COMBINATION THERAPY FOR MELANOMA

INTRODUCTION

The present invention relates to the field of oncology. More particularly, the present invention relates to a new combination therapy for use in treating melanoma.

Melanoma, a highly aggressive cancer that develops from pigment-producing cells known as melanocytes, is the most dangerous and deadly type of skin cancer. It has become a major public health concern due to its incidence steadily rising over recent decades.

Melanoma patients are typically treated either by surgery at early stages, or by a combination of surgery, chemotherapy, targeted therapy and/or immunotherapy once the cancer has disseminated. Yet, the therapeutic rate is not satisfactory across patients, especially for those with distant metastasis. While detection of the disease in early stage may be curable (in the USA, the five-year survival rate after treatment is 99% for localized disease), later-stage disease that has spread has a relatively poor prognosis, with a median survival of less than 10 months for the most advanced melanoma (in the USA, the five-year survival rate after treatment is 65% when spread to lymph nodes, and only 25% with distant spread). Even if targeted therapy and immunotherapy have improved the overall survival of melanoma patients, some patients still fail to respond or become rapidly resistant to therapy.

There is thus still a need for new therapeutic options and combinations therapies for treating melanoma that act independently of current therapies, so as to notably increase the pool of concerned patients.

Without being bound by theory, the lack of response or emerging resistance to treatment of melanoma is thought to be due to the highly dynamic nature and intra-tumor heterogeneity of this cancer. Melanoma tumors are indeed notoriously heterogeneous comprising cell populations with distinct properties and gene expression signatures. For example, BRAF and NRAS mutations, which activate the mitogen-activated protein kinase (MAPK) pathway, are frequently observed mutations in melanoma patients, yet therapies targeting these particular mutations have either proved to be toxic and/or leading to a relapse after rapid tumor regression.

Molecular characterisation of melanoma subpopulations is therefore needed to facilitate the design of more sophisticated combinatorial approaches to reduce heterogeneity and improve therapeutic response. Although rare, some vulnerabilities common to most melanoma cell states have been identified and successfully exploited to overcome therapy resistance, with a clear example being inhibition of mitochondrial function that impacts viability of multiple melanoma states irrespective of driver mutations leading to long lasting antitumor response.

Long non-coding (Lnc)RNAs, a form of non-coding RNA over 200 nucleotides in length that have low protein-coding potential, have recently emerged as important regulators of virtually every process in the cell, in particular adaptive processes involved in tumor progression and therapy resistance. Among these IncRNAs, LINC00518 (also known as LENOX) and LINC00520 have been identified as weakly expressed in healthy melanocytes yet highly upregulated in all known melanoma states (but not in other human tumors); their use as suitable diagnostic markers for melanoma and/or as suitable targets for treatment of melanoma, irrespective of phenotype or mutation status, has been reported (W02021/152005; Luan et al. Journal of Experimental & Clinical Cancer Research 2020, 39:96; Huang et al. BMC Pulm Med,2C2\, 21 :287).

However, there is a constant need of new therapeutic strategy for the treatment of melanoma.

SUMMARY OF THE INVENTION

The present Inventors are herein the first to investigate an innovative therapeutic approach aimed at targeting the two melanoma-specific IncRNAs (i) LINC00520 and (ii) LINC00518, by knocking-down their respective functional expression. To do so, the inventors assessed the efficacy of this proof-of-concept in melanoma cells that are either melanocytic or undifferentiated (mesenchymal), with different mutational status (e.g. BRAF or RNAS), using an antisense targeting LINC00520 in combination with an antisense targeting LINC00518. Testing these two types of cells is important since those that are melanocytic are typically involved in the proliferative aspect of melanoma, while those of mesenchymal phenotype are generally responsible for the invasion and resistance to therapy.

This combination therapy unexpectedly produced a synergistic improvement, thereby confirming the suitability of this approach for treating melanoma: indeed, the co-inhibition of LINC00520 and LINC00518 cooperates to induce the apoptosis and reduce the proliferation of melanoma cells, irrespective of their phenotype or mutational status (e.g. BRAF or RNAS). These results are all the more surprising given that such effects were obtained with relatively low doses of each antisense. It should nevertheless be understood that the combination of (i) any inhibitor of the functional expression of LINC00520 with (ii) any inhibitor of the functional expression of LINC00518, would achieve the same effect. The Inventors further discovered that the mechanism of action of LINC00520 underlying the promotion of melanoma cell proliferation and metastasis is not via the miR-125b-5p/ EIF5A2 pathway as alleged in the art (Luan et al. Journal of Experimental & Clinical Cancer Research 2020, 39:96), but rather via the RNA helicase DHX36.

The Inventors further discovered that co-inhibition of the functional expression of LINC00520 and of the mitogen-activated protein kinase (MAPK) pathway can effectively promote melanoma cell death in a synergistic manner, notably in melanocytic melanoma cells that are sensitive to MAPK inhibition. Similarly, to co-inhibition of LINC00520 and LINC00518, this combination therapy can allow the administration of lower doses of each inhibitor.

Thus, in a first aspect, the invention relates to (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520 for use in combination with (ii) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00518, in the treatment of a melanoma.

In a preferred embodiment, the inhibitors (each and/or in combination) increase the apoptosis and/or decrease the proliferation of melanoma cells.

In a preferred embodiment, the inhibitors (each and/or in combination) increase apoptosis and/or decrease the proliferation of melanoma cells, independent of BRAF and/or NRAS status.

In a preferred embodiment, the melanoma is an advanced melanoma or a metastatic melanoma.

In a particular embodiment, the melanoma is a melanocytic melanoma. In a particular aspect, the melanoma is a cutaneous or ocular (e.g. uveal) melanoma, especially a cutaneous or ocular (e.g. uveal) melanocytic melanoma.

In a preferred embodiment, the melanoma is a resistant melanoma, in particular a melanoma resistant to chemotherapy, targeted therapy, and/or immune checkpoint inhibitors.

In a preferred embodiment, each inhibitor is either a nucleic acid molecule interfering specifically with the expression of the long-non coding RNA, or is a genome editing system comprising a nuclease engineered to target specifically the long-non coding RNA.

In a preferred embodiment, the nucleic acid molecule interfering specifically with the expression of the long-non coding RNA is an antisense nucleic acid, an RNAi nucleic acid, or a ribozyme, preferably is an antisense nucleic acid.

In a preferred embodiment, the antisense nucleic interfering specifically with the expression of the long-non coding RNA induces a RNAse H mediated degradation. In a preferred embodiment, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA is a gapmer.

In a preferred embodiment, the antisense is an LNA antisense and/or comprises phosphothiorate linkages, preferably the gapmer is an LNA gapmer and/or comprises phosphothiorate linkages.

In a preferred embodiment, the genome editing system comprising a nuclease engineered to target specifically the long-non coding RNA is the CRISPR/Cas system, the Zinc-finger nuclease (ZFN) system, the TALEN system, or the meganuclease system, preferably is the CRISPR/Cas system such as the CRISPR/Cas9 system.

In a further aspect, the invention relates to (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520 and (ii) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00518, as described herein, as a combined preparation for simultaneous, separate or sequential use in the treatment of a melanoma.

In a further aspect, the invention relates to a pharmaceutical composition comprising (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520 and (ii) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00518, as described herein, and optionally (iii) a pharmaceutically acceptable excipient.

In a further aspect, the invention relates to an in vitro method for identifying a melanoma tumor suitable for treatment with the inhibitors of the invention, said method comprising: a) determining the respective expression level of the long-non coding RNAs (IncRNAs) LINC00520 and LINC00518, in a melanoma tumor sample; b) comparing the expression level determined in step a) with a reference expression level for each of said IncRNA, thereby identifying whether the melanoma tumor is suitable for said treatment.

In a further aspect, the invention relates to a kit for use in the in vitro method for identifying a melanoma tumor suitable for treatment with the inhibitors of the invention, said kit comprising: a) at least one reagent capable of specifically determining the expression level of the long-non coding RNA (IncRNA) LINC00520; and b) at least one reagent capable of specifically determining the expression level of the long-non coding RNA (IncRNA) LINC00518; c) optionally, instructions for performing said method. In a further aspect, the invention relates to (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520 for use in combination with (ii) an inhibitor of the mitogen-activated protein kinase (MAPK) pathway, in the treatment of a melanoma.

In a preferred embodiment, the inhibitors (each and/or in combination) increase the apoptosis and/or decrease the proliferation of melanoma cells.

In a preferred embodiment, the inhibitors (each and/or in combination) increase apoptosis and/or decrease the proliferation of melanoma cells, independent of BRAF and/or NBAS status.

In a preferred embodiment, the melanoma is an advanced melanoma or a metastatic melanoma.

In a particular embodiment, the melanoma is a melanocytic melanoma. In a particular aspect, the melanoma is a cutaneous or ocular (e.g. uveal) melanoma, especially a cutaneous or ocular (e.g. uveal) melanocytic melanoma.

In a preferred embodiment, the melanoma is a resistant melanoma, in particular a melanoma resistant to chemotherapy, targeted therapy, and/or immune checkpoint inhibitors.

In a preferred embodiment, the melanoma is not resistant to targeted inhibition of the mitogen-activated protein kinase (MAPK) pathway, especially not resistant to a BRAF inhibitor, a MEK inhibitor or any combination thereof.

In a preferred embodiment, the (i) inhibitor of the long-non coding RNA (IncRNA) LINC00520 is either a nucleic acid molecule interfering specifically with the expression of the long-non coding RNA, or is a genome editing system comprising a nuclease engineered to target specifically the long-non coding RNA.

In a preferred embodiment, the nucleic acid molecule interfering specifically with the expression of the long-non coding RNA is an antisense nucleic acid, an RNAi nucleic acid, or a ribozyme, preferably is an antisense nucleic acid.

In a preferred embodiment, the antisense nucleic interfering specifically with the expression of the long-non coding RNA induces a RNAse H mediated degradation.

In a preferred embodiment, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA is a gapmer.

In a preferred embodiment, the antisense is an LNA antisense and/or comprises phosphothiorate linkages, preferably the gapmer is an LNA gapmer and/or comprises phosphothiorate linkages.

In a preferred embodiment, the genome editing system comprising a nuclease engineered to target specifically the long-non coding RNA is the CRISPR/Cas system, the Zinc-finger nuclease (ZFN) system, the TALEN system, or the meganuclease system, preferably is the CRISPR/Cas system such as the CRISPR/Cas9 system. In a preferred embodiment, the (ii) inhibitor of the mitogen-activated protein kinase (MAPK) pathway is a BRAF inhibitor, a MEK inhibitor, a C-Kit inhibitor, or any combination thereof; preferably is a BRAF inhibitor, a MEK inhibitor or a combination thereof.

In a preferred embodiment, the BRAF inhibitor is dabrafenib, vemurafenib, or encorafenib, or any combination thereof.

In a preferred embodiment, the MEK inhibitor is trametinib, cobimetinib, binimetinib, selumetinib, or any combination thereof.

In a preferred embodiment, the MAPK inhibitor is a combination of BRAF inhibitor and a MEK inhibitor, such as a combination of dabrafenib and trametinib.

In a further aspect, the invention relates to (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520 and (ii) an inhibitor of the mitogen-activated protein kinase (MAPK) pathway, as described herein, as a combined preparation for simultaneous, separate or sequential use in the treatment of a melanoma.

In a further aspect, the invention relates to a pharmaceutical composition comprising (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520 and (ii) an inhibitor of the mitogen-activated protein kinase (MAPK) pathway, as described herein, and optionally (iii) a pharmaceutically acceptable excipient.

In a further aspect, the invention relates to an in vitro method for identifying a melanoma tumor suitable for treatment with the inhibitors of the invention, said method comprising: a) determining, in a melanoma tumor sample, (i) the expression level of the long-non coding RNA (IncRNA) LINC00520, and (ii) the response to an inhibitor of the mitogen-activated protein kinase (MAPK) pathway; b) comparing the expression level determined in step a) with a reference expression level for said IncRNA, and comparing the response determined in step a) with a reference response to said inhibitor, thereby identifying whether the melanoma tumor is suitable for said treatment.

In a further aspect, the invention relates to a kit for use in the in vitro method for identifying a melanoma tumor suitable for treatment with the inhibitors of the invention, said kit comprising: a) at least one reagent capable of specifically determining (i) the expression level of the long-non coding RNA (IncRNA) LINC00520; and b) at least one reagent capable of determining (ii) the response to an inhibitor of the mitogen-activated protein kinase (MAPK) pathway; c) optionally, instructions for performing said method. LEGENDS TO THE FIGURES

Figure 1. The LINC00520 locus is targeted by MITF and its cofactor BRG1. A. Chromatin immunoprecipitation followed by high throughput sequencing (ChlP-seq) or ATAC-seq data of 501Mel cells (melanocytic melanoma cell line with the BRAF mutation BRAF V600F) were visualized at the LINC00520 locus revealing multiple MITF and BRG1 bound sites (representative sites shown by arrows) and characterized by the active transcription marker H3K27ac. B. Regulation by LINC00520 and LINC00518 by MITF was confirmed by analysis of RNA sequencing of 501mel cells after siRNA-mediated silencing knock down for these factors. Data are expressed as log2 fold change of the siMITF or siSOXlO over the siCTRL.

Figure 2. High LINC00520 expression in melanoma. A. UCSC Xenia analysis revealed highest LINC00520 expression in cutaneous melanoma compared to normal tissues or nonmelanoma tumors. UCSC Xenia analysis is a complication of TCGA, GTEX and TARGET studies. SKCM: skin cutaneous melanoma. UVM: Uveal melanoma B-C. Mining of the GTEX (normal human tissues) and the TCGA (human tumors) databases showed only low LINC00520 expression in normal tissues and a high expression in cutaneous melanoma. D. Mining of the GSE98394 RNA-seq data revealed strong up-regulation of LINC00520 expression in melanoma compared to nevi. E. Normal skin and melanoma samples FFPE sections were analyzed for LINC00520 and MITF expression using the multiplex fluorescent RNAscope protocol. DNA was stained using DAPI.

Figure 3. High LINC00520 expression in the TCGA database of skin cutaneous melanoma data set strongly correlated with poor patent outcome. A. Overall high LINC00520 expression correlated with poor patient outcome in SKCM patients. 477 Samples; Quartiles. B-C. Separate analysis of primary and metastatic melanoma samples revealed that high LINC00520 expression was favorable in primary tumors, but associated with poor outcome in metastatic tumors. All P- values are shown on the graph.

Figure 4. A. LINC00520 RNA levels were determined in the single cell RNA seq data from the collection of uveal melanomas from Pandiani et al. (Cell Death Differ . 2021;28(6): 1990-2000). B. UMAP projection of single cells from the different tumors colored for LINC00520 expression. Tumor LH17.530 is shown by the dotted circle.

Figure 5. A. LINC00520 RNA levels were determined in a panel of melanocytic (MM117, IGR-37, SK-MEL-28, 501Mel, SK-MEL-25, MM229, M249, M249R, MM011) and undifferentiated/mesenchymal (IGR-39, SK-MEL-25R, M229R, MM029, MM099, MM047) melanoma cell lines by quantitative PCR (qPCR) using specific primers and normalized on the geometric mean of 5 housekeeping genes. The MM011 cell line bears the NRAS mutation Q61K. The SK-MEL-25(R), 229(R) and MM099 bear the BRAF mutation V600E and when indicated by R are resistant to BRAF inhibition. MM029 carries the BRAFV600K mutation, and MM047 the NRASQ61K mutation and also display moderate MAPKi resistance. B. LINC00520 expression was mined in the single cell RNA-seq data set of Rambow et al. (Cell 2018;174(4):843-855.el9 which was based on MEL001 PDX tumors. Only cells where LINC00520 RNAs was captured were included in the analysis. The % of cells analysed for each population is indicated above each bar.

Figure 6. A. The indicated melanoma cells were transfected with a negative control (CTR) or two different antisense oligonucleotides (LNA GapmeRs; GAP#1, GAP#2) targeting LINC00520 and harvested after 48 hours. LINC00520 expression was measured in total RNA by RT-qPCR and compared to the negative control by 1-way Anova (Dunnett’s multiple comparison test). B-C. Melanoma cells transfected with LINC00520 GapmerRs were stained with the CellTrace Violet dye, cultured for 72 hours and then stained using an anti-cleaved Caspase 3 antibody and analyzed by flow cytometry to determine the number of slow proliferating and apoptotic cells, respectively. Active Caspase 3 positive or cells with high CellTrace Violet fluorescence levels (low proliferative cells) were compared to control GapmeR by 1-way Anova (Dunnett’s multiple comparison test). Undifferentiated/mesenchymal MM047 melanoma cells and HEKT non-melanoma cells were used as negative controls as they do not express LINC00520. D. 501mel and MM117 melanocytic melanoma cells were transfected with LINC00520 targeting GapmeRs and after 72 hours stained with Annexin V and TOPRO-3 and analysed by flow cytometry. Single cells were defined as alive (AnnV-, TOPRO-3-), early apoptotic (AnnV+, TOPRO-3), late apoptotic (AnnV+, TOPRO-3+) and necrotic (AnnV-, TOPRO-3+). Percentages of alive cells were compared to the negative control by 1-way Anova (Dunnett’s multiple comparison test).

Figure 7. A. Expression of miR-125b-5p and LINC00520 was assessed in RNA-seq data from the indicated cell lines expressed as normalized reads per kilobase/transcript length (RPK). B. Analysis of EIF5A2 levels in the TCGA SKCM database did not show a statistically significant association with patient survival.

Figure 8. A. Immunoblots of proteins co-purified with LINC00520 or PC A3 under native of UV-crosslinked conditions. B. Immunoblots following LINC00520 or PCA3 purification from HEK293T cells ectopically expressing LINC00520 or GFP control. C. DHX36 was immunoprecipitated from 501Mel extracts and the presence of LINC00520, SAMMSON and MALAT1, a highly abundant IncRNA, was assessed by RT-qPCR. N =4 independent biological replicates.

Figure 9. A. Detection of LINC00520 by RT-qPCR analysis of RNA extracted from the cytosolic or mitochondrial fractions from MOI 17 or 501Mel cells. Data from n=3 independent biological replicates are shown as means ± SEM. B. Immunoblot detection of p32 and DHX36 in cytosolic or mitochondrial fractions from 501Mel cells.

Figure 10. A. Volcano plot showing identification of RNAs displaying increased or decreased association with DHX36 following LINC00520 silencing. B Immunoblots detecting UBE4A (Santa Cruz; sc-365904) and RBPJ (Santa Cruz; sc-271128) in cells transfected with control or LINC00520-targeting GapmeR.

Figure 11. A-B. Melanoma cells were transfected with suboptimal doses of GapmeRs targeting LINC00518 or LINC00520 as single agents or in pairwise combination. Cells were stained with the CellTrace Violet dye, cultured for 72 hours, stained with an anti -cleaved Caspase 3 antibody and analyzed by flow cytometry. Percentages of apoptotic caspase 3-positive cells (A) and low proliferative cells (B) were compared between the groups by 1-way Anova. C. Expression of LINC00518 and LINC00520 was analyzed by RT-qPCR as described above.

Figure 12. A-B. 501Mel melanoma or HeLa cells were transfected with suboptimal doses of GapmeR targeting LINC00520 in presence of DMSO or DT (100 nM each). 72 hours after transfection plates were stained with Crystal Violet and the occupied surface calculated using the FIJI software. In B, data from n=3 independent biological replicates are shown as means ± SEM and p-values were calculated using a two-way Anova test, ** indicates p<0.01 *** indicates p<0.001 and NS is non-significant.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, nomenclatures used herein, and techniques are those well-known and commonly used in the art.

The present invention may be understood more readily by reference to the following detailed description, included preferred embodiments of the invention, and examples included herein. Co-inhibition ofLINC0520 and LINC0518

The present invention aims at providing an innovative therapeutic approach for the treatment of melanoma, regardless of its phenotype, driver mutations or resistance to therapy, by inhibiting the functional expression of two melanoma-specific IncRNAs: LINC00520 and LINC00518.

In a first aspect, the present invention relates to (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520 for use in combination with (ii) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00518, in the treatment of a melanoma.

In particular, the present invention is directed to the combined use of (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520, and (ii) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00518, for the preparation of a medicament for the treatment of a melanoma.

Further provided is a method for treating a melanoma, in a subject in need thereof, said method comprising the administration, to said subject, of a therapeutically effective amount of: (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520, and (ii) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00518.

A “long-non coding RNA” (long ncRNA, or IncRNA) is generally referred as a non-coding RNA that is at least 200 nucleotides in length. A particular class of IncRNA are “long intergenic noncoding RNAs” (lincRNAs), which are sequences of IncRNA transcribed from non-coding DNA sequences between protein-coding genes.

The term “LINC00518” , also known as “LENOX" or” C6orf218”, refers to the long intergenic non-protein coding RNA 518. It is disclosed in the GeneCards database under ID GC06M0 10428, in the HGNC database under ID 28626, in the Gene database under ID 221718, in the Ensembl database under ID ENSG00000183674 and in the Genbank database under NR_027793. The LINC00518 gene encodes 8 splicing variants (i.e. isoforms), arising from up to 4 exons (see Table 1). Unless specified otherwise, the term “LINC00518” encompasses all the isoforms thereof.

Table 1. LINC00518 isoforms and exons (sequences available on Ensembl database)

The terms “LINC00520” , “long intergenic non-protein coding RNA 520", “C14orf34” or “LASSIE" refer herein to the long intergenic non-protein coding RNA 00520. It is disclosed in the Gene database under ID 645687, in the HGNC database under ID 19843, in the Ensembl database under ID ENSG00000258791. The LINC00520gene encodes 12 splicing variants (i.e. isoforms), arising from up to 5 exons (see Table 2). Unless specified otherwise, the term “LINC00520” encompasses all the isoforms thereof.

Table 2. LINC00520 isoforms and exons (sequences available on Ensembl database)

By ‘functional expression” of a gene, it is meant the transcription and/or translation of functional gene product.

An “inhibitor of functional expression" of a gene refers to a molecule or technical means capable of decreasing or even abolish the expression of said gene. For non-protein coding genes like LINC00520 and LINC00518, functional expression can be deregulated on at least two levels: first, at the DNA level, e.g. by absence or disruption of the gene, or lack of transcription taking place (in both instances preventing synthesis of the relevant gene product); second, at the RNA level, e.g. by lack of splicing or lack or decrease in the activity mediated by said IncRNA. Inhibition can be evaluated by any means known to those skilled in the art including, but not limited to, assessing the level of IncRNA transcript using e.g. quantitative PCR. Besides, in the context of the present invention, it shall be understood that the inhibitor is selective, or in other words, specific for the targeted IncRNA, in that it does not inhibit, or at least does not substantially inhibit, any other target. An inhibitor according to the invention is capable of inhibiting the functional expression of the IncRNA in vivo and/or in vitro. The inhibitor may inhibit the functional expression of the IncRNA by at least about 10%, 15% 20%, or 25%, preferably by at least about 30%, 35%, 40% or 45%, still preferably by at least about 50%, 55%, 60%, or 65%, yet preferably by at least about 70%, 75%, 80%, or 85%, more preferably by at least about 90%, or 95%.

Generally speaking, the term “treatment” or “treating” means obtaining a desired physiological or pharmacological effect depending on the degree of severity of the symptom or disorder of interest, or risks thereof, i.e. herein, depending on the degree of severity or risks of developing such symptom or disorder. In the context of cancer such as melanoma, this includes, inter alia, the alleviation of symptoms, the reduction of inflammation, the inhibition of cancer cell growth, and/or the reduction of tumor size. Furthermore, these terms are intended to encompass curing as well as ameliorating at least one symptom of the condition or disease. For example, in the case of cancer, a response to treatment includes a reduction in cachexia, increase in survival time, elongation in time to tumor progression, reduction in tumor mass, reduction in tumor burden and/or a prolongation in time to tumor metastasis, time to tumor recurrence, tumor response, complete response, partial response, stable disease, progressive disease, progression free survival, overall survival, each as measured by standards set by the National Cancer Institute and the U.S. Food and Drug Administration for the approval of new drugs. See Johnson et al., J. Clin. Oncol., 2009; 21(7): 1404-1411.

In a preferred embodiment, the term treatment refers to the inhibition or reduction of cancer cell proliferation and/or the increase in cancer cell apoptosis in a subject having the cancer, herein melanoma.

In a more preferred embodiment, the inhibition or reduction of cancer cells and/or the increase in apoptosis of cancer cells is independent of the status of tumor-associated proteins, herein independent of BRAF and/or NRAS status.

The term “status” as used herein with regard to a particular protein, specifically tumor- associated proteins (e.g. BRAF status, NRAS status), refers to the mutational status and/or the expression of these particular proteins in cells of interest (herein, melanoma cells). Typically, the term is used in the sense “irrespective of or “independent of status, meaning that an effect is observed irrespective of expression levels of, or presence of mutations in, the particular protein in the cells of interest (herein, melanoma cells).

In melanoma, BRAF mutations are more commonly observed in intermittently sun-exposed skin, and associated with KIT mutations, which are present predominantly in mucosal and acral melanomas. Examples of BRAF mutations associated with melanoma include, without limitation, R461I, I462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, V600E/K (the most prominent), 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, preferably V600E.

In melanoma, NRAS mutations are more commonly observed in older patients (above 55 years-old), with a chronic pattern of UV exposure with lesions are usually located at the extremities. Examples of NRAS mutations typically associated with melanoma include, without limitation, G12D/C, G13R, Q61K, Q61R, Q61L, Q61V, Q61H, Q61P. In a particular embodiment, the NRAS mutation is Q61K/R.

It shall nevertheless be understood that BRAF and NRAS co-mutations are not mutually exclusive.

A “therapeutically effective amount' means herein an amount that is sufficient to achieve the effect for which it is indicated, herein the treatment of a melanoma. The amount of the combination therapy of the invention to be administered can be determined by standard procedures well known by those of ordinary skill in the art. Physiological data of the patient (e.g. age, size, and weight), the routes of administration and the disease to be treated have to be taken into account to determine the appropriate dosage, optionally compared with subjects that do not suffer from a melanoma. The amount may also vary according to other components of a treatment protocol (e.g. administration of other medicaments, etc.).

A “ subtherapeutic amount” or “ subtherapeutic dose” is an amount that is not therapeutically effective if administered in absence of any other agent or therapy.

It should be further understood that the “subject” or “patients” to be treated according to the invention is one having an integumentary system. Accordingly, preferred subjects to be treated are animals, more preferably mammals, most preferably humans.

The subject to be treated has a melanoma. A “melanoma” , also known as “malignant melanoma”, refers to a type of cancer that develops from the pigment-producing cells called melanocytes. There are three main categories of melanoma: cutaneous melanoma, the most common, which corresponds to the melanoma of the skin; mucosal melanoma which can affect any mucous membrane of the body, including the nasal passages, throat, vagina, anus, or mouth; and ocular melanoma, which is a rare form of melanoma affecting the eye (also known as uveal or choroidal melanoma).

The melanoma to be treated can be at any stage. For instance, the melanoma can be at stage VII, II, III, or IV, preferably stage II, III, or IV, more preferably stage III, or IV. More specifically, the melanoma can be at stage Tla, Tib, T2a, T2b, T3a, T4a, T4b, Nl, N2, N3, Mia, Mlb, or Mlc, preferably stage T2b, T3a, T4a, T4b, Nl, N2, N3, Mia, Mlb, or Mlc. In a preferred embodiment, the melanoma is at an advanced or metastatic stage.

The melanoma can be preferably selected from the group consisting of lentigo maligna, lentigo maligna melanoma, superficial spreading melanoma, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, melanoma with small nevus-like cells, melanoma with features of a Spitz nevus, uveal melanoma, Harding-Passey melanoma, juvenile melanoma, amelanotic melanoma, Cloudman's melanoma, and vaginal melanoma.

Optionally, the melanoma is a melanocytic melanoma. Optionally, the melanoma is a cutaneous or ocular (e.g. uveal) melanoma. Optionally, the melanoma is a cutaneous or ocular (e.g. uveal) melanocytic melanoma.

The subject to be treated may have resistant melanoma. As used herein, the term “resistant melanoma’" refers to a melanoma, which does not respond to a conventional treatment for said cancer. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment or does not respond anymore. The resistance to the treatment may lead to rapid progression of metastatic of melanoma.

The melanoma can be resistant to a targeted therapy, especially inhibitors of the MAPK pathway which include BRAF inhibitors, MEK inhibitors and C-Kit inhibitors; to chemotherapy; and/or to immune checkpoint inhibitors.

In a preferred embodiment, the melanoma is resistant to a targeted therapy, especially BRAF inhibitors such as dabrafenib, vemurafenib, or encorafenib, and/or MEK inhibitors, such as trametinib, cobimetinib, binimetinib, or selumetinib, or any combination thereof. The inhibitors of functional expression according to the invention may indeed restore or increase the sensitivity of melanoma to this type of therapy.

Alternatively, the melanoma may not be resistant to a targeted therapy, especially inhibitors of the MAPK pathway which include BRAF inhibitors, MEK inhibitors and C-Kit inhibitors; to chemotherapy; and/or to immune checkpoint inhibitors.

In a preferred embodiment, the melanoma is not resistant to a targeted therapy, especially BRAF inhibitors such as dabrafenib, vemurafenib, or encorafenib, and/or MEK inhibitors, such as trametinib, cobimetinib, binimetinib, selumetinib, or any combination thereof. The inhibitors of functional expression according to the invention may indeed prevent, decrease or delay the appearance of a resistance to such therapy. In a preferred embodiment, whether the melanoma is resistant or not, the melanoma can be associated with a BRAF mutation and/or with a NRAS mutation, as described above. The inhibitors of functional expression according to the invention may indeed act independently of BRAF and/or NRAS status, as explained above.

The present invention requires a combined inhibition of two melanoma-specific IncRNAs: LINC00520 and LINC00518.

Inhibitors of functional expression of LINC00520 and LINC00518 have been reported in the scientific and patent literature (W02021/152005; Luan et al. Journal of Experimental & Clinical Cancer Research (2020) 39:96; Huang et al. BMC Pulm Med (2021) 21 :287; the contents of which are incorporated herein by reference in their entireties). Below is provided a non-exhaustive list of such inhibitors which can be used in the present invention.

Given the nature of the targeted RNA, each of the inhibitors of functional expression used in the present invention can preferably be either a nucleic acid molecule interfering specifically with the expression of the long-non coding RNA, or a genome editing system comprising a nuclease engineered to target specifically the long-non coding RNA.

A nucleic acid molecule interfering specifically with the expression of the long-non coding RNA might be preferred should one wish to inhibit functional expression at the RNA level, while a genome editing system comprising a nuclease engineered to target specifically the long-non coding RNA might be preferred should one wish to inhibit functional expression at the DNA level.

In a preferred embodiment, the inhibitor used in the present invention can be a nucleic acid molecule interfering specifically with the expression of the long-non coding RNA.

As used throughout the specification, the terms “ nucleic acid molecule", “nucleic acid', “polynucleotide" (polynucleoside) or “oligonucleotide" (oligonucleoside) refers to a succession of natural or synthetic nucleotides (or nucleosides) connected by intemucleotide (or intemucleoside) linkages, wherein each nucleotide (or nucleoside) or internucleotide (or intemucleoside) linkages may be modified or unmodified. A nucleotide is comprised of a nucleoside and a phosphate group. A nucleoside is comprised of a nucleobase and a sugar. A nucleobase comprises a modified and unmodified nucleobase. Unmodified nucleobases are well-known in the art and include adenine (A), thymine (T), cytosine (C), uracil (U) and guanine (G). An internucleotide (intemucleoside) linkage is a bond that forms a covalent linkage between adjacent nucleotides (nucleosides) in a nucleic acid, such as phosphate intemucleotide linkages that are naturally occurring. A nucleic acid may be a single-stranded or double-stranded DNA such as cDNA, genomic DNA, ribosomal DNA, and the transcription product of said DNA, such as RNA. Nucleic acids also encompass nucleic acids which can hybridize to a nucleic acid of reference. When a nucleic acid is designed to hybridize to a nucleic acid of reference, it can be chemically modified to enhance its stability, nuclease resistance, target specificity and/or improve their pharmacological properties (e.g. reduced toxicity, increased intracellular transport, etc.). For example, a nucleic acid may comprise modified nucleotide(s) and/or backbone, such as a modified sugar, a modified nucleobase, and/or a modified intemucleotide (intemucleoside) linkage. A standard modification is the replacement of native phosphates of the internucleotide (intemucleoside) linkage with phosphorothioates (PS) so as to reduce the sensitivity to nucleases and accordingly improve the stability, half-life and tissue distribution of the nucleic acid (Eckstein F., Antisense and Nucleic Acid Drug Development, 2000, 10(2): 1117-221). Another standard modification which increases the affinity of the nucleic acid towards its target is the incorporation in the sugar moiety of a methoxyethyl (MOE) or a constrained ethyl (cEt) to the 2’position, or the tethering of the 4’-carbon to the 2-hydroxyl of the ribose ring to create a bicyclic locked nucleic acid (LNA), to name a few. All these modifications are well known to the one skilled in the art (see Watts et al. J Pathol., 2012; 226(2):365-379; Seth et al., J Clin Invest., 2019; 129(3): 915-925).

Preferred nucleic acid molecules interfering specifically with the expression of the long-non coding RNA according to the invention are those capable of hybridizing specifically to the gene or transcripts of the IncRNA, at least to a part thereof; as such, these are usually non-naturally occurring nucleic acids (i.e. synthetic).

"Hybridizing" or "hydridization" means the pairing or annealing to a target, herein under physiological conditions, typically via hydrogen bonding between complementary nucleotides, such as Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding. A specific hybridization therefore means that the pairing or annealing is specific to the target (no off-targets effects, or at least no substantial off-targets effects).

"A nucleic acid molecule capable of hybridizing" to a target nucleic acid thus means that a stretch of this nucleic acid is capable of forming base pairs to another stretch of the target nucleic acid. It is thus not absolutely required that all the bases in the region of complementarity are capable of pairing with bases in the opposing strand. Mismatches may be tolerated to some extent, as long as in the circumstances, the stretch of nucleotides is capable of hybridizing to its complementary part.

The nucleic acid molecule interfering specifically with the expression of the long-non coding RNA is preferably an antisense nucleic acid, an RNAi nucleic acid, or a ribozyme.

The term "antisense nucleic acid' or "antisense oligonucleotides" (ASO) designates a synthetic single-stranded oligonucleotide of which the sequence is at least partially complementary to a target nucleic acid, such as to the RNA sequence of a target gene (Lee et al., J Cardiovasc Transl Res. 2013; 6(6):969-80; DeVos et al., Neurotherapeutics 2013; 10(3):486-972013). An antisense nucleic acid is capable of altering the expression of a specific target gene, either by splicing modification, or by recruiting RNAse H leading to RNA degradation of RNA-DNA duplexes, thus blocking the expression of the target gene. An antisense nucleic acid is typically short in length, in general 5 to 50 nucleotides in length, such as 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length, more preferably 10, 15, 20, 25, 30, 35 nucleotides in length. It is well within the skill of the person in the art to design antisense nucleic acids specific to a target, based on the knowledge of a target sequence such as introns, exons, or 5’CAP (DeVos et al., Neurotherapeutics 2013; 10(3):486-972013). Antisense nucleic acids can be prepared by methods well-known in the art, such as by chemical synthesis and enzymatic ligation reactions.

In a preferred embodiment, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA inhibits the splicing of the pre- long-non coding RNA, or in other words, it inhibits the formation of the mature long-non coding RNA. An antisense nucleic acid can indeed be designed to block a splice acceptor (SA) site and/or an exon splicing enhancer (ESE) and/or any sequence which could modulate a pre-RNA splicing, i.e. it can be designed to be complementary to a part of the pre-RNA comprising an SA, an ESE, or any sequence which could modulate its splicing.

In an alternative preferred embodiment, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA induces a RNAse H mediated degradation. RNAse H is a cellular enzyme which recognizes duplex between DNA and RNA, and enzymatically cleaves the RNA molecules. Accordingly, to induce RNAse H mediated degradation, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA comprises a region that comprises DNA or DNA-like nucleotides complementary to the targeted IncRNA which is responsible for RNAse H recruitment, ultimately leading to the cleavage of the target nucleic acid.

Regardless of its mode of action for inhibiting the functional expression of the IncRNA, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA is complementary to all or part of the IncRNA, preferably to a part thereof, such as any one of its exons or introns. Sequences of LINC00520 and LINC00518 are well known in the art, as explained above.

In a preferred embodiment, the antisense nucleic acid interfering specifically with the expression of LINC00518 comprises or consists of a contiguous nucleotide sequence of at least 10 to 30 (preferably 11 to 29, 12 to 26, 13 to 24, 14 to 22, 14 to 17, 14, 15 or 16) nucleotides in length that is complementary to a specific region of the LINC00518 sequence. Non limiting examples of such antisense nucleic acids include, without limitation, those comprising or consisting of a contiguous nucleotide sequence of at least 10 nucleotides in length of the sequence SEQ ID NO: 2 or 3, such as those comprising or consisting of the sequence SEQ ID NO: 2 or 3.

In a preferred embodiment, the antisense nucleic acid interfering specifically with the expression of LINC00520 comprises or consists of a contiguous nucleotide sequence of at least 10 to 30 (preferably 11 to 29, 12 to 26, 13 to 24, 14 to 22, 14 to 17, 14, 15 or 16) nucleotides in length that is complementary to a specific region of the LINC00520 sequence. Non limiting examples of such antisense nucleic acids include, without limitation, those comprising or consisting of a contiguous nucleotide sequence of at least 10 nucleotides in length of the sequence SEQ ID NO: 4 or 5, such as those comprising or consisting of the sequence SEQ ID NO: 4 or 5.

As described above, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA may further comprise at least one modified sugar, at least one modified internucleotide (or internucleoside) linkage, and/or at least one modified nucleobase, so as to enhance its properties, such a reduction in sensitivity to nucleases, increase in stability, half-life and/or tissue distribution, and/or enhancement of its affinity towards its target nucleic acid.

In a preferred embodiment, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA comprises at least one modified sugar, in particular in position 2’ of the nucleotide (or nucleoside) such as 2’-O-methyl (2’-0-Me), 2’fluoro (2’-F), 2’0- methoxyethyl (2’-M0E also known as MOE), 2’,4’-brigded, or cEt (constrained ethyl).

A “modified sugar” is a sugar that is non-naturally occurring. 2’,4’-brigded, which is the introduction of a methylene bridge between the 2’ and 4’ position of the nucleotide, defining locked nucleic acids (LNA), is herein particularly preferred so as to confer or increase the stability of a nucleic acid when hybridized to a complementary DNA or RNA nucleic acid.

In a preferred embodiment, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA comprises at least one modified intemucleotide (or internucleoside) linkage, such as a phosphorothioate intemucleotide (or internucleoside) linkage.

A “modified internucleotide linkage” (or modified internucleoside linkage) is an intemucleotide (or intemucleoside) linkage in a nucleic acid that is non-naturally occurring, and is thus referred as a non-phosphate linkage. A phosphorothioate linkage, which is a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom, is herein particularly preferred so as to confer or increase the resistance of a nucleic acid to nucleases. In a preferred embodiment, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA is a gapmer.

A “gapmer” refers to a nucleic acid molecule comprising an internal segment having a plurality of nucleotides (or nucleosides) that support RNase H cleavage positioned between external segments, each having one or more nucleotides (or nucleosides), wherein the nucleotide (or nucleosides) comprising the internal segment are chemically distinct from the immediately adjacent nucleotide(s) (or nucleoside(s)) comprising the external segments. The internal or central segment may be referred to as the “gap”, “gap segment” or “gap region” (G); while the external segments may be referred to as the “wings”, “flanks”, “wing segments” , “flank segments” , “wing regions” or “flank regions” (F for the 5’ flank region and F’ for the 3’ flank region). Typically, the F and F’ regions are composed of modified ribonucleotides (RNA*) which are complementary to a target nucleic acid; whereas the G region is composed of deoxyribonucleotides, i.e. DNA or DNA-like molecules, which is responsible for RNAse H recruitment which ultimately leads to the degradation of the target nucleic acid. A gapmer is therefore a chimeric antisense. Gapmers can typically comprise a gap region (G) of 5 to 15 deoxynucleotides (or deoxynucleosides) flanked by wing regions (F and F’) of 2 to 10 modified nucleotides (or nucleosides) each.

In a preferred embodiment, the gapmer interfering specifically with the expression of the long-non coding RNA comprises or consists of a contiguous nucleotide (or nucleoside) sequence corresponding to the following formula:

5’-F (RNA*) - G (DNA or DNA-like) - F’ (RNA*)-3’

It should be understood that any of the modifications that can improve the properties of the gapmer, as discussed above, can be used and combined together.

For example, the gapmer interfering specifically with the expression of the long-non coding RNA can comprise or consist of a contiguous nucleotide (or nucleoside) sequence that is complementary to a specific region of the IncNRA sequence and that corresponds to the following formula:

5’-F (RNA*) - G (DNA or DNA-like) - F’ (RNA*)-3’ wherein the gap segment (G) comprises or consists of a sequence of at least 5 to 15 nucleotides (or nucleosides) in length, wherein each wing segment (F or F’) comprises at least 2 to 10 nucleotides (or nucleosides) in length, wherein each nucleotide (or nucleoside) of each wing segment (F and F’) comprises a modified sugar, in particular in position 2’ of the nucleotide (or nucleoside) such as 2’-O-methyl (2’-O-Me), 2’fluoro (2’-F), 2’0-methoxyethyl (2’-MOE also known as MOE), 2’,4’-brigded, or cEt (constrained ethyl), more preferably 2’,4’-brigded, and wherein each intemucleotide (or intemucleoside) linkage is a modified linkage, preferably is a phosphorothioate linkage.

In a preferred embodiment, the gapmer interfering specifically with the expression of LINC00518 comprises or consists of a contiguous nucleotide (or nucleoside) sequence that is complementary to a specific region of the LINC00518 sequence and that corresponds to the following formula:

5’-F (RNA*) - G (DNA or DNA-like) - F’ (RNA*)-3’ wherein the gap segment (G) comprises or consists of a sequence of at least 5 to 15 nucleotides (or nucleosides) in length, wherein each wing segment (F or F’) comprises at least 2 to 10 nucleotides (or nucleosides) in length, wherein each nucleotide (or nucleoside) of each wing segment (F and F’) comprises a modified sugar, in particular in position 2’ of the nucleotide (or nucleoside) such as 2’-O-methyl (2’-0-Me), 2’fluoro (2’-F), 2’0-methoxyethyl (2’-M0E also known as MOE), 2’,4’-brigded, or cEt (constrained ethyl), more preferably 2’,4’-brigded, and wherein each internucleotide (or intemucleoside) linkage is a modified linkage, preferably is a phosphorothioate linkage.

Non limiting examples of gapmers interfering specifically with the expression of LINC00518 include, without limitation, gapmers comprising or consisting of any one of the following sequences: Cbs Cbs Gbs dAs mdCs mdCs dTs dGs dAs dAs dTs dTs dGs Cbs Abs Ab (SEQ ID NO: 10), or Gbs Tbs Abs dGs dAs dGs dGs mdCs dTs dAs dGs dAs dAs Cbs Tbs Gb (SEQ ID NO: 11), wherein A is an adenine, mC is a 5-methylcytosine, G is a guanine, T is a thymine, b is LNA (2',4'-methylene bridged sugar moiety), d is a 2’ -deoxyribosyl sugar moiety, and s is a phosphorothioate internucleotide (or intemucleoside) linkage.

In a preferred embodiment, the gapmer interfering specifically with the expression of LINC00520 comprises or consists of a contiguous nucleotide (or nucleoside) sequence corresponding to the following formula:

5’-F (RNA*) - G (DNA or DNA-like) - F’ (RNA*)-3’ wherein the gap segment (G) comprises or consists of a sequence of at least 5 to 15 nucleotides (or nucleosides) in length that is complementary to a specific region of the LINC00520 sequence, wherein each wing segment (F or F’) comprises at least 2 to 10 nucleotides (or nucleosides) in length, wherein each nucleotide (or nucleoside) of each wing segment (F and F’) comprises a modified sugar, in particular in position 2’ of the nucleotide (or nucleoside) such as 2’-O-methyl (2’-0-Me), 2’fluoro (2’-F), 2’0-methoxyethyl (2’-M0E also known as MOE), 2’,4’-brigded, or cEt (constrained ethyl), more preferably 2’,4’-brigded, and wherein each internucleotide (or intemucleoside) linkage is a modified linkage, preferably is a phosphorothioate linkage.

Non limiting examples of gapmers interfering specifically with the expression of LINC00520 include, without limitation, gapmers comprising or consisting of any one of the following sequences:

Tbs Tbs Tbs dGs dAs dTs dGs dAs dGs dTs dGs dAs dGs Tbs Cbs Gb (SEQ ID NO: 12)

Gbs Abs Gbs dTs dmCs dGs dmCs dTs dGs dAs dGs dAs dAs Tbs Tbs Ab (SEQ ID NO: 13) wherein A is an adenine, mC is a 5-methylcytosine, G is a guanine, T is a thymine, b is LNA (2',4'-methylene bridged sugar moiety), d is a 2’ -deoxyribosyl sugar moiety, and s is a phosphorothioate intemucleotide (or intemucleoside) linkage.

Other formats of gapmers are well-known in the art, and can be easily conceived by the skilled practitioner. See for example W02021/152005, incorporated herein in its entirety.

In another embodiment, RNAi can be used to inhibit the functional expression of the IncRNA.

“RNAi nucleic acid" refers to a nucleic acid that can inhibit expression of a target gene by RNA interference (RNAi) mechanism. By contrast to antisense nucleic acids, RNAi nucleic acids target mature RNA. RNAi nucleic acids are well-known in the art, and include short-hairpin RNA (shRNA), small interfering RNA (siRNA), double-stranded RNA (dsRNA), and single-stranded RNA (ssRNA) (Sohail et al. 2004, Gene Silencing by RNA Interference: Technology and Application 1 ^st Edition, ISBN 9780849321412; WO 99/32619; Wang et al., Pharm Res 2011, 28:2983-2995). RNA interference designates a phenomenon by which dsRNA specifically suppresses expression of a target gene at post-transcriptional level. In normal conditions, RNA interference is initiated by double-stranded RNA molecules (dsRNA) of several thousands of base pair length. In vivo, dsRNA - such as shRNA- introduced into a cell is cleaved by Dicer into a mixture of short interfering RNA called siRNA; the latter will bind to another enzyme (RISC) that will catalyze the cleavage of both the siRNA and target mRNA (Bernstein et al. Nature. 2001;409(6818):363-6). In mammalian cells, the siRNAs that are naturally produced by Dicer are typically 21-23 bp in length, with a 19 or 20 nucleotides duplex sequence, two-nucleotide 3' overhangs and 5'-triphosphate extremities (Zamore et al. Cell. 2000,101(l):25-33; Elbashir et al. Genes Dev. 2001, 15(2): 188-200; Elbashir et al. EMBO J. 2001, 20(23):6877-88). The selected siRNA or shRNA target sequence should be subjected to a BLAST search against EST database to ensure that the only desired gene is targeted. Various products are commercially available to aid in the preparation and use of synthetic siRNA or shRNA. The RNAi nucleic acid can be of at least about 10 to 40 nucleotides (or nucleosides) in length, preferably about 15 to 30 base nucleotides (or nucleosides) in length. siRNA or shRNA can comprise naturally occurring RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally- occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides (or nucleosides). Such alterations can include addition of non-nucleotide (or non-nucleoside) material, such as to the end of the molecule or to one or more internal nucleotides (or nucleoside) of the RNAi, including modifications that make the RNAi resistant to nuclease digestion, as described above.

Thus, in a preferred embodiment, the nucleic acid molecule interfering specifically with functional expression of the IncRNA is an RNAi nucleic acid, that is complementary to at least one part of the IncRNA, in particular to at least one exon thereof The RNAi nucleic acid can be a siRNA or shRNA of at least about 10 to 40 nucleotides (or nucleosides) in length, preferably of about 15 to 30 nucleotides (or nucleosides) in length.

In a preferred embodiment, the RNAi nucleic acid interfering specifically with the expression of LINC00518 comprises or consists of a contiguous nucleotide sequence of at least 10 to 40 (preferably 15 to 30, 16 to 25, 17 to 24, 20, 21, 22 or 23) nucleotides in length that is complementary to a specific region of the LINC00518 sequence. Non limiting examples of such RNAi nucleic acid include, without limitation, those comprising or consisting a contiguous nucleotide sequence of at least 10 nucleotides in length of the sequence SEQ ID NO: 15, preferably those comprising or consisting of the sequence SEQ ID NO: 15.

In a preferred embodiment, the RNAi nucleic acid interfering specifically with the expression of LINC00520 comprises or consists of a contiguous nucleotide sequence of at least 10 to 40 (preferably 15 to 30, 16 to 25, 17 to 24, 20, 21, 22 or 23) nucleotides in length that is complementary to a specific region of the LINC00520 sequence. Non limiting examples of such RNAi nucleic acid include, without limitation, those comprising or consisting a contiguous nucleotide sequence of at least 10 nucleotides in length of the sequence SEQ ID NO: 23, preferably those comprising or consisting of the sequence SEQ ID NO: 23. In another embodiment, the nucleic acid interfering specifically with the functional expression of the IncRNA is a ribozyme.

"Ribozymes" are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Ribozyme molecules specific for a target (herein a IncRNA) can be designed, produced, and administered by methods commonly known to the art (see e.g., Fanning and Symonds (2006) RNA Towards Medicine (Handbook of Experimental Pharmacology), ed. Springer p. 289-303).

In a preferred embodiment, the nucleic acid molecule interfering specifically with the functional expression of LINC00518 is a ribozyme targeting said IncRNA.

In a preferred embodiment, the nucleic acid molecule interfering specifically with the functional expression of LINC00520 is a ribozyme targeting said IncRNA.

Yet, in another embodiment, genome editing can be used to inhibit the functional expression of the IncRNA. More specifically, one can use a genome editing system comprising a nuclease engineered to target the IncRNA gene.

“Genome editing" is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases, also called molecular scissors. Nucleases create specific double-stranded break (DSBs) at desired locations in the genome, and harness the cell's endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) or non-homologous end-joining (NHEJ). There are currently four known families of nucleases suitable for genome editing: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system in particular the Cas9 system (Mali et al, Nature Methods, 2013; 10(10):957-63), or engineered meganucleases re-engineered homing endonucleases. Said nucleases can be delivered to the cells either as DNAs or mRNAs, such DNAs or mRNAs and can be engineered to target the IncRNA gene.

A particularly preferred genome editing system according to the invention is the CRISPR/Cas system in particular the Cas9 system.

In a preferred embodiment, the genome editing system interfering specifically with functional expression of LINC00518 is a CRISPR/Cas system, in particular a CRISPR/Cas9 system, targeting said IncRNA. To do so, one may use one or more single guide RNA (sgRNA) targeting specifically LINC00518. Non limiting examples of such sgRNA include, without limitation, those comprising or consisting of a contiguous nucleotide sequence of at least 10 nucleotides in length of the sequence SEQ ID NO: 16, 17 or 18, preferably those comprising or consisting of the sequence SEQ ID NO: 16, 17 or 18, or any combination thereof. In a preferred embodiment, the genome editing system is a triplet-target CRISPR system, meaning that three guide RNA are used to target LINC00518, such as those described above.

In a preferred embodiment, the genome editing system interfering specifically with functional expression of LINC00520 is a CRISPR/Cas system, in particular a CRISPR/Cas9 system, targeting said IncRNA. To do so, one may use one or more single guide RNA (sgRNA) targeting specifically LINC00520. Non limiting examples of such sgRNA include, without limitation, those comprising or consisting of a contiguous nucleotide sequence of at least 10 nucleotides in length of the sequence SEQ ID NO: 19, 20 or 21, preferably those comprising or consisting of the sequence SEQ ID NO: 19, 20 or 21, or any combination thereof. In a preferred embodiment, the genome editing system is a triplet-target CRISPR system, meaning that three guide RNA are used to target LINC00520, such as those described above.

The skilled person would readily understand that any one of the (i) inhibitors of functional expression of the long-non coding RNA (IncRNA) LINC00520, and any one of the (ii) inhibitors of functional expression of the long-non coding RNA (IncRNA) LINC00518, as described herein, can be combined for the purpose of the present invention.

In a particularly preferred embodiment, (i) the inhibitor of functional expression of the long- non coding RNA (IncRNA) LINC00520 is a nucleic acid molecule interfering specifically with the expression of said IncRNA, as described above; and (i) the inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00518 is a nucleic acid molecule interfering specifically with the expression of said IncRNA, as described above.

In a further preferred embodiment, (i) the nucleic acid molecule interfering specifically with the expression of LINC00520 is an antisense nucleic acid, as described above; and (ii) the nucleic acid molecule interfering specifically with the expression of LINC00518 is an antisense nucleic acid, as described above.

For example, (i) the nucleic acid molecule interfering specifically with the expression of LINC00520 can be an antisense nucleic acid comprising or consisting a contiguous nucleotide sequence of at least 10 nucleotides in length of the sequence SEQ ID NO: 4 or 5, such as an antisense nucleic acid comprising or consisting of the sequence SEQ ID NO: 4 or 5; and (ii) the nucleic acid molecule interfering specifically with the expression of LINC00518 can be an antisense nucleic acid comprising or consisting a contiguous nucleotide sequence of at least 10 nucleotides in length of the sequence SEQ ID NO: 2 or 3, such as an antisense nucleic acid comprising or consisting of the sequence SEQ ID NO: 2 or 3. In a more preferred embodiment, (i) the antisense nucleic acid interfering specifically with the expression of LINC00520 is a gapmer, as described above; and (ii) the antisense nucleic acid interfering specifically with the expression of LINC00518 is a gapmer, as described above.

Yet, in a further preferred embodiment, the gapmer interfering specifically with the expression of LINC00520 is an LNA gapmer and/or comprises phosphothiorate linkages, as described above; and (ii) the gapmer interfering specifically with the expression of LINC00518 is an LNA gapmer and/or comprises phosphothiorate linkages, as described above.

For example, (i) the gapmer interfering specifically with the expression of LINC00520 can comprise or consist of any one of the following sequences: Tbs Tbs Tbs dGs dAs dTs dGs dAs dGs dTs dGs dAs dGs Tbs Cbs Gb (SEQ ID NO: 12), or Gbs Abs Gbs dTs dmCs dGs dmCs dTs dGs dAs dGs dAs dAs Tbs Tbs Ab (SEQ ID NO: 13); and (ii) the gapmer interfering specifically with the expression of LINC00518 can comprise or consist of any one of the following sequences: Cbs Cbs Gbs dAs mdCs mdCs dTs dGs dAs dAs dTs dTs dGs Cbs Abs Ab (SEQ ID NO: 10), or Gbs Tbs Abs dGs dAs dGs dGs mdCs dTs dAs dGs dAs dAs Cbs Tbs Gb (SEQ ID NO: 11);; wherein A is an adenine, mC is a 5-methylcytosine, G is a guanine, T is a thymine, b is LNA (2', d'methylene bridged sugar moiety), d is a 2’-deoxyribosyl sugar moiety, and s is a phosphorothioate internucleotide (or internucleoside) linkage.

Other combination of inhibitors of the functional expression of the IncRNAs LINC00520 and LINC00518 can also be envisioned, such as:

• (i) a nucleic acid molecule interfering specifically with the expression of LINC00520, as described above; and (ii) a genome editing system comprising a nuclease engineered to target specifically LINC00518, as described above; or

• (i) a genome editing system comprising a nuclease engineered to target specifically LINC00520, as described above; and (ii) a nucleic acid molecule interfering specifically with the expression of LINC00518, as described above; or

• (i) a genome editing system comprising a nuclease engineered to target specifically LINC00520, as described above; and (ii) a genome editing system comprising a nuclease engineered to target specifically LINC00518, as described above.

It shall be further understood that any one of the (i) inhibitors of functional expression of the long-non coding RNA (IncRNA) LINC00520, and any one of the (ii) inhibitors of functional expression of the long-non coding RNA (IncRNA) LINC00518, as described herein, can be further combined with an additional therapy, especially one that is suited for melanoma. This includes, without limitation, surgery, radiotherapy, chemotherapy, targeted therapy or immune checkpoint therapy, or any combination thereof. Such therapies are well-known in the art, and therefore need not be detailed herein.

The combination of (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520, and (ii) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00518, as described herein, may be suitable for administration to a cell, tissue and/or an organ of individuals affected by or at risk of suffering from a melanoma, and may be administered in vivo, ex vivo or in vitro. Since melanoma affects melanocytes, it is preferred that said cells are melanocytes, and/or that said tissue or organ is the skin or the eye.

Whether the combination is administered simultaneously, separately or sequentially, each inhibitor can be delivered as it is to the subject, or be formulated to be compatible with their intended route of administration.

For example, the combination can be formulated in a combined pharmaceutical composition, or in separate pharmaceutical compositions, in a form suitable for parenteral, oral, transdermal or topical or intraocular administration, such as a liquid suspension, a solid dosage form (granules, pills, capsules or tablets), or a paste or gel.

For the purposes of the invention, a particularly preferred form of administration is parenteral administration, such as subcutaneous, intradermal, intravenous; transdermal administration; topical administration; or even intraocular administration.

It is within the skill of the person in the art to formulate such pharmaceutical composition(s) in accordance with standard pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). Pharmaceutical compositions according to the invention may notably be formulated to release the combination therapy immediately upon administration or at any predetermined time or time period after administration.

When formulated within a pharmaceutical composition, the combination can be further combined with a pharmaceutically acceptable excipient.

As used herein, the term a “pharmaceutically acceptable excipient” means an inactive or inert, and therefore nontoxic, component, as it has no pharmacological action itself, which can be used to improve properties of a composition, such as shelf-life, retention time at the application site, consumer acceptance, etc. It includes, without limitation, surfactants (cationic, anionic, or neutral); surface stabilizers; other enhancers, such as preservatives, wetting or emulsifying agents; solvents; buffers; salt solutions; dispersion medium; isotonic and absorption delaying agents, and the like; that are physiologically compatible.

As explained above, the combination therapy is provided in a therapeutically effective amount so as to treat the melanoma.

In a preferred embodiment, the amount of each inhibitor is a pediatric dose in monotherapy, or a dose that is subtherapeutic in monotherapy (yet, is such that it is therapeutically effective in the combination therapy of the invention). In other words, the amount of each inhibitor is a subtherapeutic dose (yet, is such that it is therapeutically effective in the combination therapy of the invention).

Those of skill in the art will recognize that such parameters are normally worked out during clinical trials.

In another aspect, the present invention relates to (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520, and (ii) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00518, as described above, as a combined preparation for simultaneous, separate or sequential use in the treatment of a melanoma.

The invention relates to the simultaneous, separate or sequential use of (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520 and (ii) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00518, as described above, for the preparation of a medicament for the treatment of a melanoma.

Further provided is therefore a method for treating a melanoma, in a subject in need thereof, said method comprising the simultaneous, separate or sequential administration, to said subject, of a therapeutically effective amount of (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520 and (ii) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00518, as described above.

Preferred embodiments, as described above, apply herein mutatis mutandis.

In another aspect, the present invention relates to a pharmaceutical composition comprising an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520 and (ii) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00518, as described above, and optionally (iii) a pharmaceutically acceptable excipient.

Preferred embodiments, as described above, apply herein mutatis mutandis. In another aspect, the present invention pertains to an in vitro method for identifying a melanoma tumor suitable for the combined treatment of the invention, said method comprising: a) determining the respective expression level of the long-non coding RNAs (IncRNAs) LINC00520 and LINC00518, in a melanoma tumor sample; b) comparing the expression level determined in step a) with a reference expression level for each of said IncRNA, thereby identifying whether the melanoma tumor is suitable for said treatment.

The term “ expression level”, as applied to a IcnRNA, refers herein to the amount or level of said IncRNA expressed in biological sample, such as a cell, tissue, or organ(s), herein preferably melanocytes or skin. The term “level” as used herein refers to an amount (e.g. relative amount or concentration) of a IncRNA that is detectable or measurable in a sample. For example, the level can be a relative amount by comparison to a reference expression level. The act of actually “determining the expression lever of a IncRNA in a biological sample refers to the act of actively detecting whether the IcnRNA is expressed in said sample or not, and notably allows to detect whether the expression of the IcnRNA is upregulated, downregulated or substantially unchanged when compared to a reference expression level.

By “reference expression lever or “control expression lever, as applied to a IcnRNA, it is meant a predetermined expression level of said IcnRNA, which can be used as a reference in the method of the invention. For example, a reference expression level can be the expression level of the IncRNA in a biological sample of a healthy subject, or the average or median expression level in a biological sample of a population of healthy subjects.

In a preferred embodiment, step a) is performed by PCR, such as quantitative PCR (qPCR).

In a preferred embodiment, an increased expression level of LINC00520 in step a) and increased expression level of LINC00518 in step a) is indicative of the suitability of said treatment, especially when the reference expression level is one of a healthy subject or population of heathy subjects.

In a further aspect, the present invention is directed to a kit for use in the in vitro method for identifying a melanoma tumor suitable for treatment with the inhibitors, said kit comprising: a) at least one reagent capable of specifically determining the expression level of the long-non coding RNA (IncRNA) LINC00520; and b) at least one reagent capable of specifically determining the expression level of the long-non coding RNA (IncRNA) LINC00518; c) optionally, instructions for performing said method. As used herein, the term “instructions ” refers to a publication, a recording, a diagram, or any other medium which can be used to communicate how to perform a method of the invention. Said instructions can, for example, be affixed to a container which contains said kit. Preferably, the instructions for using said kit include the reference expression level for each IncRNA.

The term “reagent capable of specifically determining the expression lever, as applied to a IcnRNA, designates a reagent or a set of reagents which specifically recognizes said IncRNA, and allows for the quantification of the expression level thereof. These reagents can be for example nucleotide probes (also known as primers). In the context of the present invention, such reagent is said to be “specific” for the IncRNA or “recognizes specifically” the IncRNA if it 1) exhibits a threshold level of hydridization, and/or 2) does not significantly cross-react with other targets. The hydridization capacity and cross-reactivity of a reagent can be easily determined by one skilled in the art, and thus need to be further detailed herein.

Co-inhibition ofLINC0520 and of the MAPK pathway

The present invention further aims at providing an innovative therapeutic approach for the treatment of melanoma, by inhibiting the functional expression of the melanoma-specific IncRNA LINC00520 and the mitogen-activated protein kinase (MAPK) pathway.

In a first aspect, the present invention relates to (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520 for use in combination with (ii) an inhibitor of the mitogen-activated protein kinase (MAPK) pathway, in the treatment of a melanoma.

In particular, the present invention is directed to the combined use of (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520, and (ii) an inhibitor of the mitogen-activated protein kinase (MAPK) pathway, for the preparation of a medicament for the treatment of a melanoma.

Further provided is a method for treating a melanoma, in a subject in need thereof, said method comprising the administration, to said subject, of a therapeutically effective amount of:

(i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520, and

(ii) an inhibitor of the mitogen-activated protein kinase (MAPK) pathway.

A “long-non coding RNA” (long ncRNA, or IncRNA) is generally referred as a non-coding RNA that is at least 200 nucleotides in length. A particular class of IncRNA are “long intergenic noncoding RNAs” (lincRNAs), which are sequences of IncRNA transcribed from non-coding DNA sequences between protein-coding genes. The terms “LINC00520” , “long intergenic non-protein coding RNA 520", “C14orf34” or “LASSIE" refer herein to the long intergenic non-protein coding RNA 00520. It is disclosed in the Gene database under ID 645687, in the HGNC database under ID 19843, in the Ensembl database under ID ENSG00000258791. The LINC00520gene encodes 12 splicing variants (i.e. isoforms), arising from up to 5 exons (see Table 2’). Unless specified otherwise, the term “IJNC0052(r encompasses all the isoforms thereof.

Table 2’. LINC00520 isoforms and exons (sequences available on Ensembl database) By “functional expression” of a gene, it is meant the transcription and/or translation of functional gene product.

An “inhibitor of functional expression" of a gene refers to a molecule or technical means capable of decreasing or even abolish the expression of said gene. For non-protein coding genes like LINC00520, functional expression can be deregulated on at least two levels: first, at the DNA level, e.g. by absence or disruption of the gene, or lack of transcription taking place (in both instances preventing synthesis of the relevant gene product); second, at the RNA level, e.g. by lack of splicing or lack or decrease in the activity mediated by said IncRNA. Inhibition can be evaluated by any means known to those skilled in the art including, but not limited to, assessing the level of IncRNA transcript using e.g. quantitative PCR. Besides, in the context of the present invention, it shall be understood that the inhibitor is selective, or in other words, specific for the targeted IncRNA, in that it does not inhibit, or at least does not substantially inhibit, any other target.

Such inhibitor is capable of inhibiting the functional expression of the IncRNA in vivo and/or in vitro. This inhibitor may inhibit the functional expression of the IncRNA by at least about 10%, 15% 20%, or 25%, preferably by at least about 30%, 35%, 40% or 45%, still preferably by at least about 50%, 55%, 60%, or 65%, yet preferably by at least about 70%, 75%, 80%, or 85%, more preferably by at least about 90%, or 95%.

An “inhibitor of the MAPK pathway” or “MAPK pathway inhibitor” refers to a molecule or technical means capable of decreasing or even abolish the MAPK pathway. The MAPK pathway is a signaling pathway well-known for its involvement in tumorigenesis including cancer cell proliferation, migration, invasion and survival. Inhibition of the MAPK pathway can be measured by conventional methods in the art, such as those described by Amara et al. (Eur J Cancer. 2017; 73:85-92) and/or Fernandez et al. (Cancers 2023, 15(12), 3224).

In the context of melanoma, the inhibitor of the MAPK pathway may preferably be a BRAF inhibitor, a MEK inhibitor, a C-Kit inhibitor, or any combination thereof.

A MAPK pathway inhibitor is capable of inhibiting the MAPK pathway in vivo and/or in vitro. This inhibitor may inhibit the MAPK pathway by at least about 10%, 15% 20%, or 25%, preferably by at least about 30%, 35%, 40% or 45%, still preferably by at least about 50%, 55%, 60%, or 65%, yet preferably by at least about 70%, 75%, 80%, or 85%, more preferably by at least about 90%, or 95%.

Generally speaking, the term “treatment” or “treating” means obtaining a desired physiological or pharmacological effect depending on the degree of severity of the symptom or disorder of interest, or risks thereof, i.e. herein, depending on the degree of severity or risks of developing such symptom or disorder. In the context of cancer such as melanoma, this includes, inter alia, the alleviation of symptoms, the reduction of inflammation, the inhibition of cancer cell growth, and/or the reduction of tumor size. Furthermore, these terms are intended to encompass curing as well as ameliorating at least one symptom of the condition or disease. For example, in the case of cancer, a response to treatment includes a reduction in cachexia, increase in survival time, elongation in time to tumor progression, reduction in tumor mass, reduction in tumor burden and/or a prolongation in time to tumor metastasis, time to tumor recurrence, tumor response, complete response, partial response, stable disease, progressive disease, progression free survival, overall survival, each as measured by standards set by the National Cancer Institute and the U.S. Food and Drug Administration for the approval of new drugs. See Johnson et al., J. Clin. Oncol., 2009; 21(7): 1404-1411.

In a preferred embodiment, the term treatment refers to the inhibition or reduction of cancer cell proliferation and/or the increase in cancer cell apoptosis in a subject having the cancer, herein melanoma.

In a more preferred embodiment, the inhibition or reduction of cancer cells and/or the increase in apoptosis of cancer cells is independent of the status of tumor-associated proteins, herein independent of BRAF and/or NBAS status, more preferably independent to the BRAF status.

The term “status” as used herein with regard to a particular protein, specifically tumor- associated proteins (e.g. BRAF status, NRAS status), refers to the mutational status and/or the expression of these particular proteins in cells of interest (herein, melanoma cells). Typically, the term is used in the sense “irrespective of or “independent of status, meaning that an effect is observed irrespective of expression levels of, or presence of mutations in, the particular protein in the cells of interest (herein, melanoma cells).

In melanoma, BRAF mutations are more commonly observed in intermittently sun-exposed skin, and associated with KIT mutations, which are present predominantly in mucosal and acral melanomas. Examples of BRAF mutations associated with melanoma include, without limitation, R461I, I462S, G463E, G463V, G465A, G465E, G465V, G468A, G468E, N580S, E585K, D593V, F594L, G595R, L596V, T598I, V599D, V599E, V599K, V599R, V600E/K (the most prominent), 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, preferably V600E.

In melanoma, NRAS mutations are more commonly observed in older patients (above 55 years-old), with a chronic pattern of UV exposure with lesions are usually located at the extremities. Examples of NRAS mutations typically associated with melanoma include, without limitation, G12D/C, G13R, Q61K, Q61R, Q61L, Q61V, Q61H, Q61P. In a particular embodiment, the NRAS mutation is Q61K/R.

It shall nevertheless be understood that BRAF and NRAS co-mutations are not mutually exclusive.

A “therapeutically effective amount' means herein an amount that is sufficient to achieve the effect for which it is indicated, herein the treatment of a melanoma. The amount of the combination therapy of the invention to be administered can be determined by standard procedures well known by those of ordinary skill in the art. Physiological data of the patient (e.g. age, size, and weight), the routes of administration and the disease to be treated have to be taken into account to determine the appropriate dosage, optionally compared with subjects that do not suffer from a melanoma. The amount may also vary according to other components of a treatment protocol (e.g. administration of other medicaments, etc.).

A “ subtherapeutic amount” or “ subtherapeutic dose” is an amount that is not therapeutically effective if administered in absence of any other agent or therapy.

It should be further understood that the “subject” or “patients” to be treated according to the invention is one having an integumentary system. Accordingly, preferred subjects to be treated are animals, more preferably mammals, most preferably humans.

The subject to be treated has a melanoma. A “melanoma” , also known as “malignant melanoma”, refers to a type of cancer that develops from the pigment-producing cells called melanocytes. There are three main categories of melanoma: cutaneous melanoma, the most common, which corresponds to the melanoma of the skin; mucosal melanoma which can affect any mucous membrane of the body, including the nasal passages, throat, vagina, anus, or mouth; and ocular melanoma, which is a rare form of melanoma affecting the eye (also known as uveal or choroidal melanoma).

The melanoma to be treated can be at any stage. For instance, the melanoma can be at stage VII, II, III, or IV, preferably stage II, III, or IV, more preferably stage III, or IV. More specifically, the melanoma can be at stage Tla, Tib, T2a, T2b, T3a, T4a, T4b, Nl, N2, N3, Mia, Mlb, or Mlc, preferably stage T2b, T3a, T4a, T4b, Nl, N2, N3, Mia, Mlb, or Mlc. In a preferred embodiment, the melanoma is at an advanced or metastatic stage.

The melanoma can be preferably selected from the group consisting of lentigo maligna, lentigo maligna melanoma, superficial spreading melanoma, acral lentiginous melanoma, mucosal melanoma, nodular melanoma, polypoid melanoma, desmoplastic melanoma, melanoma with small nevus-like cells, melanoma with features of a Spitz nevus, uveal melanoma, Harding-Passey melanoma, juvenile melanoma, amelanotic melanoma, Cloudman’s melanoma, and vaginal melanoma.

Optionally, the melanoma is a melanocytic melanoma. Optionally, the melanoma is a cutaneous or ocular (e.g. uveal) melanoma. Optionally, the melanoma is a cutaneous or ocular (e.g. uveal) melanocytic melanoma.

The subject to be treated may have resistant melanoma. As used herein, the term “resistant melanoma’" refers to a melanoma, which does not respond to a conventional treatment for said cancer. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment or does not respond anymore. The resistance to the treatment may lead to rapid progression of metastatic of melanoma.

The melanoma can be resistant to a targeted therapy, especially inhibitors of the MAPK pathway which include BRAF inhibitors, MEK inhibitors and C-Kit inhibitors; to chemotherapy; and/or to immune checkpoint inhibitors.

In a preferred embodiment, the melanoma is resistant to a targeted therapy, especially BRAF inhibitors such as dabrafenib, vemurafenib, or encorafenib, and/or MEK inhibitors, such as trametinib, cobimetinib, binimetinib, or selumetinib; or a combination thereof such as to the dabrafenib/ trametinib (DT) combination. The inhibitor of functional expression according to the invention may indeed restore or increase the sensitivity of melanoma to this type of therapy.

Alternatively, the melanoma may preferably not be resistant to a targeted therapy, especially inhibitors of the MAPK pathway which include BRAF inhibitors, MEK inhibitors and C-Kit inhibitors; to chemotherapy; and/or to immune checkpoint inhibitors.

In a preferred embodiment, the melanoma is not resistant to a targeted therapy, especially BRAF inhibitors such as dabrafenib, vemurafenib, or encorafenib; and/or MEK inhibitors such as trametinib, cobimetinib, binimetinib, or selumetinib; or a combination thereof such as to the dabrafenib/ trametinib (DT) combination. The inhibitor of functional expression according to the invention may indeed prevent, decrease or delay the appearance of a resistance to such therapy.

In a preferred embodiment, whether the melanoma is resistant or not, the melanoma can be associated with a BRAF mutation and/or with a NRAS mutation, as described above. The inhibitors of functional expression according to the invention may indeed act independently of BRAF and/or NRAS status, as explained above.

The present invention requires the inhibition of the melanoma-specific IncRNA LINC00520.

Inhibitors of functional expression of LINC00520 have been reported in the scientific and patent literature (Luan et al., Journal of Experimental & Clinical Cancer Research 2020, 39:96; Huang et al., BMC Pulm Med 2021, 21 :287; the contents of which are incorporated herein by reference in their entireties). Below is provided a non-exhaustive list of such inhibitors which can be used in the present invention.

Given the nature of the targeted RNA, the inhibitor of functional expression used in the present invention can preferably be either a nucleic acid molecule interfering specifically with the expression of the long-non coding RNA, or a genome editing system comprising a nuclease engineered to target specifically the long-non coding RNA.

A nucleic acid molecule interfering specifically with the expression of the long-non coding RNA might be preferred should one wish to inhibit functional expression at the RNA level, while a genome editing system comprising a nuclease engineered to target specifically the long-non coding RNA might be preferred should one wish to inhibit functional expression at the DNA level.

In a preferred embodiment, the inhibitor used in the present invention can be a nucleic acid molecule interfering specifically with the expression of the long-non coding RNA.

As used throughout the specification, the terms “ nucleic acid molecule", “nucleic acid', “polynucleotide" (polynucleoside) or “oligonucleotide" (oligonucleoside) refers to a succession of natural or synthetic nucleotides (or nucleosides) connected by intemucleotide (or intemucleoside) linkages, wherein each nucleotide (or nucleoside) or internucleotide (or intemucleoside) linkages may be modified or unmodified. A nucleotide is comprised of a nucleoside and a phosphate group. A nucleoside is comprised of a nucleobase and a sugar. A nucleobase comprises a modified and unmodified nucleobase. Unmodified nucleobases are well-known in the art and include adenine (A), thymine (T), cytosine (C), uracil (U) and guanine (G). An internucleotide (intemucleoside) linkage is a bond that forms a covalent linkage between adjacent nucleotides (nucleosides) in a nucleic acid, such as phosphate intemucleotide linkages that are naturally occurring. A nucleic acid may be a single-stranded or double-stranded DNA such as cDNA, genomic DNA, ribosomal DNA, and the transcription product of said DNA, such as RNA. Nucleic acids also encompass nucleic acids which can hybridize to a nucleic acid of reference. When a nucleic acid is designed to hybridize to a nucleic acid of reference, it can be chemically modified to enhance its stability, nuclease resistance, target specificity and/or improve their pharmacological properties (e.g. reduced toxicity, increased intracellular transport, etc.). For example, a nucleic acid may comprise modified nucleotide(s) and/or backbone, such as a modified sugar, a modified nucleobase, and/or a modified intemucleotide (intemucleoside) linkage. A standard modification is the replacement of native phosphates of the intemucleotide (intemucleoside) linkage with phosphorothioates (PS) so as to reduce the sensitivity to nucleases and accordingly improve the stability, half-life and tissue distribution of the nucleic acid (Eckstein F., Antisense and Nucleic Acid Drug Development, 2000, 10(2): 1117-221). Another standard modification which increases the affinity of the nucleic acid towards its target is the incorporation in the sugar moiety of a methoxyethyl (MOE) or a constrained ethyl (cEt) to the 2’position, or the tethering of the 4’-carbon to the 2-hydroxyl of the ribose ring to create a bicyclic locked nucleic acid (LNA), to name a few. All these modifications are well known to the one skilled in the art (see Watts et al. J Pathol., 2012; 226(2):365-379; Seth et a ., J Clin Invest., 2019; 129(3): 915-925).

Preferred nucleic acid molecules interfering specifically with the expression of the long-non coding RNA according to the invention are those capable of hybridizing specifically to the gene or transcripts of the IncRNA, at least to a part thereof; as such, these are usually non-naturally occurring nucleic acids (i.e. synthetic).

"Hybridizing" or "hydridization" means the pairing or annealing to a target, herein under physiological conditions, typically via hydrogen bonding between complementary nucleotides, such as Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding. A specific hybridization therefore means that the pairing or annealing is specific to the target (no off-targets effects, or at least no substantial off-targets effects).

‘M nucleic acid molecule capable of hybridizing" to a target nucleic acid thus means that a stretch of this nucleic acid is capable of forming base pairs to another stretch of the target nucleic acid. It is thus not absolutely required that all the bases in the region of complementarity are capable of pairing with bases in the opposing strand. Mismatches may be tolerated to some extent, as long as in the circumstances, the stretch of nucleotides is capable of hybridizing to its complementary part.

The nucleic acid molecule interfering specifically with the expression of the long-non coding RNA is preferably an antisense nucleic acid, an RNAi nucleic acid, or a ribozyme.

The term "antisense nucleic acid" or "antisense oligonucleotides" (ASO) designates a synthetic single-stranded oligonucleotide of which the sequence is at least partially complementary to a target nucleic acid, such as to the RNA sequence of a target gene (Lee et al., J Cardiovasc Transl Res. 2013; 6(6):969-80; DeVos et al., Neurotherapeutics 2013; 10(3):486-972013). An antisense nucleic acid is capable of altering the expression of a specific target gene, either by splicing modification, or by recruiting RNAse H leading to RNA degradation of RNA-DNA duplexes, thus blocking the expression of the target gene. An antisense nucleic acid is typically short in length, in general 5 to 50 nucleotides in length, such as 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length, more preferably 10, 15, 20, 25, 30, 35 nucleotides in length. It is well within the skill of the person in the art to design antisense nucleic acids specific to a target, based on the knowledge of a target sequence such as introns, exons, or 5’CAP (DeVos et al., Neurotherapeutics 2013; 10(3):486-972013). Antisense nucleic acids can be prepared by methods well-known in the art, such as by chemical synthesis and enzymatic ligation reactions.

In a preferred embodiment, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA inhibits the splicing of the pre- long-non coding RNA, or in other words, it inhibits the formation of the mature long-non coding RNA. An antisense nucleic acid can indeed be designed to block a splice acceptor (SA) site and/or an exon splicing enhancer (ESE) and/or any sequence which could modulate a pre-RNA splicing, i.e. it can be designed to be complementary to a part of the pre-RNA comprising an SA, an ESE, or any sequence which could modulate its splicing.

In an alternative preferred embodiment, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA induces a RNAse H mediated degradation. RNAse H is a cellular enzyme which recognizes duplex between DNA and RNA, and enzymatically cleaves the RNA molecules. Accordingly, to induce RNAse H mediated degradation, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA comprises a region that comprises DNA or DNA-like nucleotides complementary to the targeted IncRNA which is responsible for RNAse H recruitment, ultimately leading to the cleavage of the target nucleic acid.

Regardless of its mode of action for inhibiting the functional expression of the IncRNA, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA is complementary to all or part of the IncRNA, preferably to a part thereof, such as any one of its exons or introns. Sequences of LINC00520 are well known in the art, as explained above.

In a preferred embodiment, the antisense nucleic acid interfering specifically with the expression of LINC00520 comprises or consists of a contiguous nucleotide sequence of at least 10 to 30 (preferably 11 to 29, 12 to 26, 13 to 24, 14 to 22, 14 to 17, 14, 15 or 16) nucleotides in length that is complementary to a specific region of the LINC00520 sequence. Non limiting examples of such antisense nucleic acids include, without limitation, those comprising or consisting a contiguous nucleotide sequence of at least 10 nucleotides in length of the sequence SEQ ID NO: 4 or 5, such as those comprising or consisting of the sequence SEQ ID NO: 4 or 5.

As described above, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA may further comprise at least one modified sugar, at least one modified internucleotide (or internucleoside) linkage, and/or at least one modified nucleobase, so as to enhance its properties, such a reduction in sensitivity to nucleases, increase in stability, half-life and/or tissue distribution, and/or enhancement of its affinity towards its target nucleic acid. In a preferred embodiment, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA comprises at least one modified sugar, in particular in position 2’ of the nucleotide (or nucleoside) such as 2’-O-methyl (2’-O-Me), 2’fluoro (2’-F), 2’0- methoxyethyl (2’-M0E also known as MOE), 2’,4’-brigded, or cEt (constrained ethyl).

A “modified sugar” is a sugar that is non-naturally occurring. 2’,4’-brigded, which is the introduction of a methylene bridge between the 2’ and 4’ position of the nucleotide, defining locked nucleic acids (LNA), is herein particularly preferred so as to confer or increase the stability of a nucleic acid when hybridized to a complementary DNA or RNA nucleic acid.

In a preferred embodiment, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA comprises at least one modified intemucleotide (or internucleoside) linkage, such as a phosphorothioate intemucleotide (or internucleoside) linkage.

A “modified internucleotide linkage” (or modified internucleoside linkage) is an intemucleotide (or intemucleoside) linkage in a nucleic acid that is non-naturally occurring, and is thus referred as a non-phosphate linkage. A phosphorothioate linkage, which is a modified phosphate linkage in which one of the non-bridging oxygen atoms is replaced with a sulfur atom, is herein particularly preferred so as to confer or increase the resistance of a nucleic acid to nucleases.

In a preferred embodiment, the antisense nucleic acid interfering specifically with the expression of the long-non coding RNA is a gapmer.

A “gapmer” refers to a nucleic acid molecule comprising an internal segment having a plurality of nucleotides (or nucleosides) that support RNase H cleavage positioned between external segments, each having one or more nucleotides (or nucleosides), wherein the nucleotide (or nucleosides) comprising the internal segment are chemically distinct from the immediately adjacent nucleotide(s) (or nucleoside(s)) comprising the external segments. The internal or central segment may be referred to as the “gap”, “gap segment” or “gap region” (G); while the external segments may be referred to as the “wings”, “flanks”, “wing segments” , “flank segments” , “wing regions” or “flank regions” (F for the 5’ flank region and F’ for the 3’ flank region). Typically, the F and F’ regions are composed of modified ribonucleotides (RNA*) which are complementary to a target nucleic acid; whereas the G region is composed of deoxyribonucleotides, i.e. DNA or DNA-like molecules, which is responsible for RNAse H recruitment which ultimately leads to the degradation of the target nucleic acid. A gapmer is therefore a chimeric antisense. Gapmers can typically comprise a gap region (G) of 5 to 15 deoxynucleotides (or deoxynucleosides) flanked by wing regions (F and F’) of 2 to 10 modified nucleotides (or nucleosides) each. In a preferred embodiment, the gapmer interfering specifically with the expression of the long-non coding RNA comprises or consists of a contiguous nucleotide (or nucleoside) sequence corresponding to the following formula:

5’-F (RNA*) - G (DNA or DNA-like) - F’ (RNA*)-3’

It should be understood that any of the modifications that can improve the properties of the gapmer, as discussed above, can be used and combined together.

For example, the gapmer interfering specifically with the expression of the long-non coding RNA can comprise or consist of a contiguous nucleotide (or nucleoside) sequence that is complementary to a specific region of the IncNRA sequence and that corresponds to the following formula:

5’-F (RNA*) - G (DNA or DNA-like) - F’ (RNA*)-3’ wherein the gap segment (G) comprises or consists of a sequence of at least 5 to 15 nucleotides (or nucleosides) in length, wherein each wing segment (F or F’) comprises at least 2 to 10 nucleotides (or nucleosides) in length, wherein each nucleotide (or nucleoside) of each wing segment (F and F’) comprises a modified sugar, in particular in position 2’ of the nucleotide (or nucleoside) such as 2’-O-methyl (2’-0-Me), 2’fluoro (2’-F), 2’O-methoxyethyl (2’-M0E also known as MOE), 2’,4’-brigded, or cEt (constrained ethyl), more preferably 2’,4’-brigded, and wherein each internucleotide (or intemucleoside) linkage is a modified linkage, preferably is a phosphorothioate linkage.

In a preferred embodiment, the gapmer interfering specifically with the expression of LINC00520 comprises or consists of a contiguous nucleotide (or nucleoside) sequence corresponding to the following formula:

5’-F (RNA*) - G (DNA or DNA-like) - F’ (RNA*)-3’ wherein the gap segment (G) comprises or consists of a sequence of at least 5 to 15 nucleotides (or nucleosides) in length that is complementary to a specific region of the LINC00520 sequence, wherein each wing segment (F or F’) comprises at least 2 to 10 nucleotides (or nucleosides) in length, wherein each nucleotide (or nucleoside) of each wing segment (F and F’) comprises a modified sugar, in particular in position 2’ of the nucleotide (or nucleoside) such as 2’-O-methyl (2’-0-Me), 2’fluoro (2’-F), 2’O-methoxyethyl (2’-M0E also known as MOE), 2’,4’-brigded, or cEt (constrained ethyl), more preferably 2’,4’-brigded, and wherein each internucleotide (or intemucleoside) linkage is a modified linkage, preferably is a phosphorothioate linkage.

Non limiting examples of gapmers interfering specifically with the expression of LINC00520 include, without limitation, gapmers comprising or consisting of any one of the following sequences:

Tbs Tbs Tbs dGs dAs dTs dGs dAs dGs dTs dGs dAs dGs Tbs Cbs Gb (SEQ ID NO: 12)

Gbs Abs Gbs dTs dmCs dGs dmCs dTs dGs dAs dGs dAs dAs Tbs Tbs Ab (SEQ ID NO: 13) wherein A is an adenine, mC is a 5-methylcytosine, G is a guanine, T is a thymine, b is LNA (2',4'-methylene bridged sugar moiety), d is a 2’ -deoxyribosyl sugar moiety, and s is a phosphorothioate intemucleotide (or intemucleoside) linkage.

Other formats of gapmers are well-known in the art, and can be easily conceived by the skilled practitioner.

In another embodiment, RNAi can be used to inhibit the functional expression of the IncRNA.

“RNAi nucleic acid" refers to a nucleic acid that can inhibit expression of a target gene by RNA interference (RNAi) mechanism. By contrast to antisense nucleic acids, RNAi nucleic acids target mature RNA. RNAi nucleic acids are well-known in the art, and include short-hairpin RNA (shRNA), small interfering RNA (siRNA), double-stranded RNA (dsRNA), and single-stranded RNA (ssRNA) (Sohail et al. 2004, Gene Silencing by RNA Interference: Technology and Application 1 ^st Edition, ISBN 9780849321412; WO 99/32619; Wang et al., Pharm Res 2011, 28:2983-2995). RNA interference designates a phenomenon by which dsRNA specifically suppresses expression of a target gene at post-transcriptional level. In normal conditions, RNA interference is initiated by double-stranded RNA molecules (dsRNA) of several thousands of base pair length. In vivo, dsRNA - such as shRNA- introduced into a cell is cleaved by Dicer into a mixture of short interfering RNA called siRNA; the latter will bind to another enzyme (RISC) that will catalyze the cleavage of both the siRNA and target mRNA (Bernstein et al. Nature. 2001;409(6818):363-6). In mammalian cells, the siRNAs that are naturally produced by Dicer are typically 21-23 bp in length, with a 19 or 20 nucleotides duplex sequence, two-nucleotide 3' overhangs and 5'-triphosphate extremities (Zamore et al. Cell. 2000,101(l):25-33; Elbashir et al. Genes Dev. 2001, 15(2): 188-200; Elbashir et al. EMBO J. 2001, 20(23):6877-88). The selected siRNA or shRNA target sequence should be subjected to a BLAST search against EST database to ensure that the only desired gene is targeted. Various products are commercially available to aid in the preparation and use of synthetic siRNA or shRNA. The RNAi nucleic acid can be of at least about 10 to 40 nucleotides (or nucleosides) in length, preferably about 15 to 30 base nucleotides (or nucleosides) in length. siRNA or shRNA can comprise naturally occurring RNA, synthetic RNA, or recombinantly produced RNA, as well as altered RNA that differs from naturally- occurring RNA by the addition, deletion, substitution and/or alteration of one or more nucleotides (or nucleosides). Such alterations can include addition of non-nucleotide (or non-nucleoside) material, such as to the end of the molecule or to one or more internal nucleotides (or nucleoside) of the RNAi, including modifications that make the RNAi resistant to nuclease digestion, as described above.

Thus, in a preferred embodiment, the nucleic acid molecule interfering specifically with functional expression of the IncRNA is an RNAi nucleic acid, that is complementary to at least one part of the IncRNA, in particular to at least one exon thereof The RNAi nucleic acid can be a siRNA or shRNA of at least about 10 to 40 nucleotides (or nucleosides) in length, preferably of about 15 to 30 nucleotides (or nucleosides) in length.

In a preferred embodiment, the RNAi nucleic acid interfering specifically with the expression of LINC00520 comprises or consists of a contiguous nucleotide sequence of at least 10 to 40 (preferably 15 to 30, 16 to 25, 17 to 24, 20, 21, 22 or 23) nucleotides in length that is complementary to a specific region of the LINC00520 sequence. Non limiting examples of such RNAi nucleic acid include, without limitation, those comprising or consisting of a contiguous nucleotide sequence of at least 10 nucleotides in length of the sequence SEQ ID NO: 23, preferably those comprising or consisting of the sequence SEQ ID NO: 23.

In another embodiment, the nucleic acid interfering specifically with the functional expression of the IncRNA is a ribozyme.

"Ribozymes" are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region. Ribozyme molecules specific for a target (herein a IncRNA) can be designed, produced, and administered by methods commonly known to the art (see e.g., Fanning and Symonds (2006) RNA Towards Medicine (Handbook of Experimental Pharmacology), ed. Springer p. 289-303).

In a preferred embodiment, the nucleic acid molecule interfering specifically with the functional expression of LINC00520 is a ribozyme targeting said IncRNA.

Yet, in another embodiment, genome editing can be used to inhibit the functional expression of the IncRNA. More specifically, one can use a genome editing system comprising a nuclease engineered to target the IncRNA gene. “Genome editing is a type of genetic engineering in which DNA is inserted, replaced, or removed from a genome using artificially engineered nucleases, also called molecular scissors. Nucleases create specific double-stranded break (DSBs) at desired locations in the genome, and harness the cell's endogenous mechanisms to repair the induced break by natural processes of homologous recombination (HR) or non-homologous end-joining (NHEJ). There are currently four known families of nucleases suitable for genome editing: zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system in particular the Cas9 system (Mali et al, Nature Methods, 2013 ; 10(10):957-63), or engineered meganucleases re-engineered homing endonucleases. Said nucleases can be delivered to the cells either as DNAs or mRNAs, such DNAs or mRNAs and can be engineered to target the IncRNA gene.

A particularly preferred genome editing system according to the invention is the CRISPR/Cas system in particular the Cas9 system.

In a preferred embodiment, the genome editing system interfering specifically with functional expression of LINC00520 is a CRISPR/Cas system, in particular a CRISPR/Cas9 system, targeting said IncRNA. To do so, one may use one or more single guide RNA (sgRNA) targeting specifically LINC00520. Non limiting examples of such sgRNA include, without limitation, those comprising or consisting of a contiguous nucleotide sequence of at least 10 nucleotides in length of the sequence SEQ ID NO: 19, 20 or 21, preferably those comprising or consisting of the sequence SEQ ID NO: 19, 20 or 21, or any combination thereof. In a preferred embodiment, the genome editing system is a triplet-target CRISPR system, meaning that three guide RNA are used to target LINC00520, such as those described above.

The present invention further requires the inhibition of the MAPK pathway.

Inhibitors of the MAPK pathway have been reported in the scientific and patent literature, and include BRAF inhibitors, MEK inhibitors and/or C-Kit inhibitors, which can be useful for treating melanoma.

Thus, in a preferred embodiment, the (ii) inhibitor of the mitogen-activated protein kinase (MAPK) pathway is a BRAF inhibitor, a MEK inhibitor, a C-Kit inhibitor, or any combination thereof; preferably is a BRAF inhibitor, a MEK inhibitor or a combination thereof.

BRAF inhibitors are well known in the art. They have notably been described in e.g. WO 2005/062795, WO 2007/002325, WO 2007/002433, WO 2008/079903, and WO 2008/079906, the disclosure thereof being incorporated by reference in its entirety. In a particular embodiment, the inhibitor of BRAF can be vemurafenib or other inhibitors as disclosed in US8470818, US8470818, US8143271, US7863288, US9447089, US7504509 or US8741920, the disclosure thereof being incorporated by reference in its entirety. Vemurafenib also known as PLX4032, RG7204 or R05185426 and commercialized by Roche as zelboraf. Alternatively, the inhibitor of BRAF can be Dabrafenib also known as tafinlar, which is commercialized by Novartis. Other inhibitors of BRAF can be encorafenib, or also LGX818 (Novartis), TAK-632 (Takeda), MLN2480 (Takeda/Millennium), or PLX-4720 (Plexxikon).

In a preferred embodiment, the BRAF inhibitor is dabrafenib, vemurafenib, or encorafenib, or any combination thereof.

MEK inhibitors are also well known in the art. In a particular embodiment, the inhibitor of MEK can be Trametinib also known as mekinist, which is commercialized by GSK. In another particular embodiment, the inhibitor of MEK can be Cobimetinib also known as cotellic commercialized by Genentech. In a further particular embodiment, the inhibitor of MEK can be Binimetinib also known as MEK162, ARRY-162 is developed by Array Biopharma. Other MEK inhibitors can be AZD6244 (AstraZeneca/ Array BioPharma), R05126766 (Roche/Chugai), GDC- 0623 (Genentech/Chugai), PD0325901 (Pfizer), and Selumetinib.

In a preferred embodiment, the MEK inhibitor is trametinib, cobimetinib, binimetinib, selumetinib, or any combination thereof.

In a preferred embodiment, the MAPK inhibitor is a combination of BRAF inhibitor and a MEK inhibitor, such as a combination of dabrafenib and trametinib.

Example 1 described below notably provides evidence that the combination of the ASO targeting LINC00520 with a combination of a BRAF inhibitor and a MEK inhibitor act in a synergistic manner to increase the apoptosis of melanoma cells, even at suboptimal dose.

The skilled person would readily understand that any one of the (i) inhibitors of functional expression of the long-non coding RNA (IncRNA) LINC00520, and any one of the (ii) inhibitors of the MAPK pathway, as described herein, can be combined for the purpose of the present invention.

In a particularly preferred embodiment, (i) the inhibitor of functional expression of the long- non coding RNA (IncRNA) LINC00520 is a nucleic acid molecule interfering specifically with the expression of said IncRNA, as described above; and (i) the inhibitor of the MAPK pathway is a BRAF inhibitor, a MEK inhibitor, a C-Kit inhibitor, or any combination thereof, as described above.

In a further preferred embodiment, (i) the nucleic acid molecule interfering specifically with the expression of LINC00520 is an antisense nucleic acid, as described above; and (ii) the inhibitor of the MAPK pathway is a BRAF inhibitor, a MEK inhibitor, or any combination thereof, as described above.

For example, (i) the nucleic acid molecule interfering specifically with the expression of LINC00520 can be an antisense nucleic acid comprising or consisting a contiguous nucleotide sequence of at least 10 nucleotides in length of the sequence SEQ ID NO: 4 or 5, such as an antisense nucleic acid comprising or consisting of the sequence SEQ ID NO: 4 or 5; and (ii) the BRAF inhibitor is dabrafenib, vemurafenib, or encorafenib, or any combination thereof, and/or the MEK inhibitor is trametinib, cobimetinib, binimetinib, selumetinib, or any combination thereof.

In a more preferred embodiment, (i) the antisense nucleic acid interfering specifically with the expression of LINC00520 is a gapmer as described above, such as an LNA gapmer which may comprise phosphothiorate linkages; and (ii) the BRAF inhibitor is dabrafenib and/or the MEK inhibitor is trametinib, as described above.

For example, (i) the gapmer interfering specifically with the expression of LINC00520 can comprise or consist of any one of the following sequences: Tbs Tbs Tbs dGs dAs dTs dGs dAs dGs dTs dGs dAs dGs Tbs Cbs Gb (SEQ ID NO: 12), or Gbs Abs Gbs dTs dmCs dGs dmCs dTs dGs dAs dGs dAs dAs Tbs Tbs Ab (SEQ ID NO: 13); and (ii) the MAPK inhibitor can be a combination of dabrafenib and a trametinib.

Other combination of (i) inhibitors of the functional expression of the IncRNAs LINC00520 and (ii) inhibitors of the MAPK pathway can also be envisioned, such as: (i) a genome editing system comprising a nuclease engineered to target specifically LINC00520, as described above and (ii) a BRAF inhibitor, a MEK inhibitor, a C-Kit inhibitor, or any combination thereof, as described above.

It shall be further understood that any one of the (i) inhibitors of functional expression of the long-non coding RNA (IncRNA) LINC00520, and any one of the (ii) inhibitors of the MAPK pathway, as described herein, can be further combined with an additional therapy, especially one that is suited for melanoma. This includes, without limitation, surgery, radiotherapy, chemotherapy, other targeted therapy or immune checkpoint therapy, or any combination thereof. Such therapies are well-known in the art, and therefore need not be detailed herein.

The combination of (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520, and (ii) an inhibitor of the MAPK pathway, as described herein, may be suitable for administration to a cell, tissue and/or an organ of individuals affected by or at risk of suffering from a melanoma, and may be administered in vivo, ex vivo or in vitro. Since melanoma affects melanocytes, it is preferred that said cells are melanocytes, and/or that said tissue or organ is the skin or the eye.

Whether the combination is administered simultaneously, separately or sequentially, each inhibitor can be delivered as it is to the subject, or be formulated to be compatible with their intended route of administration.

For example, the combination can be formulated in a combined pharmaceutical composition, or in separate pharmaceutical compositions, in a form suitable for parenteral, oral, transdermal or topical or intraocular administration, such as a liquid suspension, a solid dosage form (granules, pills, capsules or tablets), or a paste or gel.

For the purposes of the invention, a particularly preferred form of administration is parenteral administration, such as subcutaneous, intradermal, intravenous; transdermal administration; topical administration; or even intraocular administration.

It is within the skill of the person in the art to formulate such pharmaceutical composition(s) in accordance with standard pharmaceutical practice (see, e.g., Remington: The Science and Practice of Pharmacy (20th ed.), ed. A. R. Gennaro, Lippincott Williams & Wilkins, 2000 and Encyclopedia of Pharmaceutical Technology, eds. J. Swarbrick and J. C. Boylan, 1988-1999, Marcel Dekker, New York). Pharmaceutical compositions according to the invention may notably be formulated to release the combination therapy immediately upon administration or at any predetermined time or time period after administration.

When formulated within a pharmaceutical composition, the combination can be further combined with a pharmaceutically acceptable excipient.

As used herein, the term a “pharmaceutically acceptable excipient” means an inactive or inert, and therefore nontoxic, component, as it has no pharmacological action itself, which can be used to improve properties of a composition, such as shelf-life, retention time at the application site, consumer acceptance, etc. It includes, without limitation, surfactants (cationic, anionic, or neutral); surface stabilizers; other enhancers, such as preservatives, wetting or emulsifying agents; solvents; buffers; salt solutions; dispersion medium; isotonic and absorption delaying agents, and the like; that are physiologically compatible.

As explained above, the combination therapy is provided in a therapeutically effective amount so as to treat the melanoma.

In a preferred embodiment, the amount of each inhibitor is a pediatric dose in monotherapy, or a dose that is subtherapeutic in monotherapy (yet, is such that it is therapeutically effective in the combination therapy of the invention). In other words, the amount of each inhibitor is a subtherapeutic dose (yet, is such that it is therapeutically effective in the combination therapy of the invention).

Those of skill in the art will recognize that such parameters are normally worked out during clinical trials.

In another aspect, the present invention relates to (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520, and (ii) an inhibitor of the MAPK pathway, as described above, as a combined preparation for simultaneous, separate or sequential use in the treatment of a melanoma.

The invention relates to the simultaneous, separate or sequential use of (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520 and (ii) an inhibitor of the MAPK pathway, as described above, for the preparation of a medicament for the treatment of a melanoma.

Further provided is therefore a method for treating a melanoma, in a subject in need thereof, said method comprising the simultaneous, separate or sequential administration, to said subject, of a therapeutically effective amount of (i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520 and (ii) an inhibitor of the MAPK pathway, as described above.

Preferred embodiments, as described above, apply herein mutatis mutandis.

In another aspect, the present invention relates to a pharmaceutical composition comprising

(i) an inhibitor of functional expression of the long-non coding RNA (IncRNA) LINC00520, and

(ii) an inhibitor of the MAPK pathway, as described above, and optionally (iii) a pharmaceutically acceptable excipient.

Preferred embodiments, as described above, apply herein mutatis mutandis.

In another aspect, the invention pertains to an in vitro method for identifying a melanoma tumor suitable for treatment with the inhibitors of the invention, said method comprising: a) determining, in a melanoma tumor sample, (i) the expression level of the long-non coding RNA (IncRNA) LINC00520, and (ii) the response to (ii) an inhibitor of the mitogen-activated protein kinase (MAPK) pathway; b) comparing the expression level determined in step a) with a reference expression level for said IncRNA, and comparing the response determined in step a) with a reference response to said inhibitor, thereby identifying whether the melanoma tumor is suitable for said treatment.

The term “ expression level”, as applied to a IcnRNA, refers herein to the amount or level of said IncRNA expressed in biological sample, such as a cell, tissue, or organ(s), herein preferably melanocytes or skin. The term “level” as used herein refers to an amount (e.g. relative amount or concentration) of a IncRNA that is detectable or measurable in a sample. For example, the level can be a relative amount by comparison to a reference expression level. The act of actually “determining the expression lever of a IncRNA in a biological sample refers to the act of actively detecting whether the IcnRNA is expressed in said sample or not, and notably allows to detect whether the expression of the IcnRNA is upregulated, downregulated or substantially unchanged when compared to a reference expression level.

By “reference expression lever or “control expression lever, as applied to a IcnRNA, it is meant a predetermined expression level of said IcnRNA, which can be used as a reference in the method of the invention. For example, a reference expression level can be the expression level of the IncRNA in a biological sample of a healthy subject, or the average or median expression level in a biological sample of a population of healthy subjects.

In a preferred embodiment, the expression level is performed by PCR, such as quantitative PCR (qPCR).

The term "response" or “sensitivity”, as applied to a tumor subjected to a cancer therapy, means herein that said tumor is either “resistant' to the therapy or “non-resistant” thereto, as described above.

By “reference response”, as applied to a tumor sample subjected to a cancer therapy, it is meant a predetermined response of a tumor sample of the same tissue to that therapy, which can be used as a reference in the method of the invention. For example, a reference response can be the response in a biological sample of a diseased subject, or the average or median reference response in a biological sample of a population of diseased subjects.

In a preferred embodiment, the response can be determined by performing an apoptosis assay and/or proliferation assay on the tumor sample.

In a preferred embodiment, an increased expression level of LINC00520 in step a) and non- resistance to the inhibitor of the MAPK pathway in step a) is indicative of the suitability of said treatment, especially when the reference expression level is one of a healthy subject or population of heathy subjects. In a further aspect, the invention relates to a kit for use in the in vitro method for identifying a melanoma tumor suitable for treatment with the inhibitors, said kit comprising: a) at least one reagent capable of specifically determining the (i) expression level of the long-non coding RNA (IncRNA) LINC00520; and b) at least one reagent capable of determining (ii) the response to an inhibitor of the mitogen-activated protein kinase (MAPK) pathway; c) optionally, instructions for performing said method.

As used herein, the term “instructions” refers to a publication, a recording, a diagram, or any other medium which can be used to communicate how to perform a method of the invention. Said instructions can, for example, be affixed to a container which contains said kit. Preferably, the instructions for using said kit include the reference expression level for the IncRNA and/or the reference response to the inhibitor of the MAPK pathway.

The term “reagent capable of specifically determining the expression lever, as applied to a IcnRNA, designates a reagent or a set of reagents which specifically recognizes said IncRNA, and allows for the quantification of the expression level thereof. These reagents can be for example nucleotide probes (also known as primers). In the context of the present invention, such reagent is said to be “specific” for the IncRNA or “recognizes specifically” the IncRNA if it 1) exhibits a threshold level of hydridization, and/or 2) does not significantly cross-react with other targets. The hydridization capacity and cross-reactivity of a reagent can be easily determined by one skilled in the art, and thus need to be further detailed herein.

The term “reagent capable of determining the response", as applied to a tumor sample subjected to a cancer therapy, designates a reagent or a set of reagents which can quantify the response thereto. Such reagents are well-known in the art, and thus need not be further detailed herein.

The present invention will be better understood in the light of the following detailed experiments. Nevertheless, the skilled artisan will appreciate that the present examples are not limitative and that various modifications, substitutions, omissions, and changes may be made without departing from the scope of the invention. EXAMPLES

EXAMPLE 1

MATERIALS AND METHODS

Cell culture and

Melanoma cell lines Sk-mel-25, Sk-mel-25R, Sk-mel-28 and 501mel were grown in RPMI 1640 medium supplemented with 10% Fetal Calf Serum (FCS) and gentamicine; IGR-37 and IGR- 39 in RPMI 1640 medium supplemented with 15% FCS and gentamicine. MM011, MM117, MM047, MM099 were grown in HAM-F10 medium supplemented with 10% FCS, 5.2 mM glutamax, 25 mM Hepes and penicillin/streptomycin (7.5 ug/ml). M229, M229R, M249, M249R were grown in DMEM medium supplemented with glucose (4.5 g/1), 5% FCS and penicillin/streptomycin (7.5 ug/ml). HEK293T cells were grown in DMEM medium supplemented with glucose (lg/1), 10% FCS and penicillin/streptomycin (7.5 ug/ml).

For GapmeR knockdown experiments cells were transfected using Lipofectamine RNAiMAX (Invitrogen) with 20 nM of GapmeR (Qiagen) and harvested 48 or 72 hours as indicated. For combination GapmeR experiments cells were transfected with 15nM of LINC00518 GAP#2 and/or LINC00520 GAP#2, using control GapmeR to obtain a constant 30 nM in each transfection.

RNAs for LINC00520 and MITF were detected with the RNAscope assay (Advanced Cell Diagnostics, ACD, Hayward, CA) according to the manufacturer’s protocols. Briefly, patient sections were de-paraffinized, incubated with hydrogen peroxide at room temperature for 10 min, boiled with target retrieval reagent for 15 min, and then treated with protease plus reagent at 40°C for 30 min. The sections were hybridized with Hs-MITF probe (ACD, Cat. No. 310951), or custom designed LINC00520 probe. Hybridization signals were amplified and visualized with RNAscope Multiplex Fluorescent Reagent Kit v2 (ACD, Cat. No. 323100). Images were captured with a confocal (Leica DMI6000) microscope.

RNA extraction and quantitative PCR

Total mRNA isolation was performed using Trizol and isopropanol precipitation. Total was treated with DNAsel following the TurboDnase free kit instructions (Thermofisher) and reversed transcribed using Superscript IV reverse transcriptase (Thermofisher) following manufacturer instructions. qRT-PCR was carried out with SYBR Green I (Roche) and monitored by a LightCycler 480 (Roche). Target gene expression was normalized using TBP, HBMS, GAPDH, ACTB, RPL13A as reference genes.

Proliferation and apoptosis analyses by flow cytometry

To assess apoptosis and proliferation after GapmeR-mediated knockdown, cells were stained with Cell Trace Violet (Invitrogen) on the day of transfection harvested after 72 hours and stained with the active caspase 3 kit (BD Biosciences) following manufacturer instructions. Where indicated, cells were stained with Annexin-V (Biolegend) and TOPRO-3 (Invitrogen) following manufacturer instructions. Cells were analysed on a LSRII Fortessa (BD Biosciences) and data were analysed with Flowjo software (Tree Star).

Oligonucleotide-mediated LINC00520 Purification

501Mel cells were grown in 15cm petri dishes, harvested by trypsinization, washed, pelleted, re-suspended in lysis buffer (TrisHCl 20mM pH8, NaCl 200mM, MgC12 2.5mM, Triton 0.05%, DEPC water) supplemented with fresh DTT (ImM), protease and phosphatase inhibitor cocktail (Thermofisher) and RNAsin (Thermofisher) and kept 20 minutes on ice. Membranes were pelleted 3000g for 3 minutes at 4°C and supernatant pre-cleared for 1 hour at 4°C with streptavidin coated sepharose beads. The lysate was incubated 2 hours with streptavidin coated beads and 400pmol anti-PCA3 or LINC00520 specific DNA biotinylated oligonucleotides. Beads were pelleted for 3 min at 3000g and washed 3 times with lysis buffer. After final wash beads were divided for RNA and protein extraction. RNA was purified by Trizol and isopropanol precipitation, digested with DNAse, reverse transcribed and analyzed by qPCR for LENOX and TINCR. Proteins were eluted by boiling beads in Laemli sample buffer and separated on NuPAGE® Novex 4-12% gradient gels. Three independent experiments were performed and the entire lane was excised in seven consecutive bands and subjected to “in-gel” digestion. Mass spectrometry was performed at the Harvard University proteomics facility. For UV crosslinking, cells were were washed with PBS and irradiated using a UVP CL-1000 Crosslinker (Analytik Jena) at 400 mJ/cm ² prior to cell harvesting.

Protein extraction and western blotting

Whole cell extracts were prepared by freeze-thaw technique using LSDB 500 buffer (500 mM KC1, 25 mM Tris at pH 7.9, 10% glycerol (v/v), 0.05% NP-40 (v/v), 16mM DTT, and protease inhibitor cocktail). Lysates were subjected to SDS-polyacrylamide gel electrophoresis (SDS- PAGE) and proteins were transferred onto a nitrocellulose membrane. Membranes were incubated with primary antibodies in TBS+ 5% BSA + 0.01% Tween-20. Overnight at 4 °C. The membrane was then incubated with HRP -conjugated secondary antibody (Jackson ImmunoResearch) for Ih at room temperature, and visualized using the ECL detection system (GE Healthcare).

DHX36 immunoprecipitation and RNA-sequencing

501Mel cells were transduced with lentiviruses expressing either a Doxycycline-inducible control shRNA or a LINC00520 targeting shRNA (see annex below). Each cell line was grown in 15cm petri dishes and treated for 48 hours Doxycycline with before harvesting and preparation of cytoplasmic protein extracts as described above. For immunoprecipitation, extracts were precleared for 1 hour at 4°C with protein G magnetic beads. Lysate was quantified by Bradford protein quantification assay (Biorad) and incubated overnight at 4°C with anti-DHX36a antibody (13159-1-AP Proteintech) or control IgG. Protein G magnetic beads were added for 3 hours at 4°C to isolate RNA-protein complexes and washed 5 times in lysis buffer. After final wash RNA was purified by Trizol and isopropanol precipitation and proteins eluted by boiling beads in Laemli sample buffer. Immunoprecipitation and subsequent RNA-seq was performed in 3 biological replicates. Briefly, ccomplementary DNA was generated and subjected to ribo-depletion with Ribo-Zero Plus kit and the library prepared using the llumina Stranded Total RNA Prep Ligation before sequencing on an Illumina Hi-Seq2000. Analyses was performed by using the Wald test for differential expression and implemented in the Bioconductor package DESeq2 version 1.16.1. Genes with high Cook’s distance were filtered out and independent filtering based on the mean of normalized counts was performed, - values were adjusted for multiple testing using the Benjamini and Hochberg method. RNA showing differential association with DHX36 in control shRNA lines versus LINC00520 shRNA lines were defined as genes with log2(FoldChange) >1 or <-l and adjusted - value <0.05.

Mitochondria fractionation

Mitochondria were isolated with the Mitochondria Isolation kit (Thermofisher) following manufacturer instructions. Harvested cells were washed and pelleted, resuspended in buffer A and incubated 2 minutes on ice. Buffer B was added for 5 minutes, vortexing every minute and diluted with buffer C. Nuclei were pelleted 10 minutes at 700g and supernatant centrifuged for 15 minutes at 3000g. Purified mitochondria were washed once in buffer C and used for RNA (Trizol- isopropanol precipitation) or protein (TBS+CHAPS 2%) extraction. Table 3. Gapmers, shRNA and primers used in the present invention

RESULTS

Chromatin immunoprecipitation coupled to high throughput sequencing in melanocytic type 501Mel melanoma cells showed that the LINC00520 locus comprised multiple prominent binding sites for transcription factor MITF, the master regulator of melanoma cell identity (Figure 1A). Silencing of MITF leads to strongly reduced LINC00520 expression showing that it is a direct MITF target gene (Figure IB). Mining of public databases showed that LINC00520 expression was higher in cutaneous melanoma compared to normal tissues and other non-melanoma cancers (Figure 2A). Further mining of the public GTEX database revealed that LINC00520 displayed very low expression (>0.15 TPM) in several normal tissues with highest expression in oesophageal mucosa (Figure 2B). In the TCGA tumour collection, LINC00520 showed highest expression in cutaneous melanoma (40-250 TPM, thus more than 100-fold greater than in normal tissues), but lower expression in uveal melanoma and was expressed at low levels in several other tumour types (Figure 2C). Moreover, its expression increased in melanoma compared to nevi (Figure 2D). RNAscope confirmed the absence of LINC00520 expression in keratinocytes, its low expression in normal MITF-expressing melanocytes with much higher expression in primary melanoma (Figure 2E).

Importantly, overall high LINC00520 expression showed a strong association with poor patient outcome in the TCGA cutaneous melanoma dataset (SKCM) (Figure 3A). However, when the TCGA cutaneous melanoma dataset was divided into primary and metastatic melanoma patients, high LINC00520 expression was associated with favorable outcome in primary melanoma, but poor outcome in metastatic melanoma (Figures 3B-C). This means that primary tumors with high levels of melanocytic LINC00520-expressing cells (see below) are less likely to metastasize as this requires transition to a more mesenchymal state associated with low LINC00520 expression. By contrast, metastatic tumor growth appears to be favored by the presence of LINC00520-expressing melanocytic cells contributing to poor patient outcome in the metastatic disease.

Uveal melanoma is a highly aggressive form of melanoma. While overall LINC00520 expression was lower in uveal melanoma than in cutaneous melanoma (Figures 2A-C), reanalyses of single cell RNA-sequencing data from a collection of uveal melanoma tumors published by Pandiani et al. (Cell Death Differ. 2021;28(6): 1990-2000) revealed one uveal melanoma tumor with LINC00520 expression that further displayed poor patient outcome (Figures 4A-B).

Melanoma cells adopt multiple phenotypes with specific gene expression signatures. RT- qPCR on RNA from a collection of melanocytic and de-differentiated/mesenchymal lines showed that LINC00520 was highly expressed in melanocytic cell lines, irrespective of driver mutation, but to trace levels in MM099 and MM047 and absent in Vemurafenib-resistant SK-MEL-25R and M229R or MM029, all of which were of the de-differentiated cell state (Figure 5A). Mining of scRNA-seq of a human melanoma PDX treated with a combination of the MAP Kinase inhibitors Dabrafinib and Trametinib (DT) (Rambow et al., Cell 2018;174(4):843-855.el9) showed that LINC00520 was expressed in the MITF expressing proliferative, hypo-metabolic and pigmented cells, but also in the MAPKi-resistant interferon active/inflamed cells, but not in the neural-crest like and invasive/undifferentiated cells displaying minimal expression of MITF the major activator of its expression in melanocytic cells (Figure 5B).

To assess the requirement for LINC00520 in melanoma cells, the inventors used 2 different locked nucleic acid oligonucleotides (GapmeRs) to induce its degradation compared to a control non-targeting GapmeR (CTR). Transfection of two LINC00520-targeting GapmeRs (Gapl and Gap2) strongly reduced its expression in each tested melanocytic melanoma cell line (Figure 6A) leading to a strong increase in the number of low proliferating cells and in apoptotic cells staining for activated Caspase 3 (Figures 6B-C). No effect was seen in undifferentiated MM047 melanoma cells and in HEK293T cells where LINC00520 was not expressed. Apoptosis was confirmed using Annexin V staining (Figure 6D). These results showed that LINC20050 was essential for normal proliferation and survival of melanocytic melanoma cells irrespective of the driver mutation.

It has previously been proposed that LINC00520 promotes melanoma cell proliferation and metastasis via competitive binding to miR-125b-5 which in turn promotes Eukaryotic initiation factor 5A2 (EIF5A2) expression (Luan et al., J Exp Clin Cancer Res. 2020;39(l):96). To investigate this allegation, the Inventors assessed miR-125b-5p expression in a collection of melanoma cells of melanocytic or mesenchymal type using normalized RNA-seq data. While, as shown in Figure 5A, LINC00520 was expressed in melanocytic cells (MM074, MM074VR, 501Mel, Mel 888), miR-125b-5p was expressed only in mesenchymal type cells that do not express LINC00520 (MM074R, MM047, MM047R, MM099 (Figure 7A). Moreover, no association of EIF5A2 levels was observed with patient outcome, contrary to LINC00520 (Figure 7B). These data are incompatible with a mechanism where LINC00520 drives a miR-125b-5p EIF5A2 axis in melanoma. LINC00520 must therefore act in a mechanism independent of miR-125b-5p.

To identify the real mechanism of action of LINC00520 in melanoma, the Inventors used a set of biotinylated oligonucleotides complementary to LINC00520 to purify it from 501Mel melanoma cells using oligonucleotides complementary to LincRNA PCA3, not expressed in melanoma cells, as a negative control. Proteins that were enriched after LINC00520 purification compared to PCA3 were determined by mass-spectrometry and confirmed by immunoblot analyses. The major enriched protein identified in this experiment was the RNA helicase DHX36. Immunoblot analysis showed that DHX36 was strongly enriched by LINC00520 pulldown compared to PC A3 in both native conditions and after UV protein RNA crosslinking (Figure 8A). To confirm this interaction, the Inventors performed LINC00520 pulldown in HET293T cells ectopically expressing LINC00520, a cell line with no endogenous expression of this lincRNA. DHX36 was selectively captured in HEK293T cells expressing LINC00520 but not in the control GFP-expressing cells (Figure 8B). In a converse experiment, immunoprecipitation of DHX36 led to enrichment on LINC00520, but not of SAMMSON orMALATl, an abundant lincRNA (Figure 8C). Moreover, LINC00520 does not interact with CARF or RAP2, which are the respective effector proteins of the melanoma-specific lincRNAs SAMMSON (Vendramin et al., Nat Struct Mol Biol. 2018;25(11): 1035-1046) and LINC00518 (Gambi et al., Cancer Res. 2022;82(24):4555- 4570) (Figure 8A). To conclude, LINC00520 interacts selectively with DHX36 indicating, which is a completely distinct mechanism of action from those described for SAMMSON or LINC00518. In addition, the Inventors examined the intracellular localization of LINC00520 and its DHX36 effector protein. Biochemical purification of the cytosol and mitochondria indicated that LINC00520 partitioned between the cytosolic and mitochondrial fractions with mild enrichment in the mitochondrial fraction (Figure 9A). Similarly, immunoblot analyses indicated that DHX36 was also mildly enriched in the mitochondrial fraction whose purity was confirmed using the mitochondrial specific protein p32 (Figure 9B).

DHX36 is an RNA helicase that unwinds G4 quadruplex structures in RNA molecules facilitating their homeostasis and translation. To examine whether LINC00520 regulates association of mRNAs with DHX36 and their translation, the Inventors immunoprecipitated DHX36 from 501 Mel melanoma cells (N = 3) expressing a control shRNA or an shRNA targeting LINC00520. The DHX36-associated RNAs were identified by RNA sequencing and RNA displaying differential association with DHX36 identified using DESEQ2 software. 260 RNAs showed reduced association with DHX36 after LINC00520 silencing, whereas 131 showed increased association (Figure 10A). Messenger RNAs for UBE4A or RBPJ showed prominent differential association with DHX36. To assess if LINC00520 silencing affected their translation, the Inventors performed immunoblots on extracts from cells transfected with control or LINC00520 targeting GapmeR. UBE4A protein levels were down-regulated following LINC00520 silencing whereas those of RBPJ were increased (Figure 10B). These results indicate that mRNA whose association with DHX36 are reduced following LINC00520 silencing were less well translated whereas the opposite was seen for mRNAs with increased association.

LINC00520 acts therefore by regulating protein translation in melanoma cells at least in part through modulating mRNA interaction with DHX36. Its mechanism of action therefore does not involve miR-125b-5p that is not co-expressed with LINC00520 and is distinct from that of the previously described melanoma-specific lincRNAs SAMMSON and LINC00518.

Since LINC00518 and LINC00520 were both found to be expressed in melanocytic type melanoma cells, yet act via distinct mechanisms of action (RAP2C and DRP1 for LINC00518 and DHX36 for LINC00520), the Inventors asked if they could cooperate with one another notably in the promotion of melanoma cell proliferation and survival. To do so, experiments were conducted using suboptimal concentrations of GapmeRs for each LincRNA alone or in combination using CTR GapmeR to ensure a constant amount of GapmeR in each condition. Compared to each GapmeR separately, each combination of LincRNA knockdown led to a synergistic increase in apoptotic Caspase 3-expressing cells (Figure 11A) and a potent reduction in the proliferation of 501Mel and MM011 cells (Figure 11B). The effectiveness of the knockdown was confirmed by RT-qPCR, which showed a specific reduction in the expression of each LincRNA in the presence of its respective GapmeR (Figure 11C). Thus, these findings show that LINC00518 and LINC00520 act cooperatively to promote melanoma cell proliferation and survival.

Finally, to assess cooperativity between GapmeR-mediated LINC00520 silencing and the clinically used MAP kinase inhibitor DT combination in melanoma cells, the Inventors transfected 501Mel cells with GapmeR targeting LINC00520 in presence of the drugs or DMSO as control. Cell viability was impacted in presence of GapmeR or the DT combination alone, while the combination resulted in a potent cooperative reduction in cell viability with essentially complete eradication of the melanoma cells (Figure 12A). This cooperative effect was not seen in HeLa cells that do not express LINC00520 (Figure 12 B). These data showed that targeting of LINC00520 acted cooperatively with MAP kinase inhibitors to promote melanoma cell death.

Thus, the present data strongly support the notion that co-targeting of LINC00520 and LINC00518, or targeting LINC00520 in the presence of MAP kinase inhibitors such as DT, could serve as promising therapeutic strategies for effectively eliminating melanoma.

Combinatorial targeting of LINC00520 and LINC00518, would especially allow the synergistic targeting of melanocytic cells that form the majority of tumors cells and play a crucial role in metastasis, while still also targeting the smaller populations of mesenchymal and neural crest cells via LINC00518.

All these approaches hold great potential in combating melanoma and improving treatment outcomes.

EXAMPLE 2

A similar experiment to EXAMPLE 1 (double LINC knockdown) is conducted with a different type of IncRNA inhibitors. This time, individual knockdown of LINC00520 or LINC00518, as well as combinatorial knockdown thereof, are performed in a melanoma cell line using RNAi nucleic acids targeting each IncRNA. A control (CTR) RNAi nucleic acid is also used to ensure a constant amount of RNAi nucleic acids in each condition.

To do so, melanoma cells are transfected with suboptimal concentrations of shRNA or siRNA for each IncRNA, used either alone or in combination, using a control (CTR) shRNA to ensure a constant amount of sh/siRNA in each condition. Table 4. RNAi nucleic acids for individual and combinatorial knockdown

Individual and combinatorial inhibition is also performed with an RNAi nucleic acid targeting LINC00520 from Table 4, and the dabrafenib-trametinib combination.

EXAMPLE 3 A similar experiment to EXAMPLE 1 (double LINC knockdown) is conducted with a different type of IncRNA inhibitors. This time, individual knockdown of LINC00520 or LINC00518, as well as combinatorial knockdown, are performed in a melanoma cell line using vectors encoding the CRISPR/dCAS9-KAPl fusion protein with or without (CTR) sgRNA targeting it to the LINC518 or LINC00520 promoter region. To do so, melanoma cells are co-transfected using Fugene 6 (Promega) with a plasmid expressing dead Cas9 protein fused to the Kruppel-associated box (KRAB) domain-containing KAP1 (dCas9-KAPl) and the red fluorescent protein mScarlet (pX-dCas9-KRAB-Scarlet), together with a plasmid expressing GFP and three single guide RNAs targeting the transcription start site of LINC00518 or LINC00520 or a control plasmid expressing GFP only (pCMV-GFP). Double Scarlet-GFP positive cells are sorted 24 hours after co-transfection, stained with Cell Trace Violet and kept in culture for additional 96 hours. Cells are harvested to prepare total RNA and stained with AnnexinV-APC (Biolegend). Cells are analysed on a LSRII Fortessa (BD Biosciences) and data were analysed with Flowjo software (Tree Star).

Table 5. Single guide RNAs for individual and combinatorial knockdown Individual and combinatorial inhibition is also performed with an sgRNA targeting LINC00520 from Table 5, and the dabrafenib-trametinib combination.

EXAMPLE 4

A similar experiment to EXAMPLE 1 (double LINC knockdown) is conducted in a melanoma cell line for knockdown of LINC00520 and LINC00518, by using:

• a GapmeR for one IncRNA, combined with a RNAi nucleic acid for the other IncRNA;

• a GapmeR for one IncRNA, combined with a vector encoding the CRISPR/dCAS9-KAPl fusion protein with sgRNA targeting the promoter region of the other IncRNA;

• a RNAi nucleic acid for one IncRNA, combined with vector encoding the CRISPR/dC AS9- KAP1 fusion protein with sgRNA targeting the promoter region of the other IncRNA.