, Mean +/- SEM are plotted on the graph. Student’s t-test was performed on the endpoint values.). B. Transwell migration quantified the migration of A375 cells upon TRIM24 knockdown. The cells were submitted to a FB S gradient to stimulate migration through the membrane. Quantification was performed using the ImageJ software. (n=3, Student’s t-test was performed.). C and D. IC50 to PLX-4032 was calculated for C.09-10 (G.) and A375 (I.) cells upon TRIM24 knock-down. IC50 values were calculated using GraphPad Prism 10. (n=3, Mean +/- SEM are plotted on the graphs. Student’s t-test was performed.). E and F. Sensitivity of C.09-10 (H.) and A375 (J.) cells to PLX-4032 was assessed by live-cell imaging using an Incucyte device. Cells were incubated with or without the drug, in presence of propidium iodide. Fluorescent dead cells were quantified using the Incucyte companion software. (n=3 for C.09-10, n=2 for A375. Mean +/- SEM are plotted on the graphs.)

Figure 3. High TRIM24 protein expression is associated with a somber prognosis for melanoma patients. A. Schematic representation of the sample processing pipeline. B. TRIM24 protein expression in the tumor and the stroma on FFPE sections, as defined by tissue segmentation algorithms on the inform software. (n=52. Paired Wilcoxon’s test was performed.). C. Distribution of Responder and Non-responder patients to anti-PDl immunotherapy according to their TRIM24 status. (n=52, Chi-2 test was performed). D. Survival analysis of the stage III melanoma patients according to their TRIM24 status. (n=52, Log-Rank test was performed).

Figure 4. TRIM24-associated gene signature is preferentially expressed by dedifferentiated cells in public melanoma datasets. A. Expression of the TRIM24 signature in the melanoma cell lines described in Tsoi et al., 2018 according to their differentiation phenotype. (Mean +/- SEM are plotted on the graph. One-way ANOVA and Tukey’s multiple comparisons test were performed.). B. Expression of the TRIM24 signature in the mouse melanoma single-cell RNAseq dataset published by Karras et al., 2022 according to their differentiation phenotype, (each point represents a tumor cell.). C. Expression of the TRIM24 signature in the human melanoma single-cell RNAseq dataset published by Pozniak et al., 2023 according to their differentiation phenotype. D. Expression of the TRIM24 signature in the human melanoma single-cell RNAseq dataset published by Pozniak et al., 2023 according to the patient’s response to anti-PDl immunotherapy, (each point represents a tumor cell.)

Figure 5. TRIM24 targeting resensitizes melanoma cells to BRAF inhibitors. A.Western Blot analyses showing TRIM24 and TRIM28 protein levels upon treatment with increased doses of dTRIM24 in C.09-10 (A) and A375 (B). (n=2). B. Western Blot analyses showing TRIM24 protein levels upon treatment with dTRIM24 in 25F4 and N6.1 murine cell lines (n=l). C. IC50 to PLX-4032 was calculated for A375 and GOKA cells upon treatment with dTRIM24. IC50 values were calculated using GraphPad Prism 10. (n=l, Mean +/- SEM are plotted on the graphs.). D. Evaluation of dTRIM24 efficiency in vivo by oral administration. A375 cells were grafted in nude mice, and TRIM24 level in tumors was assessed by immunochemistry after 6 administrations.

Figure 6. TRIM24 binds to promoter and enhancer regions associated with specific histone patterns. A. Localization of TRIM24 peaks in the genome in A375 cells as defined by TRIM24 CUT & Tag analysis. B. Enrichment of melanoma cell phenotype signatures on genes associated to a TRIM24 peak in A375 cells.

Figure 7. TRIM24 knock-down impairs tumor growth, remodels the immune infiltrate and resensitizes to immunotherapy in immunocompetent mice. A. Hallmark pathways enriched in H3K4me3 Cut & Tag data in A375 cells upon TRIM24 knock-down. B. Tumor growth after injection of N.6.1 shCTL (black) and shTRIM24 (green) cells in C57BL6/J immunocompetent mice. The mean tumor volume and standard deviation are plotted. n=9 mice per group. C. Boxplot showing the percentage of CD4+ lymphocytes (among CD45+ cells) in TRIM24-knock-down tumors compared to control. D. Total CD45+ infiltration among total tumor cells. P values were determined by a Student’s t-test at final timepoint (B), and Mann- Whitney test (C). Differences were considered statistically significant at *P < 0.05, **P < 0.01 and ***P < 0.001. ns (non-significant) means P > 0.05.

Figure 8. TRIM24 mediates activating and repressive histone marks remodelling. Localization of H3K27me3 marks in A375 cells (left) and its distance to TSS (right).

Figure 9. TRIM24 localisation to chromatin is associated with major epigenetic and transcriptomic alterations. Localisation of TRIM24 peaks in the genome in C.09-10 cells as defined by TRIM24 Cut & Tag analysis.

MATERIAL AND METHODS

Human tumor samples immunofluorescence analyses

Melanoma tumor samples were obtained through the Biological Resource Center of the Lyon Sud Hospital (Hospices Civils de Lyon) and were used with the patient’s written informed consent. This study was approved by a regional review board (Comite de Protection des Personnes He de France XI, Saint-Germain-en-Laye, France, number 12027) and is registered in ClinicalTrial.gov (MelBase, NCT02828202). 52 primary cutaneous melanoma patients were used for multi-immunofluorescence analyses (Supplementary Table 1).

3-pm tissue sections were cut from formalin-fixed paraffin-embedded human melanoma specimens. The sections underwent immunofluorescence staining using the OPAL™ technology (Akoya Biosciences) on a Leica Bond RX. DAPI was used for nuclei detection. TRIM24 (HPA043495, RRID: AB 10962915, Sigma-Aldrich), SOXIO (sc-365692, RRID: AB_1084400, Santa Cruz). Sections were digitized with a Vectra Polaris scanner (Perkin Elmer, USA). Using the Inform software (Perkin Elmer), an autofluorescence treatment of images was carried out and tissue segmentation was performed to identify epidermis, stroma and tumor. Cell segmentation was then applied to analyze the expression of each marker in each cell. The matrix of phenotype containing the X- and Y- positions of each cell as well as the mean nuclear intensities of each fluorescence staining was then further analyzed using the R software. Tumors were spatially reconstructed using the R plot() function.

Cell culture and treatments

The A375 human melanoma cell line was purchased from ATCC and cultured in DMEM complemented with 10% fetal bovine serum (FBS) (Cambrex) and 100 U/ml penicillinstreptomycin (Invitrogen). In order to authenticate the cell lines, the expected major genetic alterations were verified by NGS sequencing. The absence of Mycoplasma contamination was verified every 3 weeks with the MycoAlert detection kit (Lonza). Previously described patient- derived C-09.10 cells, established from BRAFV600 metastatic melanoma, was grown in RPMI complemented with 10% FBS and 100 U/ml penicillin-streptomycin (Durand et al., 2024). Previously described BrafV600-mutated Br25F4 and NRasQ61 -mutated NR6.1 mouse melanoma cell lines were cultured in RPMI 1640 Glutamax (61870044, Life Technologies) complemented with 10% FBS (Cambrex) and 100 U/mL penicillin-streptomycin (15140148, Gibco) (Plaschka et al., 2022). The BRAF inhibitor PLX4032/vemurafenib was purchased from Selleck Chemicals (Houston, TX, USA) and reconstituted in DMSO.

Viral infections

For shRNA-Trim24 knock-down in human (A375 and C-09.10 cells) and murine (Br25F4 and NR6.1) melanoma cell lines, human embryonic kidney 293T cells (4 x 106) were transfected with lentiviral expression constructs (10 pg) in combination with GAG-POL (5 pg) and ENV expression vectors (10 pg). The constructs allowed the insertion of shRNA targeting TRIM24 in a lentiviral plasmid vector pLKO. l-puro (MISSION, Sigma) for human (SHCLNG- NM_003852) and mouse (SHCLNG-NM_145076) or a non-target shRNA control plasmid (SHC016-1EA). Viral supernatants were collected 48 h post-transfection, filtered (0.45 pm membrane), and placed in contact with 2 x 106 melanoma cells for 8 h in the presence of 8 pg/mL polybrene. The cells were selected in the presence of puromycin (1 pg/mL) (Invitrogen) 48h post infection.

Immunoblot analyses

Cells were washed twice with phosphate buffered saline (PBS) containing CaC12 and then lysed in a 100 mM NaCl, 1% NP40, 0.1% SDS, 50 mM Tris pH 8.0 RIPA buffer supplemented with a complete protease inhibitor cocktail (Roche, Mannheim, Germany) and phosphatase inhibitors (Sigma-Aldrich). A Trim24 antibody was used (Sigma-Aldrich, HPA04349). Loading was controlled using anti-GAPDH (Sigma- Aldrich, Cat# ABS16 (RRID: AB 10806772). Horseradish peroxidase-conjugated goat anti-rabbit polyclonal antibodies (Glostrup) were used as secondary antibodies. Western blot detections were conducted using the Luminol reagent (Santa Cruz). Western Blot Digital Imaging was performed with the ChemiDoc™ MP Imager (Bio-Rad).

RT-Q-PCR

Total RNA was isolated using the RNeasy Kit (QIAGEN) and reverse-transcribed using a high cDNA capacity reverse transcription kit (Maxima First Strand cDNA synthesis Kit, Thermoscientific) following the manufacturer's instructions using 1000 ng of RNA as a reverse transcription template in a 20 pL final volume. The samples were incubated for 10 minutes at 25°C, followed by 15 minutes at 50°C and 5 minutes at 85°C in T 100 Thermal Cycler (1861096, Bio-Rad). Real-time qPCR reactions were performed using OneGreen® FAST qPCR Premix (OZYA008-200XL, Ozyme) according to the manufacturers protocol. Reactions were done using 15ng of cDNA template and IpM of each primer. All reactions, including no-template controls and RT controls were performed in triplicate on an Azure Ciel real Time PCR (Ozyme) pre-denaturation 3 minutes at 95°C then 40 cycles at 95°C for 5s followed by 30s at 60°C. Results were analyzed with the Azure Cielo manager software. Human GAPDH was used for normalization. Exon-spanning probes were designed using the primer-blast (NCBI).

Wound healing assays

Wound healing assays were performed using incucyte assay (Sartorius). Briefly, A375 cells were trypsinized and seeded at 60000 cells per well in a 96 well-plate. The day after, the cells were treated with 10 pg/ml of mitomycin and 2h after the scratch module was used to create the wound. EssenBioScience IncuCyte Zoom System was used to acquire images and measure the wound in real-time every 2 h for 48h.

Rezasurin and Incucyte viability assays

Viability was measured using rezasurin assays. 500 A375 and 2000 C.09-10 cells were plated per well in 96-well plates. Cells were treated for 7 days and the medium was renewed 3 times a week with indicated PLX4032 concentration (Selleckchem) and the same DMSO volume for control. The relative viability was assessed by measuring resorufin fluorescence on the ClarioSTAR plate reader (BMG LABTECH).

For incucyte assays, 2 000 A375 and 20 000 C.09-10 cells were seeded onto a 24-well plate. After 24 h, cell medium was renewed with indicated treatments, as well as PLX4032 and propidium iodide (Sigma-Aldrich, 1/3000). The EssenBioScience IncuCyte Zoom Live-Cell Analysis System was used to measure and analyze real-time cell mortality every 2 h. Dead cells were marked with propidium iodide. Data were then converted into prism in order to draw graphs.

Sphere formation assay

Sphere formation assay was performed by seeding 5, 10 and 20 A375 cells per well of a 96 low adherence culture plate in DMEM-F12 medium supplemented with FGF7 (5 ng/mL, Peprotech), FGF10 (20 ng/mL, Peprotech), FGF10 (20 ng/mL, Peprotech), and IX B27 (Gibco). The medium was renewed two times a week. The sphere were allowed to grow for 3 weeks and images were acquired with EssenBioScience IncuCyte Zoom Live-Cell Analysis System to determine the number of well containing a melanoma sphere.

RNA-seq analyses

RNA libraries were prepared with the TrueSeq poly-A+ kit from Illumina and sequenced on the genomic platform of the CRCL, on an Illumina NovaSeq 6000 sequencing machine with a paired-end protocol (2x75bp, 32Mp reads). Raw sequencing reads were aligned on the human genome (GRCh38) with STAR (v2.7.8a), with the annotation of known genes from gencode v37. RNA quality control metrics were computed using RSeQC (v4.0.0). Gene expression was quantified with Salmon (1.4.0) on the raw sequencing reads, using the annotation of protein coding genes from gencode v37 as index.

Starting from raw counts, principal component analyses were completed with the R package ade- selecting only the top 10% most variant genes (defined by the 10%-trimmed variance) as input data. Differential expression analyses were performed through the R package DESeq2 (vl.34.0), using Wald test, sequencing batches correction and apeglm shrinkage estimator (vl.14.0). Heatmaps were generated with the R package ComplexeHeatmap (v2.10.0). Unsupervised hierarchical clustering was performed with Euclidean distance metric and Ward.D2 clustering algorithm. Gene Set Enrichment Analyses (GSEAs) were carried out using fgsea R package (vl.20.0) and gene lists were pre-ranked using Signal2Noise metric. Single sample GSEA (ssGSEA) scores were computed on TPM normalized data through gsva R package.

TCGA data of melanoma tumors were analysed and retrieved from cbioportal.

Bulk RNA-seq data of melanoma cell lines from (Tsoi et al., 2018) were retrieved from GEO, with accession number GSE80829. Single-cell RNAseq data of melanoma cell lines from (Wouters et al., 2020) were retrieved from GEO (GSE134432) and from (Pozniak et al., 2024) and (Karras et al., 2022) from the KU Leuven Research Data Repository. Single-cell RNAseq data were analysed and visualized using Seurat (4.3.0) and SCpubr (1.1.2) packages.

CUT&Tag analyses

CUT&Tag assays were carried out using iDeal CUT&Tag Kit for Histones for chromatin profiling (Diagenode) following manufacturer’s recommendations. Each condition was performed on 150 000 cells. Chromatin fragments were immunoprecipitated with antibodies directed against TRIM24 (1 pg, BETHYL-A300-815) or IgG (1 pg, Bio-Rad, PRABP01, RRID: AB321631) as negative control. Antibodies targeting H3K4me3 (C15200152), H3K27me3 (Cl 5200181-50), H3K23Ac (Cl 5410344), H3K27Ac (Cl 5200184-50), H3K4un (C15200149), H3K9Me (C15410193) were all CUT&Tag validated and acquired from Diagenode. Immunoprecipitated DNA was purified and dissolved into 25 pl of DNA elution buffer. DNA libraries were prepared with the Diagenode MicroPlex Library Preparation Kit v2, and sequenced on an Illumina Novaseq sequencing machine (paired-end protocol, 75bp, 80 M reads) on the genomic platform of the CRCL. The library was verified using Bioanalyzer (Agilent). Two biological replicates from two independent experiments were performed for each CUT&Tag condition.

For the analysis, after adapter trimming using Trimgalore, Fastq files were aligned with BWA to the human reference genome GRCh38. Reads were then filtered out in order to avoid blacklisted regions (form ENCODE), duplicates, unmapped, multiple locations, > 4 mismatches, insert size > 2kb, different chromosomes and other than FR orientation mappings. Normalized BigWig (scaled to 1 million mapped reads) were generated and peaking calling was performed with MACS2 for TRIM24, H3K4me3 and K27ac marks (v2.2.7.1) (Zhang et al., 2008) and EPIC2 (v.0.0.52) (Stovner and Ssetrom, 2019) was used for H3K4un, H3K9me3, H3K23ac and H3K27me3 marks, independently on each sample, considering IgG immunoprecipitation as control.

Genomic localization of called peaks was performed through assignChromosomeRegion function from ChIPpeakAnno R package (v3.28.1). Distance to closest TSS was defined using annotatePeaklnBatch function with the output set as "nearesll.ocalion". Finally, peak-to-gene assignment was conducted through the annotatePeaklnBatch function using the following options: output="overlapping", bindingRegion=c(-1000, 500), FeatureLocForDistance="TSS", select="all". Annotation data were obtained from TxDb.Hsapiens.UCSC.hg38.knownGene (v3.14.0) using "transcript" as feature. Motif enrichment analysis was conducted using fmdMotifsGenome function, from HOMER software (v4.11.1) (Heinz et al., 2010). The motifs were searched 400-bp regions centered on each peak summit. Read density heatmaps and clustering analysis were performed using Profileplyr package.

All analyses and statistical tests were carried out with the R software (v4.1.0) and plots were generated with ggplot2 (v3.4.3). All statistical tests were two-tailed and p-values were corrected, when indicated, with the Benjamini -Hochberg method. Enrichment of lists of genes in specific biological pathways were tested using clusterProfiler (v4.2.2) and msgidbr (v7.5.1) R packages. Pathway lists originated from MSigDB (Molecular Signatures Database) Hallmark (H) gene sets (Liberzon et al., 2015) together with previously published melanoma signatures. ATAC-seq

ATAC-seq was performed using ATAC-seq kit for open chromatin assessment kit (Diagenode) following manufacturer’s instructions. Each condition was performed in duplicate using 1.5 x 105 cells as an input.

Mouse injections

Experiments using mice were performed in accordance with the animal care guidelines of the European Union and French laws and were validated by the local Animal Ethic Evaluation Committee and the French MESRI (APAFIS #43229; APAFIS #46228; APAFIS #38245). Mice were housed and bred in a specific pathogen-free animal facility “ AniCan” at the CRCL, Lyon, France. Single cell suspensions of Br25F4 and NR6.1 cell models (1-3 x 106 cells), in PBS/Matrigel (BD Biosciences, Oxford, UK) (1 : 1) were injected subcutaneously into the flank of six-week-old male C57BL/6J mice (Charles River laboratories). 1-3 x 106 A375 cells were injected subcutaneously into the flank of six-week-old female Swiss Nude (Charles River laboratories). n=5 mice were included in each experimental group, in separate cages. No randomization was performed. Tumor growth was monitored for 2-6 weeks post-injection. Tumors grew up to 1.5 cm in diameter, at which point animals were euthanized. For anti-PD-1 treatment, 5 days after injection, mice were treated with intra-peritoneal injection of 200 pg of anti-PD-1 rat anti -mouse PD-1 clone RMP1-14 (BP0146, Bio X Cell) or with the control isotype 3 times every 2 days.

IHC staining analyses of mouse tumor samples

Tumors were embedded in paraffin and TRIM24 staining was performed using the anti- TRIM24 antibody (Sigma-Aldrich, HPA04349) and purple chromogen (for heavily pigmented tumors) detection and counterstaining with hematoxylin. Images were digitalized with a 3DHistech Pannoramic SCAN2 scanner on the Research Pathology Platform. Quantification was done with HALO™ Image Analysis Software (Indica Labs).

Mouse tumors sample immunophenotyping using flow cytometry

Tumors were dissociated, digested 20 minutes at 37°C in a digestion medium composed of DNasel type II (D4527, 10 pg/mL, Sigma), Collagenase A (COLLA-RO 11088793001, 2 mg/mL, Sigma) in RPMI complemented with 2% FBS and filtered using MACS SmartStrainer 70 pm (Miltenyi). 1.106 cells per condition were stained with a 30-marker spectral flowcytometry panel characterizing the whole immune infiltrate by the cytometry platform (TCRgd, Ly6C, C38, CD3, FoxP3, CD4, IgD, IA-IE, EpCAM, SiglecH, IgM, Siglec F, CD62L, CD45, CD103, CD49b, CD64, CD44, XCR1, CD25, CD127, CD172a, CD27, CDl lc, CDl lb, CD8, Ly6G, B220, CD 19, Viability). After staining, the labeling acquisition was performed on the Cytek AURORA spectral Flow Cytometer. Quality control (QC) procedures were carried out in accordance with the manufacturer's recommendations utilizing "spectroflo" beads lot 2004. The data were cleaned with PeacoQC algorithm (Emmaneel et al., 2022) and not aligned because acquired during the same day. Data were analyzed using FlowJo VlO.lO.O software.

Data availability

The data reported in this paper will be deposited in the Gene Expression Omnibus (GEO) database.

Statistical analyses

To ensure adequate power and decreased estimation error, we performed multiple independent repeats and experiments were conducted at least in triplicate. Data are presented as mean ±s.d. or ± s.e.m as specified in the figure legends. Statistical analyses were performed using GraphPad Prism 10 software (GraphPad Software, Inc., San Diego, USA) or R software (v4.1.0) and plots were generated with ggplot2 (v3.3.5). All statistical tests were two-tailed and p-values were corrected, when indicated, with the Benjamini -Hochberg method. Unpaired Student’s /-tests were used to compare the means of two groups. To determine significant differences between two groups, student’ s t tests or Mann Whitney tests were used as indicated in the figure legends.

EXAMPLE 1 :

Results:

Utilizing an in silico approach and comprehensive literature curation, we identified TRIM24/TIFla as a frequently altered and amplified epigenetic regulator in melanoma (Figure 1A). TRIM24 encodes for the tripartite RING/B-Box/Coil-coiled transcriptional coactivator, which interacts with specific histone marks, thus modulating gene expression by recruiting coactivators. Although TRIM24 has been demonstrated to interact with nuclear receptors (AR, ERa) and other transcription factors (STAT3) to promote sternness and progression in carcinomas, its function in the context of melanoma remains largely unknown. In the TCGA cutaneous melanoma cohort, we observed a strong correlation between elevated TRIM24 expression and metastatic disease (Figure IB). We conducted shRNA-mediated TRIM24 knockdown experiments in vitro using two melanoma cell lines: the C.09-10 and the A375, respectively harbouring a melanocytic and a neural-crest-like phenotype. The inhibition of TRIM24 in C.09-10 and A375 cell lines (Figure 1C&1D) led to major transcriptomic alteration in melanoma cells (Figure 1E&1F) characterized by the induction of a differentiation program with an elevated melanocytic signature and the repression of both undifferentiated and neural- crest-like signatures (Figure 1G&H). We confirmed this TRIM24-mediated cell identity remodelling effect on specific melanoma cell state markers at both RNA and protein level (Figure 1H&1I). Notably, TRIM24 inhibition resulted in the upregulation of differentiation markers MITF and SOXIO while concurrently reducing the expression of the undifferentiated and neural-crest-like markers NGFR, SOX9 and ZEB 1.

The TRIM24-mediated global transcriptomic alterations were further associated with modifications in melanoma cell properties. TRIM24 knockdown led to decreased in migration capacities in migratory A375 melanoma cells, as evidenced by both wound healing and transwell assays (Figure 2A&2B). Morevover, in both A375 and C.09-10 cell lines, TRIM24- inhibited cells exhibited increased mortality when treated with the small molecule BRAF inhibitor (PLX4032) with both rezasurin and incucyte assays. The IC50 values were nearly twofold reduced in TRIM24 inhibited cells when treated with BRAF inhibitor (Figure 2C&2D). Subsequently, we investigated the expression of TRIM24 using spatial multiimmunofluorescence stains in a stage 3 melanoma cohort treated with adjuvant anti-PDl immunotherapy (n=52) (Figure 3A). We examined the expression of TRIM24 and measured SOXIO expression to label tumor cells. We performed whole slide image analyses using the inForm software, revealing higher levels of TRIM24 protein in the tumor compared to the stroma (Figure 3B). By stratifying the patients in TRIM24high (top 25%) or TRIM241ow (bottom 75%), we found that and elevated TRIM24 protein level is associated with an adverse outcome to PD-1 therapy in the cohort with most responders displaying a TRIM241ow status (Figure 3C). Furthermore, TRIM24 expression was also linked to survival in this study with TRIM24high patients presenting a statistically significant twofold worse relapse-free survival probability (Figure 3D).

We then defined the melanoma-specific TRIM24 signature by selecting the intersection of the genes differentially expressed in RNAseq data in TRIM24 knock-downed C.09-10 and A375 (data not shown). We used this signature of genes to explore published melanoma datasets and further confirm the role of TRIM24 in melanoma. In RNAseq data from melanoma cell lines with characterized phenotype (Tsoi et al, 2018), the TRIM24 signature was found to be more highly expressed in undifferentiated and neural-crest-like cell lines compared to transitory and melanocytic ones (Fig 4A). Similar results were observed when investigating single-cell transcriptomic data, with TRIM24 being highly expressed in undifferentiated and neural-crest subpopulations in a murine melanoma model (Karras et al., 2022) (Fig 4B). Additionally, the TRIM24 signature was upregulated in the mesenchymal and neural-crest-like subpopulations of human metastatic melanoma tumors (Pozniak et al.) (Fig 4C).

Interestingly, the TRIM24 signature is upregulated in patient samples which are resistant for immune checkpoint blockade in the same dataset (Fig 4D).

Finally, we investigated the effect of dTRIM24, a proteolysis targeting chimera (PROTAC) molecule which ubiquitinylates TRIM24 and induces its proteasome-mediated degradation (Gechijian et al, 2018). The treatment of C.09-10 and A375 cells with dTRIM24 induced a dose-dependent degradation of TRIM24 with no degradation of its homolog TRIM28, as assessed by Western Blot (Figure 5A&5B). The efficiency of murine TRIM24 protein degradation was also assessed in murine melanoma cell line (Figure 5B). Furthermore, we confirmed the efficacy of dTRIM24 in degrading TRIM24 protein in the BRAF inhibitorresistant melanoma cell line GOKA (Figure 5C). Treating the A375 and GOKA melanoma cells with 5 pM dTRIM24 prior to BRAFi treatment increased their sensitivity to targeted therapy (Figure 5C). The A375 cells, which display a basal sensitivity to BRAFi, have a BRAFi IC50 decrease comparable to our shRNA-mediated knock-down when pretreated with dTRIM24. Meanwhile the GOKA cells, which are resistant to BRAFi at baseline, were fully resensitized to BRAFi when concurrently treated with dTRIM24. Moreover, we demonstrate the efficacy of TRIM24 PROTAC-mediated degradation in vivo with oral administration of dTRIM24 in nude mice which results in a decrease of TRIM24 protein level assessed by IHC after subcutaneous injection of A375 cells (Figure 5D).

EXAMPLE 2 :

Results

Next to characterize the epigenome reconfiguration associated with TRIM24-mediated transcriptome reprogramming, the pattern of histone modifications was analyzed by CUT&Tag for both active (trimethyl-H3K4) (Data not shown) and repressive (trimethyl-H3K27) (Data not shown) histone marks in A375 and C.09-10 cells (Figure 8 & Data not shown). Interestingly, TRIM24 knockdown induced an opposite modification of these two histone marks, with respectively increased H3K4me3 and decreased H3K27me3 signals. Consistent with RNA-seq results, H3K4me3 marks gained after TRIM24 knock-down were associated with genes related to the proliferative/melanocytic signature, whereas decreased H3K4me3 marks were observed on genes related to the invasive and NCL phenotypes (Data not shown). Enrichment of the repressive H3K27me3 mark was observed at proliferative/melanocytic genes in the shCTL condition (Data not shown) and associated with the invasive signature in the shTRIM24 condition (Data not shown). Specifically investigating histone marks at TRIM24- mediated differentially expressed genes (DEGs) confirmed the link between TRIM24-induced transcriptomic alterations and histone marks remodeling. Within the genes unregulated upon TRIM24 knock-down, a majority (55%) did not show any significant H3K4me3 variation. Importantly, 22% of these genes showed a gain in H3K4me3 mark upon TRIM24 knock-down. Interestingly, although found on fewer genes, the repressive H3K27me3 mark was specifically present in only 0, 1% of these genes in the shTRIM24 condition compared to 2,5% in the control condition.

Overall, we demonstrate that TRIM24-knock-down induces a transcriptional reprogramming from a NCL- and invasive program towards a melanocytic/proliferative programs, associated with H3K4me3 and H3K27me3 histone marks remodeling.

To further elucidate TRIM24 mechanisms of action at the chromatin level, CUT&Tag analyses were performed in the A375 (Figures 6) and C-09,10 (Figure 9 & Data not shown) cell lines with a TRIM24 antibody. A total of 7080 binding peaks were evidenced in A375 and 3675 in C-09, 10 (Figure 9), The majority of TRIM24 binding peaks were located in promoter regions (52%), centered on the transcriptional start site (Figure 6A, 9 & Data not shown). Of note, 38% of TRIM24-occupied sites were observed in introns and intergenic regions. When looking at promotors-associated peaks, most of the TRIM24 peaks observed in C.09-10 were also present in A375 (n=1976), still 49% and 17% of the peaks were specific to A375 and C.09- 10 cells, respectively (Data not shown). TRIM24 was bound to genes regulating melanoma cell phenotype and was preferentially bound to invasive genes in A375 (Figure 6B) and to proliferative genes in C.09-10 (Data not shown), most likely reflecting their different phenotype, invasive/NCL and melanocytic, respectively.

We next performed additional CUT&Tag analyses for histone marks described in the literature as associated with TRIM24 binding, namely unmethylated H3K4 (H3K4un) and acetylated-H3K23 (H3K23ac) (Tsai et al., 2010), as well as histones marks well-known for governing gene expression, H3K27ac (enhancers) and H3K9me3 (heterochromatin) (Talbert and Henikoff, 2021), We also analyzed chromatin accessibility by ATAC-seq and integrated these data altogether. We compared the binding profile of TRIM24 with the profiles of the various histone marks mentioned above (Data not shown). First, TRIM24 presented a binding profile highly similar to H3K23ac, and was also located to accessible chromatin regions as defined by ATAC-seq signal. However, in contrast to previous studies (Tsai et al., 2010), TRIM24 binding did not correlate with H3K4un profile in our models, this mark being rather associated with heterochromatin regions, exhibiting enriched H3K9me3 signal (cluster 1), Importantly, TRIM24 binding was not only observed in gene promotors close to the TSS with high levels of H3K4me3 marks, but also in enhancers regions, in introns, and in more distal intergenic regions presenting an enriched H3K27ac3 signal. Altogether, we precisely defined the binding pattern of TRIM24 protein, which is located on H3K23ac histones marks at accessible regions of both gene promotors and distal enhancers.

We next focused on the epigenetic remodelling of genes that are deregulated upon TRIM24 knock-down. By focusing on gene promotors where TRIM24 is physically located, we observed that 39% of these genes had a significantly altered RNA expression upon TRIM24 knock-down, with equal distribution between up and down-regulated genes, 19% being up- and 20% being down-regulated (Data not shown). Specifically, TRIM24 is located at H7VX45 and FIGNL2 loci, which are the most down- and up-regulated genes upon TRIM24 knock-down, respectively (Data not shown). Consistently, TRIM24 knock-down was associated with a gain of the repressive mark H3K27me3 at ANXA5 locus, while a gain of the activating H3K4me3 mark combined with a loss of H3K27me3 mark was observed at the FIGNL2 locus. These data suggest that TRIM24 may participate in both transcriptional activation and repression.

Due to the fact that TRIM24 recognizes histone marks and not DNA sequences, we hypothesized that its effect on transcription might differ depending on the recruited coactivators and/or co-repressors. To identify transcription factors that may cooperate with TRIM24 to regulate gene expression, we performed DNA-binding motifs enrichment analysis on TRIM24-occupied sites with HOMER. Motif enrichment analysis evidenced an enrichment for the AP-1 complex when considering a 200 bp region centered on the TRIM24 peaks (Data not shown) in both models. Activator Protein-1 (AP-1) complex members JUN and FOS are major regulators of the mesenchymal state (Verfaillie et al„ 2015), Interestingly, when inspecting separately the motif enriched in the regions bound by TRIM24 in the up-regulated and down-regulated genes upon TRIM24 knock-down, while the AP-1 complex motif remained the most enriched in the unregulated genes, the motifs enriched in the down-regulated genes were altered and rather highlighted the presence of SP1-2-5 and KLF3-6 binding motifs. These are members of the Specificity protein/Kruppel-like factor (Sp/KLF) family which are deregulated in cancer and melanoma (Tetreault et al., 2013; Huh et al„ 2010), This observation therefore suggests a selective recruitment of co-activators or co-repressors with TRIM24 in this context. Altogether, integrative transcriptomic, epigenomic and TRIM24 binding analyses allowed us to define the critical role of TRIM24 in controlling melanoma cell phenotype. TRIM24 activates NCL- and invasive genes while repressing melanocytic/proliferative genes through direct localization on gene promotors, in turn inducing alteration of the surrounding epigenetic landscape and transcriptomic activity.

The enrichment in TRIM24 expression/signature in IT-resistant melanoma patients prompted us to investigate the putative role of TRIM24 in regulating immune evasion and response to IT, Of note, in parallel to melanoma phenotype switching signatures, hallmark pathway enrichment analyses in H3K4me3 CUT&Tag data highlighted the activation of IFN alpha/gamma response signatures upon TRIM24 knockdown (Figure 7 A). We thus evaluated the role of TRIM24 in regulating endogenous immune response by monitoring tumor growth in melanoma mouse models deficient for TRIM24, To do so, we established TRIM24-knocked- down cells in two syngeneic mouse models, that we already described as responding or not to anti-PDl (N6, 1 and Br25f4 respectively) (Plaschka et al., 2022), Decreased growth of TRIM24- knocked-down cells was observed upon subcutaneous grafting in immunocompetent C57BL6/J mice (Figure 7B), TRIM24 knock-down was confirmed in the tumors at the protein level by H4C (Data not shown). We then performed a deep characterization of immune cell composition by spectral flow cytometry and demonstrated that despite similar total CD45+ cells infiltration (Figure 7D), TRIM24 knocked-down tumors presented an increased proportion of T lymphocytes, more specifically CD4+ effector lymphocytes, suggesting an increased immune response upon TRIM24-knock-down (Figure 7C & Data not shown). These data suggest that high TRIM24 expression may foster immune evasion at least in part by decreasing CD4+ T cells activity. Finally, we evaluated the putative synergistic effect of TRIM24-knock-down in combination with anti-PDl (Data not shown). Our results suggest that TRIM24 depletion in tumor cells may further potentiate the efficacy of anti-PDl in immunocompetent mice, as compared to monotherapy, by increasing the probability of survival.

Conclusion

Altogether, these results show that TRIM24 orchestrates the plasticity of melanoma cells and governs an undifferentiated/neural-crest-like phenotype. An elevated expression of TRIM24 is associated with increased invasiveness and resistance to targeted therapies in melanoma. Our study reveals a greater TRIM24 expression in the tumor compared to surrounding tissues, and high expression of TRIM24 is associated with unfavorable prognoses and a negative response to therapy in melanoma patients. Our results firmly establish TRIM24 as a robust marker of aggressiveness in melanoma. Furthermore, we successfully and specifically degraded TRIM24 in melanoma cells which increases the sensitivity to BRAFi and restore responsiveness in resistant cell line, positioning TRIM24 as a promising targetable protein for melanoma treatment in combination with the current therapies available.

Our integrative analyses of RNA-seq, ATAC-seq and CUT&Tag data (for TRIM24 and histone marks) allowed to decipher how TRIM24 transcriptional complex epigenetically reprograms melanoma cells. We show that TRIM24, which is a non-DNA binding epigenetic regulator, reads out the local acetylation state of H3K23, but not H3K4un, in chromatin accessible regions. TRIM24 is physically located in gene promoters and more distal enhancers and is significantly enriched in the vicinity of genes regulating melanoma cell phenotype. Additional regulation of super-enhancers remains to be analyzed (Mendelson et al., 2024). TRIM24 recruitment induces an epigenetic remodelling and exerts a dual effect on gene transcription, with concomitant enhancement of a NCL/invasive gene program and repression of the melanocytic/proliferative gene program. This epigenetic and transcriptomic reprogramming finally induces a switch in melanoma cell properties.

TRIM24 has been described as an oncogenic transcriptional activator of the androgen receptor (AR) in prostate cancer (Groner et al., 2016) while it acts as a co-repressor complex that suppresses murine hepatocellular carcinoma (Herquel et al., 2011), highlighting cell-type specific functions that may rely on differential interactions with specific TF and co-factors, either co-activators and/or repressors. DNA-binding motifs enrichment analysis on TRIM24- occupied sites identified a co-localisation with the well-known API complex, suggesting a putative cooperative effect. While TRIM24 has been shown to interact with nuclear receptors (AR, ERa) (Tsai et al., 2010; Groner et al., 2016), STAT3 (Lv et al., 2017) or p53 (Allton et al., 2009; Isbel et al., 2023) in carcinoma models, our data suggest that it could potentially colocalize and cooperate with other transcription factors (such as API or the Spl/KLF family) in a melanoma-specific context. Furthermore, the motifs enriched in the vicinity of TRIM24 binding seem modified depending on whether the genes are down- or up-regulated by the latter, suggesting that the protein-protein interaction domain of TRIM24 might recruit different coactivators or co-repressors.

Our data additionally support the role of TRIM24 in fostering immune evasion, by inducing a remodelling of the immune microenvironment as previously described for other epigenetic regulators (Benboubker et al., 2022). Although our data in mouse models currently highlight a preferential impact on T cells, namely decreased CD4+ lymphocytes (Borst et al., 2018) in TRIM24high tumors, a putative involvement of eosinophils or other myeloid cells cannot be excluded and should be further characterized, both in mouse models and human samples. Regarding TRIM24-mediated molecular mechanisms of immune escape, a more in- depth investigation of the interferon pathways activation should be carried out in the future. Indeed, our data are reminiscent of previously described functions of TRIM24 in retroviral restriction and antiviral defense (Herquel et al., 2013). TRIM24 complexes were shown to mediate endogenous retrovirus (ERV) repression: a derepression of VL30 class of retrotransposons was observed in Trim24-knockout murine hepatocytes leading to activation of interferon response. We essentially focused our study on promoter regions, more specifically related to melanoma phenotype switching, but our data show that about 17% of TRIM24 binding peaks are located in intergenic regions. A more precise investigation of these regions, notably looking for ERV should be performed in the future.

Overall, our results firmly establish TRIM24 as a robust marker of aggressiveness and resistance to immunotherapy in melanoma, representing a promising druggable target for melanoma treatment in combination with the current therapies available. We provide the proof- of-concept that TRIM24 knock-down may resensitize tumors to immune checkpoint blockade. In this respect, a proteolysis targeting chimera (PROTAC) molecule, which ubiquitinylates TRIM24 and induces its proteasome-mediated degradation, dTRIM24, was developed (Gechijian et al., 2018), that will be evaluated in combination with immunotherapy in our melanoma models.

Claims

CLAIMS:

1. An in vitro method for predicting the survival time of a patient suffering from melanoma comprising i) determining the level of Tripartite motif-containing 24 (TRIM24) from a sample obtained from the patient, ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the patient will have a short survival time when the level determined at step i) is higher than the predetermined reference value or concluding that the patient will have a long survival time when the level determined at step i) is lower than the predetermined reference value.

2. An in vitro method of predicting the risk of relapse in a subj ect suffering from melanoma i) comprising determining the level of TRIM24 in a sample obtained from the subject ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the subject is at high risk of relapse when the level of TRIM24 determined at step i) is higher than the predetermined reference value.

3. An in vitro method for predicting the response to treatment in a patient suffering from melanoma, comprising the step of determining in a sample obtained from said patient the level of TRIM24, wherein a low level of TRIM24 indicates that the subject is at high risk of achieving a response and a high level of TRIM24 indicates that the subject is at high risk of not achieving a response.

4. The method according to claim 3, wherein the treatment is BRAF inhibitors, MEK inhibitors or immune checkpoint inhibitors.

5. The method according to claim 4, wherein the immune checkpoint inhibitor is anti-PDl inhibitors.

6. An in vitro method for predicting the risk of having a resistant melanoma in a subject suffering from melanoma comprising i) determining the level of TRIM24 in a sample obtained from the subject ii) comparing the level determined at step i) with a predetermined reference value and iii) concluding that the subject is at high risk of having a resistant melanoma when the level of TRIM24 determined at step i) is higher than the predetermined reference value and concluding that the subject is at low risk of having a resistant melanoma when the level of TRIM24 determined at step i) is lower than the predetermined reference value.

7. A method for treating melanoma in a subject in need thereof comprising administering a therapeutically effective amount of TRIM24 inhibitors.

8. The method according to claim 7, wherein the TRIM24 inhibitors is i) an inhibitor of TRIM24 activity or an inhibitor of TRIM24 expression.

9. The method according to claim 8, wherein the TRIM24 inhibitor is a proteolyse targeting chimeric molecule (PROTAC).

10. The method according to claim 7 to 9, wherein the melanoma is a resistant melanoma.

11. The method according to claim 10, wherein the melanoma is resistant to BRAF inhibitors, MEK inhibitors or immune checkpoint inhibitors.

12. The method according to claim 7 to 11, wherein the TRIM24 inhibitor is administered in combination with a compound selected in the group consisting from BRAF inhibitor, MEK inhibitor and immune checkpoint inhibitor.

13. The method according to claim 7 to 12, wherein the subject has been identified with a poor prognosis according to claim 1, or at high risk of having a resistant melanoma according to 3 to 6.

14. A pharmaceutical composition comprising a TRIM24 inhibitor according to the invention and a pharmaceutically acceptable carrier for use in the treatment of melanoma.