WO2024245951A1 - Combination of slc8a1 inhibitor and mitochondria-targeted antioxidant for treating melanoma - Google Patents

Abstract

Metastatic uveal melanomas are highly resistant to all existing treatments. Here, a kinome-wide CRISPR-Cas9 knockout screen, revealed that the LKB1-SIK2 module plays a critical role in constraining uveal melanoma cell tumorigenesis. The inventors' results demonstrate that a combination of SLC8A1 inhibitor and mitochondria-targeted antioxidant has an enhanced cell death efficacy in LKB1 and SIK2-negative uveal melanoma cells. They also designed a LKB1 loss gene signature that is predictive of patient survival and treatment response. Their data thus identify new prognosis markers, and metabolic vulnerability, thereby providing a therapeutic strategy for these subtypes of metastatic uveal melanomas. The present invention relates to a method for treating melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of a combination of SLC8A1 inhibitor and mitochondria-targeted antioxidant.

Description

COMBINATION OF SLC8A1 INHIBITOR AND MITOCHONDRIA-TARGETED ANTIOXIDANT FOR TREATING MELANOMA

FIELD OF THE INVENTION:

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

BACKGROUND OF THE INVENTION:

Uveal melanoma, the main primary intraocular malignancy in adult, is an aggressive and deadly neoplasm, which develops from melanocytes mainly in the choroid. At diagnosis, only 1-3% of the patients have detectable metastases (Carvajal et al., 2017). However, despite successful treatment of the primary tumor, up to 50% of patients develop metastases, predominantly to the liver (Garg et al., 2022). Uveal melanoma metastases are highly refractory to all therapies, even those that improve the clinical outcomes of patients with cutaneous melanoma, because they are biologically and genetically different tumors (Pandiani et al., 2017). Recently, tebentafusp, a novel immunotherapy, has been shown for the first time to improve the overall survival of patients with metastatic uveal melanomas (Nathan et al., 2021). However, tebentafusp treatment is limited to HLA-A*02:01 positive patients and demonstrated a benefit in only a few of them. To date, ninety percent of patients with metastatic uveal melanoma still die within 6 months after diagnosis of metastases, highlighting the unmet clinical needs.

Uveal melanoma is driven by oncogenic mutations in the heterotrimeric G protein subunit a (GNAQ) and in its paralog GNA11, which share >90% peptide sequence identity and strikingly similar effects (Onken et al., 2008; Van Raamsdonk et al., 2009, 2010). The most frequent GNAQ and GNA11 mutation is the substitution of glutamine at position 209 by proline or leucine (GNAQ/11Q209P/L) that results in loss of GTPase activity producing constitutive activation of GNAQ/GNA11. Although the GNAQ/GNA11 signaling pathway is activated in virtually all uveal melanomas, additional rare mutations in CYSLTR2 and PLCB4, which function upstream and downstream of GNAQ/GNA11 respectively, have also been identified, demonstrating the importance of this pathway in uveal melanoma oncogenesis (Robertson et al., 2017). GNAQ/11 mutations are coupled to mutations that are almost mutually exclusive with each other and prognostically significant of the metastatic risk, the most frequent being a loss of function in BRC Al -associated protein 1 (BAP1), which is associated with a high metastatic risk and a poor prognosis. Uveal melanomas are also associated with chromosomal imbalances, including monosomy of chromosome 3 and amplification of 8q (Field et al., 2018; Shain et al., 2018).

Over the past decades, advances into the understanding of the molecular mechanisms underlying uveal melanomas have been made. Oncogenic GNAQ, through ADP ribosylation factor 6 (ARF6), promotes activation of multiple downstream signaling pathways such as phospholipase C-b (PLC-P)/protein kinase C (PKC)/extracellular signal regulated kinase (ERK), trio Rho guanine nucleotide exchange factor (Trio)/RHO/RAC/yes-associated protein (YAP) (Chen et al., 2017; Pandiani et al., 2017; Yoo et al., 2016). Oncogenic GNAQ triggers activation of other cascades such as the phosphatidylinositol 3 -kinase (PI3K)/protein kinase B (Akt)/mammalian target of rapamycin (mTOR) module and P-catenin (Saraiva et al., 2005; Yoo et al., 2016). Clinical studies have evaluated different drugs targeting these signaling pathways, alone or in combination with limited, if any, efficacy generally observed (Carvajal et al., 2023). Thus, advances did not translate into effective therapeutic targets to prevent or eliminate metastasis so far.

Hence, although uveal melanomas with GNAQ/11 mutations have constitutively active growth signals, how they sustain their proliferation and survival remains to be fully understood.

The inventors performed a CRISPR-Cas9 kinome screen in metastatic uveal melanoma to identify exploitable vulnerabilities. Their data identify a novel kinase cascade that plays a key role in the control of metastatic uveal melanoma cell proliferation and survival, exerting its effects via the regulation of calcium and reactive oxygen species metabolism. Their work also identifies a prognostic molecular signature for patient survival that is also predictive of cellular response to a combination of drugs that affect calcium and ROS metabolism. Thus, we unveil here new potential therapeutic targets and a method to stratify the patients most likely to respond to these treatments.

SUMMARY OF THE INVENTION:

The present invention relates to a method for treating melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of a combination of SLC8A1 inhibitor and mitochondria-targeted antioxidant. In particular, the invention is defined by the claims. DETAILED DESCRIPTION OF THE INVENTION:

Metastatic uveal melanomas are highly resistant to all existing treatments. Here, a kinome-wide CRISPR-Cas9 knockout screen, revealed that the LKB1-SIK2 module plays a critical role in constraining uveal melanoma cell tumorigenesis. Functionally, LKB1 loss strongly enhances proliferation and survival through SIK2 suppression and up-regulation of the sodium/calcium (Na+/Ca2+) exchanger SLC8A1. This signaling cascade promotes increased level of intracellular calcium and mitochondrial reactive oxygen species, two hallmarks of cancers. The inventors’ results demonstrate that a combination of SLC8A1 inhibitor and mitochondria- targeted antioxidant has an enhanced cell death efficacy in LKB1 and SIK2 -negative uveal melanoma cells. They also designed a LKB1 loss gene signature that is predictive of patient survival and treatment response. Their data thus identify new prognosis markers, and metabolic vulnerability, thereby providing a therapeutic strategy for these subtypes of metastatic uveal melanomas.

Method for treating melanoma

Accordingly, in a first aspect, the present invention relates to a method for treating melanoma in a subject in need thereof comprising a step of administering said subject with a combination of SLC8A1 inhibitor and mitochondria-targeted antioxidant.

More particularly, the present invention relates to a combination of SLC8A1 inhibitor and mitochondria-targeted antioxidant for use in the treatment of melanoma in a subject in need thereof.

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

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

In a particular embodiment, the melanoma is uveal melanoma.

In some embodiment, the present invention relates to a method for treating uveal melanoma in a subject in need thereof comprising a step of administering said subject with a combination of SLC8A1 inhibitor and mitochondria-targeted antioxidant.

More particularly, the invention relates to an a combination of a SLC8A1 inhibitor and mitochondria-targeted antioxidant for use in the treatment of uveal melanoma in a subject in need thereof.

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

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

As used herein the terms "administering" or "administration" refer to the act of injecting or otherwise physically delivering a substance as it exists outside the body (e.g. SLC8A1 inhibitor and/or mitochondria-targeted antioxidant) into the subject, such as by topical, intravitreal, mucosal, intradermal, intravenous, subcutaneous, intramuscular delivery and/or any other method of physical delivery described herein or known in the art. When a disease, or a symptom thereof, is being treated, administration of the substance typically occurs after the onset of the disease or symptoms thereof. When a disease or symptoms thereof, are being prevented, administration of the substance typically occurs before the onset of the disease or symptoms thereof.

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

A “therapeutically effective amount” is intended for a minimal amount of active agent which is necessary to impart therapeutic benefit to a subject. For example, a "therapeutically effective amount" to a subject is such an amount which induces, ameliorates or otherwise causes an improvement in the pathological symptoms, disease progression or physiological conditions associated with or resistance to succumbing to a disorder. It will be understood that the total daily usage of the compounds of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the disorder being treated and the severity of the disorder; activity of the specific compound employed; the specific composition employed, the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidential with the specific compound employed; and like factors well known in the medical arts. For example, it is well within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient (e.g. SLC8A1 inhibitor or mitochondria-targeted antioxidant) for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, preferably from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

As used herein, the term “mitochondria” has its general meaning in the art and refers to an organelle found in the cells of most eukaryotes, such as animals, plants and fungi. Mitochondria have a double membrane structure and implicated in many vital processes in animal cells, including energy production, fatty-acid oxidation and the Tricarboxylic Acid (TCA) cycle, calcium signaling, permeability transition, apoptosis and heat production. The main function of mitochondria is to produce Adenosine Triphosphate (ATP). In the cell, the necessary energy in the form of ATP is produced in two ways: in the cytosol as a product of glycolysis, and in the mitochondria as a product of oxidative phosphorylation (OXPHOS). The substrates, in the form of fatty acids and pyruvate, are oxidized via fatty acid P-oxidation and the TCA cycle respectively. The Nicotinamide Adenine Dinucleotide (NADH) and flavin adenine dinucleotide (FADH2) produced by these reactions are used by the electron transport chain to generate ATP.

As used herein, the term “mitochondria-targeted” refers to pharmacological targeting of the mitochondria since its intracellular organelle has a number of vital functions and mitochondrial damage is crucial for the development of many diseases. The vast majority of synthesized mitochondria-targeted drug fall into one of the following categories: antioxidants, uncouplers of oxidative phosphorylation and respiration (which lower A m and ATP production), poisons (mitotoxic and cytotoxic compounds inducing cell death, mainly apoptosis) and probes and sensors for detection of reactive oxygen, nitrogen and sulfur species.

As used herein, the term “mitochondria-targeted antioxidant” refers to an antioxidant that can accumulate inside mitochondria and scavenge and/or inactivate one or several reactive oxygen species (ROS). Non-limitative examples of mitochondria-targeted antioxidants suitable for implementing the invention are disclosed by, e.g. , Jiang et al. (2020) and Fock and Pamova (2021). Others example of mitochondria-targeted antioxidant include but are not limited to MitoQ, MitoTEMPO, MitoTEMPOL, MitoE, MitoVitE, MitoSOD, MitoSNO, SKQ1, SKQR1, SKQ2, SKQ3, SKQ4, SKQ5, SKQBerb, SKQPalm, C12TPP, melatonin, dimethyl malonate, methylene blue, Mn-porphyrin-oligopeptide conjugate, M40401, SS20, SS31, XJB-5-125, XJB-5-131 and XJB-5-197.

In some embodiments, the mitochondria-targeted antioxidant is MitoQ.

As used herein, the term “Mitoquinone mesylate”(MitoQ mesylate) or “Mitoquinone” (MitoQ) refers to mitochondrially targeted antioxidant and has the following formula C38H47O7PS. Mitoquinone mesylate has the following CAS number : 845959-50-4 and structure in the art:

In some embodiments, the mitochondria-targeted antioxidant is a SKQ1.

As used herein, the term “SkQl” or “SKQ1” or “Visomitin” refer to mitochondria-targeted antioxidant and has the following formula CAFheBrCFP. SkQl has the following CAS number : 934826-68-3 and structure in the art :

As used herein, the terms “sodium-calcium exchanger”, “Na+/Ca2+ exchanger” or “NCX” relate to an antiporter membrane protein that removes calcium from cells. It uses the energy that is stored in the electrochemical gradient of sodium (Na+) by allowing Na+ to flow down its gradient across the plasma membrane in exchange for the counter transport of calcium ions (Ca2+). A single calcium ion is exported for the import of three sodium ions. The exchanger exists in many different cell types and animal species. The NCX is considered one of the most important cellular mechanisms for removing Ca2+. The exchanger is usually found in the plasma membranes and the mitochondria and endoplasmic reticulum of excitable cells. The Na+/Ca2+ exchanger comprise a family of three genes NCX1 (or SLC8A1),NCX2 (or SLC8A2) and NCX3(or SLC8A3).

As used herein, the term “SLC8A1” or “solute carrier family 8 (sodium-calcium exchanger), member 1” or “NCX1” is a exchanger or channel that mediates the exchange of one Ca2+ ion against three to four Na+ ions across the cell membrane, and thereby contributes to the regulation of cytoplasmic Ca2+ levels and Ca2+-dependent cellular processes. In a first phase, voltage-gated channels mediate the rapid increase of cytoplasmic Ca2+ levels due to release of Ca2+ stores from the endoplasmic reticulum. SLC8A1 mediates the export of Ca2+ from the cell during the next phase, so that cytoplasmic Ca2+ levels rapidly return to baseline. SLC8A1 has the following NCBI Entrez Gene number : 6546 and has the following UniProt number : P32418.

As used herein the term "SLC8A1 inhibitor" refers to an agent (i.e. a molecule) which inhibits or blocks the activity of SLC8A1. For instance, an antagonist of SLC8A1 refers to a molecule which inhibits or blocks the activity of the SLC8A1 exchanger or channel. Preferably, the SLC8A1 antagonists according to the invention act through direct interaction with the SLC8A1 exchanger or channel. In a particular embodiment, the SLC8A1 inhibitor is a peptide, peptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide. The term “peptidomimetic” refers to a small protein-like chain designed to mimic a peptide.

In a particular embodiment, the SLC8A1 inhibitor is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

In a particular embodiment, SLC8A1 inhibitor is a small organic molecule. The term “small organic molecule” refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e.g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da.

In a particular embodiment, the SLC8A1 inhibitor is a KB-R7943. Typically, KB-R7943 also known as KB-R7943 Mesylate or 2-[2-[4-(4-Nitrobenzyloxy)phenyl]ethyl]isothiourea mesylate has the following formula CieHnNsChS’CTUSCh. KB-R7943 has the following CAS number : 182004-65-5 and structure in the art:

In a particular embodiment, the SLC8A1 inhibitor is SN-6. Typically, SN-6 also known as 2- [[4-[(4-Nitrophenyl)methoxy]phenyl]methyl]-4-thiazolidinecarboxylic acid ethyl ester has the following formula : C20H22N2O5S. SN-6 has the following CAS number : 415697-08-4 and structure in the art:

In a particular embodiment, the SLC8A1 inhibitor is ORM-11372. Typically, ORM-11372 has the following formula: C17H15FN2O. ORM-11372 has the following CAS number : 2376217- 14-8 and structure in the art:

In a particular embodiment, the SLC8A1 inhibitor is SEA0400. Typically, SEA0400 also known as 2-[4-[(2,5-Difluorophenyl)methoxy]phenoxy]-5-ethoxybenzenamine has the following formula: C21H19F2NO3. SEA0400 has the following CAS number: 223104-29-8 and structure in the art:

In a particular embodiment, the SLC8A1 inhibitor is Aprindine. Typically, Aprindine has the following formula: C22H30N2. Aprindine has the following CAS number: 37640-71-4 and the structure in the art :

In a particular embodiment, the SLC8A1 inhibitor is SAR296968. Typically, SAR2968 has the following formula: C22H22N2O4S. SAR296968 has the following CAS number: 1426899-28-6 and the structure in the art :

In a particular embodiment, the SLC8A1 inhibitor is SAR340835. Typically, SAR340835 has the following formula : C22H2iN2Na2O?PS and the structure in the art :

In some embodiments, the SLC8A1 inhibitor is a short hairpin RNA (shRNA), a small interfering RNA (siRNA) or an antisense oligonucleotide which inhibits the expression of metabolites involved in SLC8A1 metabolism.

In a particular embodiment, the SLC8A1 inhibitor is siRNA. A short hairpin RNA (shRNA) is a sequence of RNA that makes a tight hairpin turn that can be used to silence gene expression via RNA interference. shRNA is generally expressed using a vector introduced into cells, wherein the vector utilizes the U6 promoter to ensure that the shRNA is always expressed. This vector is usually passed on to daughter cells, allowing the gene silencing to be inherited. The shRNA hairpin structure is cleaved by the cellular machinery into siRNA, which is then bound to the RNA-induced silencing complex (RISC). This complex binds to and cleaves mRNAs that match the siRNA to which it is bound. Small interfering RNA (siRNA), sometimes known as short interfering RNA or silencing RNA, are a class of 20-25 nucleotide-long double- stranded RNA molecules that play a variety of roles in biology. Most notably, siRNA is involved in the RNA interference (RNAi) pathway whereby the siRNA interferes with the expression of a specific gene.

In a particular embodiment, the SLC8A1 inhibitor is an anti-sense oligonucleotides (ASO). Anti-sense oligonucleotides include anti-sense RNA molecules and anti-sense DNA molecules, would act to directly block the translation of the targeted mRNA by binding thereto and thus preventing protein translation or increasing mRNA degradation, thus decreasing the level of the targeted protein, and thus activity, in a cell. For example, antisense oligonucleotides of at least about 15 bases and complementary to unique regions of the mRNA transcript sequence can be synthesized, e.g., by conventional phosphodiester techniques. Methods for using antisense techniques for specifically inhibiting gene expression of genes whose sequence is known are well known in the art (e.g. see U.S. Pat. Nos. 6,566,135; 6,566,131; 6,365,354; 6,410,323; 6,107,091; 6,046,321; and 5,981,732). Antisense oligonucleotides, siRNAs, shRNAs of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid to the cells and typically mast cells. Typically, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rous sarcoma virus; adenovirus, adeno- associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

In some embodiments, the SLC8A1 inhibitor is an endonuclease. In the last few years, staggering advances in sequencing technologies have provided an unprecedentedly detailed overview of the multiple genetic aberrations in cancer. By considerably expanding the list of new potential oncogenes and tumor suppressor genes, these new data strongly emphasize the need of fast and reliable strategies to characterize the normal and pathological function of these genes and assess their role, in particular as driving factors during oncogenesis. As an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, the new technologies provide the means to recreate the actual mutations observed in cancer through direct manipulation of the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the errorprone nonhomologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).

In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR/Cas9 system has been described in US 8697359 Bl and US 2014/0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038/cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707-714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534/genetics.l 13.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836- 843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038/cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA-directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR/Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13). In some embodiments, the SLC8A1 inhibitor is an antibody. As used herein, the term “antibody” is used in the broadest sense and specifically covers monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments so long as they exhibit the desired biological activity. The term includes antibody fragments that comprise an antigen binding domain such as Fab', Fab, F(ab')2, single domain antibodies (DABs), TandAbs dimer, Fv, scFv (single chain Fv), dsFv, ds-scFv, Fd, linear antibodies, minibodies, diabodies, bispecific antibody fragments, bibody, tribody (scFv-Fab fusions, bispecific or trispecific, respectively); sc-diabody; kappa(lamda) bodies (scFv-CL fusions); BiTE (Bispecific T-cell Engager, scFv-scFv tandems to attract T cells); DVD-Ig (dual variable domain antibody, bispecific format); SIP (small immunoprotein, a kind of minibody); SMIP ("small modular immunopharmaceutical" scFv-Fc dimer; DART (ds-stabilized diabody "Dual Affinity ReTargeting"); small antibody mimetics comprising one or more CDRs and the like. The techniques for preparing and using various antibody -based constructs and fragments are well known in the art (see Kabat et al., 1991, specifically incorporated herein by reference). Diabodies, in particular, are further described in EP 404, 097 and WO 93/1 1 161; whereas linear antibodies are further described in Zapata et al. (1995). Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, Fv, dsFv, Fd, dAbs, TandAbs, ds-scFv, dimers, minibodies, diabodies, bispecific antibody fragments and other fragments can also be synthesized by recombinant techniques or can be chemically synthesized. Techniques for producing antibody fragments are well known and described in the art. For example, each of Beckman et al., 2006; Holliger & Hudson, 2005; Le Gall et al., 2004; Reff & Heard, 2001 ; Reiter et al., 1996; and Young et al., 1995 further describe and enable the production of effective antibody fragments. In some embodiments, the antibody is a “chimeric” antibody as described in U.S. Pat. No. 4,816,567. In some embodiments, the antibody is a humanized antibody, such as described U.S. Pat. Nos. 6,982,321 and 7,087,409. In some embodiments, the antibody is a human antibody. A “human antibody” such as described in US 6,075,181 and 6,150,584. In some embodiments, the antibody is a single domain antibody such as described in EP 0 368 684, WO 06/030220 and WO 06/003388.

In a particular embodiment, the SLC8A1 inhibitor is a monoclonal antibody. Monoclonal antibodies can be prepared and isolated using any technique that provides for the production of antibody molecules by continuous cell lines in culture. Techniques for production and isolation include but are not limited to the hybridoma technique, the human B-cell hybridoma technique and the EBV-hybridoma technique.

In a particular, the SLC8A1 inhibitor is an intrabody. As used herein, the term "intrabody" generally refer to an intracellular antibody or antibody fragment. Antibodies, in particular single chain variable antibody fragments (scFv), can be modified for intracellular localization. Such modification may entail for example, the fusion to a stable intracellular protein, such as, e.g., maltose binding protein, or the addition of intracellular trafficking/localization peptide sequences, such as, e.g., the endoplasmic reticulum retention. In some embodiments, the intrabody is a single domain antibody. In some embodiments, the antibody according to the invention is a single domain antibody. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb.

Method for treating resistant melanoma

In a second aspect, the invention relates to a method for treating resistant melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of a combination of SLC8A1 inhibitor and mitochondria-targeted antioxidant.

As used herein, the term “resistant melanoma” refers to melanoma which does not respond to a treatment. The cancer may be resistant at the beginning of treatment, or it may become resistant during treatment. The resistance to drug leads to rapid progression of metastatic of melanoma.

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

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

In some embodiments, the melanoma is resistant to MEK inhibitors. MEK refers to Mitogen- activated protein kinase kinase, also known as MAP2K, MEK, MAPKK. It is a kinase enzyme which phosphorylates mitogen-activated protein kinase (MAPK). MEK is activated in melanoma.

In some embodiments, the melanoma is resistant to NRAS inhibitors. The NRAS gene is in the Ras family of oncogene and involved in regulating cell division. NRAS mutations in codons 12, 13, and 61 arise in 15-20 % of all melanomas.

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

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

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

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

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

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

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

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

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

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

The term "small organic molecule" as used herein, refers to a molecule of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macro molecules (e. g. proteins, nucleic acids, etc.). Typically, small organic molecules range in size up to about 5000 Da, more preferably up to 2000 Da, and most preferably up to about 1000 Da. Typically, the small organic molecules interfere with transduction pathway of A2AR, B7-H3, B7-H4, BTLA, CTLA-4, CD277, IDO, KIR, PD-1, LAG-3, TIM-3 or VISTA.

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

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

In a particular embodiment, the inhibitor of IDO is Epacadostat, (INCB24360, INCB024360) has the following chemical formula in the art and refers to -N-(3-bromo-4-fluorophenyl)-N'- hydroxy-4-{[2-(sulfamoylamino)-ethyl]amino}-l,2,5-oxadiazole-3 carboximidamide :

In a particular embodiment, the inhibitor is BGB324, also called R428, such as described in W02009054864, refers to lH-l,2,4-Triazole-3,5-diamine, l-(6,7-dihydro-5H- benzo[6,7]cyclohepta[l,2-c]pyridazin-3-yl)-N3-[(7S)-6,7,8,9-tetrahydro-7-(l-pyrrolidinyl)- 5H-benzocyclohepten-2-yl]- and has the following formula in the art:

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

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

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

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

In a particular embodiment, the melanoma is metastatic uveal melanoma.

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

In a particular embodiment, the resistant melanoma is uveal resistant melanoma. As used herein, the term “uveal melanoma resistant” refers to uveal melanoma which does not respond to a treatment. The cancer may be resistant at the beginning of treatment or it may become resistant during treatment. The resistance to drug leads to rapid progression of metastatic of uveal melanoma.

The resistance of cancer for the medication is caused by mutations in the gene which are involved in the proliferation, divisions or differentiation of cells.

In a particular embodiment, the uveal melanoma resistant has at least one mutation in the five following genes: BAP L EIF1AX, GNA17, GNAQ, and/or SF3B1.

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

Combined preparation

In a third aspect, the present invention relates to i) SLC8A1 inhibitor, ii) and mitochondria- targeted antioxidant and iii) a classical treatment as a combined preparation for use in the treatment of melanoma and/or resistant melanoma.

In a particular embodiment, the present invention relates to i) SLC8A1 inhibitor, ii) and mitochondria-targeted antioxidant and iii) a classical treatment as a combined preparation for use in the treatment of uveal melanoma and/or uveal resistant melanoma.

In a particular embodiment, the SLC8A1 inhibitor is KB-R7943.

In a particular embodiment, the mitochondria-targeted antioxidant is mitoquinol (MitoQ).

In a further embodiment, the invention relates to i) KB-R7943, ii) mitoquinol (MitoQ) and iii) a classical treatment as a combined preparation for use in the treatment of melanoma and/or resistant melanoma.

In a particular embodiment, the invention relates to i) KB-R7943, ii) mitoquinol (MitoQ) and iii) a classical treatment as a combined preparation for use in the treatment of uveal melanoma. In a particular embodiment, the invention relates to i) KB-R7943, ii) mitoquinol (MitoQ) and iii) a classical treatment as a combined preparation for use in the treatment of uveal resistant melanoma.

In a particular embodiment, the invention i) KB-R7943, ii) mitoquinol (MitoQ) and iii) a classical treatment as a combined preparation for simultaneous, separate or sequential use in the treatment of melanoma.

In a particular embodiment, the invention i) KB-R7943, ii) mitoquinol (MitoQ) and iii) a classical treatment as a combined preparation for simultaneous, separate or sequential use in the treatment of uveal melanoma and/or uveal resistant melanoma.

In a particular embodiment, the mitochondria-targeted antioxidant is SKQ1.

In a further embodiment, the invention relates to i) KB-R7943, ii) SKQ1 and iii) a classical treatment as a combined preparation for use in the treatment of melanoma and/or resistant melanoma.

In a particular embodiment, the invention relates to i) KB-R7943, ii) SKQland iii) a classical treatment as a combined preparation for use in the treatment of uveal melanoma.

In a particular embodiment, the invention relates to i) KB-R7943, ii) SKQ1 and iii) a classical treatment as a combined preparation for use in the treatment of uveal resistant melanoma.

In a particular embodiment, the invention i) KB-R7943, ii) SKQ1 and iii) a classical treatment as a combined preparation for simultaneous, separate or sequential use in the treatment of melanoma.

In a particular embodiment, the invention i) KB-R7943, ii) SKQ1 and iii) a classical treatment as a combined preparation for simultaneous, separate or sequential use in the treatment of uveal melanoma and/or uveal resistant melanoma.

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

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

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

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

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

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

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

In a particular embodiment, the invention relates to i) KB-R7943, ii) mitoquinol (MitoQ) and iii) an histone deacetylase inhibitor as a combined preparation for simultaneous, separate or sequential use in the treatment of uveal melanoma and/or uveal resistant melanoma.

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

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

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

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

In a particular embodiment the HDAC inhibitor is Belinostat also called Beleodaq (PXD-101) has received the FDA approval on 2014 (Ja et al 2003; Valente et al 2014). In a particular embodiment the HD AC inhibitor is Entinostat (as SNDX-275 or MS-275). This molecule has the following chemical formula (C21H20N4O3) and has structure as described in Valente et al 2014.

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

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

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

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

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

In a particular embodiment the HDAC inhibitor is CHR-3996 having the following chemical formula (C20H19FN6O2) (Moffat D et al 2010; Banerji et al 2012).

In a particular embodiment the HDAC inhibitor is AR-42 having the following chemical formula (C18H20N2O3) (Lin et al 2012).

Pharmaceutical composition

The combination of SLC8A1 inhibitor and mitochondria-targeted antioxidant for use according to the invention combined with classical treatment as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. Accordingly, in a fourth aspect, the invention relates to a pharmaceutical composition comprising a combination of SLC8A1 inhibitor and mitochondria-targeted antioxidant for use in the treatment of melanoma.

In a particular embodiment, the pharmaceutical composition according to the invention for use in the treatment of uveal melanoma and/or uveal resistant melanoma.

In a particular embodiment, the pharmaceutical composition according to the invention comprising a combination of KB-R7943 and mitoquinol (MitoQ) for use in the treatment of melanoma and/or resistant melanoma.

In a particular embodiment, the pharmaceutical composition according to the invention comprising a combination of KB-R7943 and mitoquinol (MitoQ) for use in the treatment of uveal melanoma and/or uveal resistant melanoma.

In a particular embodiment, the pharmaceutical composition according to the invention comprising i) SLC8A1 inhibitor, ii) mitochondria-targeted antioxidant and iii) a classical treatment, as a combined preparation for use in the treatment of melanoma and/or resistant melanoma.

In a particular embodiment, the pharmaceutical composition according to the invention comprising i) KB-R7943, ii) mitoquinol (MitoQ) and iii) a classical treatment, as a combined preparation for use in the treatment of melanoma and/or resistant melanoma.

In a particular embodiment, the pharmaceutical composition according to the invention comprising i) SLC8A1 inhibitor, ii) mitochondria-targeted antioxidant and iii) a classical treatment, as a combined preparation for use in the treatment of uveal melanoma and/or uveal resistant melanoma.

In a particular embodiment, the pharmaceutical composition according to the invention comprising i) KB-R7943, ii) mitoquinol (MitoQ) and iii) a classical treatment, as a combined preparation for use in the treatment of uveal melanoma/and or uveal resistant melanoma. The SLC8A1 inhibitor, the mitochondria-targeted antioxidant and the combined preparation as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intravitreal administration, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation capable of being injected. These may be in particular isotonic, sterile, saline solutions (monosodium or disodium phosphate, sodium, potassium, calcium or magnesium chloride and the like or mixtures of such salts), or dry, especially freeze-dried compositions which upon addition, depending on the case, of sterilized water or physiological saline, permit the constitution of injectable solutions. The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations including sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. Solutions comprising compounds of the invention as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The polypeptide (or nucleic acid encoding thereof) can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetables oils. The proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminium monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active polypeptides in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuumdrying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. For parenteral administration in an aqueous solution, for example, the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this connection, sterile aqueous media which can be employed will be known to those of skill in the art in light of the present disclosure. For example, one dosage could be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.

Method of screening

In a fifth aspect, the present invention relates to a method of screening a drug suitable for the treatment of melanoma, aggressive/invasive melanoma, metastatic melanoma, or melanoma resistant, uveal melanoma or uveal resistant melanoma comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression and/or activity of SIK2 metabolism.

Typically, such test compound is able to inhibit the the expression and/or activity of inhibitor of SIK2.

Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit SIK2. In some embodiments, the assay first comprises determining the ability of the test compound to bind to SIK2 metabolism . In particular, the effect triggered by the test compound is determined relative to that of a population of immune cells incubated in parallel in the absence of the test compound or in the presence of a control agent either of which is analogous to a negative control condition. The term "control substance", "control agent", or "control compound" as used herein refers a molecule that is inert or has no activity relating to an ability to modulate a biological activity or expression. It is to be understood that test compounds capable of inhibiting the activity of SIK2 metabolism, as determined using in vitro methods described herein, are likely to exhibit similar modulatory capacity in applications in vivo. Typically, the test compound is selected from the group consisting of peptides, petptidomimetics, small organic molecules, aptamers or nucleic acids. For example the test compound according to the invention may be selected from a library of compounds previously synthesised, or a library of compounds for which the structure is determined in a database, or from a library of compounds that have been synthesised de novo. In some embodiments, the test compound may be selected form small organic molecules.

Method for predicting the patient survival and treatment response.

In a sixth aspect, the present invention relates to a method for predicting the survival time of a patient suffering from a melanoma comprising i) determining in a tumor sample obtained from the patient the loss gene expression level of LKB1 ii) comparing the expression level determined at step i) with a predetermined reference value and iii) providing a good prognosis when the loss expression level of LKB1 determined at step i) is lower than the predetermined reference value, or providing a bad prognosis when the loss expression level of LKB1 determined at step i) is higher than the predetermined reference value.

In a particular embodiment, the present invention relates to a method for predicting the survival time of a patient suffering from uveal melanoma comprising i) determining in a tumor sample obtained from the patient the loss gene expression level of LKB1 ii) comparing the expression level determined at step i) with a predetermined reference value and iii) providing a good prognosis when the loss expression level of LKB1 determined at step i) is lower than the predetermined reference value, or providing a bad prognosis when the loss expression level of LKB1 determined at step i) is higher than the predetermined reference value.

In a particular embodiment, the present invention relates to a method for predicting the survival time of a patient suffering from uveal melanoma resistant comprising i) determining in a tumor sample obtained from the patient the loss gene expression level of LKB1 ii) comparing the expression level determined at step i) with a predetermined reference value and iii) providing a good prognosis when the loss expression level of LKB1 determined at step i) is lower than the predetermined reference value or providing a bad prognosis when the loss expression level of LKB1 determined at step i) is higher than the predetermined reference value.

The present invention also relates to a method for determining whether a patient suffering from melanoma will respond to a combination treatment of SLC8A1 inhibitor and mitochondria- targeted antioxidant comprising i) determining in a tumor sample obtained from the patient the loss gene expression level of LKB1 ii) comparing the loss expression level determined at step i) with a predetermined reference value and iii) concluding that the patient will significantly respond to the treatment when the loss expression level of LKB1 determined at step i) is higher than the predetermined reference value. In a particular embodiment, the present invention relates to a method for determining whether a patient suffering from uveal melanoma will respond to a combination treatment of SLC8A1 inhibitor and mitochondria-targeted antioxidant of comprising i) determining in a tumor sample obtained from the patient the loss gene expression level of LKB1 ii) comparing the loss expression level determined at step i) with a predetermined reference value and iii) concluding that the patient will significantly respond to the treatment when the expression level of LKB1 determined at step i) is higher than the predetermined reference value.

In a particular embodiment, the present invention relates to a method for determining whether a patient suffering from uveal melanoma resistant will respond to a combination treatment of SLC8A1 inhibitor and mitochondria-targeted antioxidant comprising i) determining in a tumor sample obtained from the patient the loss gene expression level of LKB1 ii) comparing the loss expression level determined at step i) with a predetermined reference value and iii) concluding that the patient will significantly respond to the treatment when the expression level of LKB1 determined at step i) is higher than the predetermined reference value.

As used herein, the term “tumor sample” means any tissue tumor sample derived from the patient. Said tissue sample is obtained for the purpose of the in vitro evaluation. The sample can be fresh, frozen, fixed (e.g., formalin fixed), or embedded (e.g., paraffin embedded). In a particular embodiment the tumor sample may result from the tumor resected from the patient. In another embodiment, the tumor sample may result from a biopsy performed in the primary tumour of the patient or perfomed in metastatic sample distant from the primary tumor of the patient. In a particular embodiment, the tumor sample is a melanoma sample, particularly a uveal melanom.

As used herein, the term “LKB1” also known as Serine/threonine kinase 11 (STK11) or renal carcinoma antigen NY-REN-19 refers to a protein kinase that in humans is encoded by the STK11 gene. LKB1 is a primary upstream kinase of adenosine monophosphate-activated protein kinase (AMPK), a necessary element in cell metabolism that is required for maintaining energy homeostasis. LKB1 has the following NCBI Entrez Gene number : 6794 and has the following UniProt number : QI 5831. Measuring the expression level of a gene can be performed by a variety of techniques well known in the art.

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

Other methods of Amplification include ligase chain reaction (LCR), transcription-mediated amplification (TMA), strand displacement amplification (SDA) and nucleic acid sequence based amplification (NASBA).

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

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

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

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

Additional labels include, for example, radioisotopes (such as ³ H), metal chelates such as DOTA and DPTA chelates of radioactive or paramagnetic metal ions like Gd3+, and liposomes.

Detectable labels that can he used with nucleic acid molecules also include enzymes, for example horseradish peroxidase, alkaline phosphatase, acid phosphatase, glucose oxidase, betagalactosidase, beta-glucuronidase, or beta-lactamase.

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

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

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

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

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

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

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

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

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

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

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

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

Predetermined reference values used for comparison may comprise “cut-off’ or “threshold” values that may be determined as described herein. Each reference (“cut-off’) value for each gene of interest may be predetermined by carrying out a method comprising the steps of a) providing a collection of tumor tissue samples from patients suffering of cancer; b) determining the expression level of the gene for each tumour tissue sample contained in the collection provided at step a); c) ranking the tumor tissue samples according to said expression level d) classifying said tumour tissue samples in pairs of subsets of increasing, respectively decreasing, number of members ranked according to their expression level, e) providing, for each tumour tissue sample provided at step a), information relating to the actual clinical outcome for the corresponding cancer patient (i.e. the duration of the disease- free survival (DFS) or the overall survival (OS) or both); f) for each pair of subsets of tumour tissue samples, obtaining a Kaplan Meier percentage of survival curve; g) for each pair of subsets of tumour tissue samples calculating the statistical significance (p value) between both subsets h) selecting as reference value for the expression level, the value of expression level for which the p value is the smallest.

For example the expression level of a gene X has been assessed for 100 cancer samples of 100 patients. The 100 samples are ranked according to their expression level. Sample 1 has the best expression level and sample 100 has the worst expression level. A first grouping provides two subsets: on one side sample Nr 1 and on the other side the 99 other samples. The next grouping provides on one side samples 1 and 2 and on the other side the 98 remaining samples etc., until the last grouping: on one side samples 1 to 99 and on the other side sample Nr 100. According to the information relating to the actual clinical outcome for the corresponding cancer patient, Kaplan Meier curves are prepared for each of the 99 groups of two subsets. Also for each of the 99 groups, the p value between both subsets was calculated.

The reference value is selected such as the discrimination based on the criterion of the minimum p value is the strongest. In other terms, the expression level corresponding to the boundary between both subsets for which the p value is minimum is considered as the reference value. It should be noted that the reference value is not necessarily the median value of expression levels.

In routine work, the reference value (cut-off value) may be used in the present method to discriminate tumour samples and therefore the corresponding patients.

Kaplan-Meier curves of percentage of survival as a function of time are commonly to measure the fraction of patients living for a certain amount of time after treatment and are well known by the man skilled in the art.

The man skilled in the art also understands that the same technique of assessment of the expression level of a gene should of course be used for obtaining the reference value and thereafter for assessment of the expression level of a gene of a patient subjected to the method of the invention.

Such predetermined reference values of expression level may be determined for any gene defined above.

Intermediate conclusions may also be provided when at least the loss gene SKB1 is lower than its corresponding predetermined reference value. Every time that the loss expression level of SKB1 is lower than its predetermined reference value, better will be the response of the patient to the treatment. The invention will be further illustrated by the following figures and examples. However, these examples and figures should not be interpreted in any way as limiting the scope of the present invention.

FIGURES:

Figure 1. LKB1 loss impact on calcium metabolism and SLC8A1 expression. Overall survival stratified by SLC8A1 mRNA expression (median) from UM-TCGA dataset (tumors n=80). Low SLC8A1 mRNA level in blue and high SLC8A1 mRNA level in red. P-value, log rank test.

Figure 2. Inhibition of SLC8A1 and mtROS decreases malignant phenotypes in LKB1- KO OMM1.3 cells in vivo. A. Quantification of tumor volume in nude mice bearing xenograft tumors of LKB1-KO cells treated with MitoQ (5 mg/kg), KB-R7943 (5 mg/kg), a combination of both or vehicle three times per week. Mann-Whitney test was performed for comparison between groups. Data are mean ± SEM. *p < 0.05. n=4. B. Kaplan-Meier analysis of the LKB1- KO signature in UM-TCGA dataset. C. Receiver operating characteristic (ROC) curves show the sensitivity and specificity of our LKB1 signature compared to the gene expression profiling signature (GEP) (Onken et al., 2004), for predicting the patient disease specific survival (UM- TCGA cohort). D. Time-dependent Receiver operating characteristic (ROC) curves show the sensitivity and specificity of the LKB1 signature compared to the gene expression profiling signature (GEP) (Onken et al., 2004), for predicting the progression free survival (Laurent et al., 2011). E. Representative box and whiskers plots of the LKB1 loss signature score based on LKB1 mRNA expression level (low and high) from the UM-TCGA dataset. Mann-Whitney test was performed for comparison between groups. *p=0.0487. All points are represented.

Figure 3. Inhibition of SLC8A1 and mitochondrial ROS trigger tumor regression. Tumor weights of the indicated xenografts at the endpoint (12 days) are shown as the mean ± SD (n = 4 mice, each group). Mann-Whitney test was performed for comparison between groups. *p < 0.05.

EXAMPLE:

Material & Methods Cell cultures

Human uveal melanoma cell lines OMM1.3 (GNAQQ209P) (Chen et al., 1997), 92.1 (GNAQQ209L) (De Waard - Siebinga et al., 1995) and OMM2.5 (GNAQQ209P) (Chen et al., 1997) were grown in RPMI supplemented with 10% FBS at 37° C in a humidified atmosphere containing 5%CO2 (Griewank et al., 2012). Cell lines are regularly tested for mycoplasma and are mycoplasma-free.

Biochemicals

KB-R7943 and H89 were obtained from MedChem. MitoQ was obtained from Cayman Chemical. EDTA and EGTA were from Sigma. ORM10103 was from Fisher Scientific.

Pooled CRISPR library details

In total, 3,052 unique sgRNAs targeting 763 human kinome genes for 4 guides per target were used for pooled CRISPR screens (Addgene #75312). Libraries were amplified following the Broad Institute protocol. To ensure library diversity, colonies were collected from 15 bacterial plates after transformation of STBL4 electrocompetent cells (New England Biolabs). The pool of plasmids was prepared for infection using an endotoxin-free Maxi prep kit (Qiagen).

CRISPR-Cas9 screen

Human 0MM1.3 uveal melanoma cells were first infected with the lentiCas9-Hygro (LCH) (Addgene # 104995) and selected with hygromycin (10 pg/mL). Cells were then infected with the sgRNA library at a low MOI (<1) to ensure a single sgRNA vector per cell. After 48 hours of infection, cells were treated with 0.5pg of puromycin for 72 hours and <20% of cells were selected, corresponding to single vector copy. Cells were next expanded for 10 days. A fraction of cells was collected at day 0 to ensure a proper coverage of sgRNAs. Medium was changed every 3 days. At day 35, cells from all conditions were collected and genomic DNA was extracted. Since melanin pigment may interfere with DNA-and/or RNA-based molecular profiling (Lagonigro et al., 2004), we purified the samples using the OneStepTM PCR inhibitor Removal Kit (Zymo Research). The integrated sgRNAs were then amplified by PCR with primers containing multiplexing barcodes and adaptors and sequenced on the Illumina NextSeq500. Hits were selected based on the log2 fold change of sgRNA reads at day 35. Analyses and plots of the sequencing data were conducted using Prism 6 software (GraphPad Software) and Rank Products Analysis to determine P values. Data were analysed using the software Mageck which calculates a score based on a fold change where either sgRNA is depleted or enriched compared to the control condition.

RNA-sequencing

Reads were preprocessed in order to remove adapter and low - quality sequences (Phred quality score below 20). After this preprocessing, reads shorter than 40 bases were discarded for further analysis. These preprocessing steps were performed using cutadapt version 1.10. Reads were mapped to rRNA sequences using bowtie version 2.2.8, and reads mapping to rRNA sequences were removed for further analysis. Reads were mapped onto the hg38 assembly of Homo sapiens genome using STAR version 2.5.3a. Gene expression quantification was performed from uniquely aligned reads using htseq - count version 0.6. Ipl, with annotations from Ensembl version 99 and “union” mode. Only non - ambiguously assigned reads have been retained for further analyses. Read counts have been normalized across samples with the median - of - ratios method (Anders and Wolfgang, 2010). Differential gene expression analysis was performed using the methodology implemented in the Bioconductor package DESeq2 version 1.16.1 (Love et al., 2014). P - values were adjusted for multiple testing by the method proposed by Benjamini and Hochberg (Benjamini and Hochberg, 1995). Deregulated genes were defined as genes with log2(foldchange) > 1 or < -1 and adjusted P - value < 0.05.

Transient transfection of siRNA and infection of shRNA

Briefly, a single pulse of 50 nM of control or SLC8A1 siRNA (Dharmacon J-007620-08-0010 and Sigma SASI_Hs02_00325535) was administered to the cells at 50% confluency through transfection with 5 pl of LipofectamineTM RNAiMAX in Opti-MEM medium (Invitrogen, San Diego, CA, USA) as described (Leclerc et al., 2019). LKB1 wild-type and kinase dead mutant were purchased from Addgene. mRNA preparation and real-time/quantitative PCR

The mRNAs were prepared using TRIzol (Fisher Scientific, 15596026T) according to a standard procedure. QRT-PCR was performed using SYBR® Green I (Fisher Scientific, 4368708) and Multiscribe Reverse Transcriptase (Applied Biosystems) and subsequently monitored using the StepOnePlus Real-Time PCR Systems (Applied Biosystems, Foster City, CA). The detection of the ACTIN gene was used to normalize the results. Primer sequences for each cDNA were designed using either Primer bank (https://pga.mgh.harvard.edu/primerbank/). Sequences are available upon request.

Western blot assays

Briefly, cell lysates (30 pg) were separated using SDS-PAGE, transferred onto a PVDF membrane and subsequently exposed to the appropriate antibodies, anti-SLC8Al (1/1,000) and anti-pan phospho-threonine SIK (1/1,000), from ABCAM, anti-LKBl (D60C5; 1/1,000) and anti-SIK2 (D28G3; 1/1,000) from CST, anti-actin (1/1,000) and anti-HSP90 (1/1,000) from Santa Cruz Biotechnology. The proteins were visualized using the ECL system (Amersham). Detection of SLC8A1 was conducted after membrane enrichment using the Mem-PER™ Plus Membrane Protein Extraction Kit (Thermofisher Scientific). The western blots shown are representative of at least 3 independent experiments.

Colony formation assay

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

Immunohistochemistry and RNAscope staining

For immunohistochemical stainings, the cool immunohistochemistry machine was used. Dako Target Retrieval Solution pH9 was used for all stainings. LKB1 (catalog number sc-374334, Santa Cruz Biotechnology, Dallas, TX) was used. mRNAs for SLC8A1 in sections from human metastatic uveal melanomas were detected with RNAscope assay (Biotechne) according to the manufacturer's protocols. Images were captured with a spinning disk confocal microscope (Nikon).

Uveal melanoma metastases were a gift from the University Research Priority Program biobank Zurich, University Hospital Zurich, Switzerland. All patients included in this study have signed a patient release form, which has been approved by an ethics committee and assigned the number EK647 and EK800.

Intracellular Ca2+ measurements Cells were plated in 96 well plates at 20000 cells per well 24 h before the experiment. Adherent cells were loaded for 45 min at 37 °C with the ratiometric dye Fura2-AM (5 pM) then washed by PBS solution supplemented with 2mM Ca2 +. During the experiment, cells were incubated with Physiologic Saline Solution PSS Ca2+. Fluorescence emission was measured at 510 nm using the FlexStation-3 (Molecular Devices, San Jose, CA, USA) with excitation at 340 and 380 nm.

Mitochondrial Ca2+ and ROS measurements

To measure mitochondrial Ca2+ using Rhod-2 AM (543 nm/580-650 nm) dye, the cells were cultured at 50-60% confluency. The cells were washed with media without FBS and antibiotic- antimycotic agents. Then, the cells were incubated in media containing 3 pM Rhod-2 AM (without FBS and antibiotic-antimycotic agents) at 37°C for 45 min. The cells were washed and kept in PSS (HEPES-buffered saline solution (140 mM NaCl, 1.13 mM MgC12, 4.7 mM KC1, 2 mM CaC12, 10 mM D-glucose, and 10 mM HEPES, adjusted to pH 7.4 with NaOH)) containing 2mM CaC12 for imaging. Mitochondrial ROS were measured after cell incubation in a FACS buffer (PBS lx, 1% BSA, 2 mM EDTA) containing 5 pM dihydrorhodamine 123 for 30 min. The cells were washed and kept in FACS buffer.

Animal experimentation

Animal experiments were performed in accordance with French law and approved by a local institutional ethical committee. The animals were maintained on a 12-h light/dark cycle in a temperature-controlled facility at 22°C and provided free access to food (standard laboratory chow diet). Human 0MM1.3 LKB1-WT and LKB1-KO melanoma cells (3x106 cells) were subcutaneously inoculated into 8-wk-old female immune-deficient athymic nude FOXNlnu mice (Janvier Laboratory). When the tumor reached 100 mm3, KB-R7943 (5 mg/kg), MitoQ (5 mg/kg), a combination of both or vehicle (0.5% methylcellulose + 0.1% Tween-80 molecular grade sterile water) was administered three times per week for up to 12 days by intraperitoneal injection. The growth tumor curves were determined after measuring the tumor volume using the equation V = (L x W2)/2 as previously reported (Ohanna et al., 2018).

Statistical analyses

Statistical significance between groups was determined by the Mann-Whitney U test or Unpaired t test with Welsch’s correction if not indicated. *p-value <0.05; **p-value<0.01; * * *p-value<0.001; * * * *p-value<0.0001. Results

A kinome genetic CRISPR-Cas9 screen identifies LKB1 and SIK2 as key drivers of uveal melanoma cell proliferation.

To identify new genes/pathways involved in metastatic uveal melanoma proliferation and survival, we conducted a CRISPR-Cas9 knockout screen using the Human Kinome Brunello pooled sgRNAs library targeting -760 kinases (Doench et al., 2016). Representative 0MM1.3 uveal melanoma cells, originally derived from liver metastasis and harboring a GNAQQ209P mutation, were engineered to stably express Cas9, transduced with the single-guide RNA (sgRNA) library (4 sgRNAs per gene encoded in pLKO.l) and subjected to puromycin selection.

After expansion, genomic DNA was isolated from cell culture at day 0, which represents the library distribution prior to the screen, and at day 35, the abundance of each sgRNA was determined using next-generation sequencing. MaGeck software, which calculates a score based on a fold change where either sgRNAs are depleted (left part of the volcano plot) or enriched (right part of the volcano plot) compared to the control condition, was used to analyze the CRISPR-Cas9 screen dataset.

The screen yielded a number of valuable candidates, among the top hits was LKB1 (Data not shown), a well-established tumor suppressor gene. The functional impact of LKB1 loss on proliferation was validated by introducing individual sgRNAs and deriving clonal cell lines. Partial or total LKB1 inhibition in pool 0MM1.3 cells was confirmed by immunoblot (Data not shown) and resulted in a substantially increased colony -forming capacity (Data not shown). Clonal LKB1 knockout (KO) 0MM1.3 cell lines (LKB1-KO1, LKB1-KO2 and LKB1-KO3) illustrated by complete LKB 1 deficiency in western blot (Data not shown) resulted in an even more pronounced effect showing increased growth rate (Data not shown) and colony -forming capacity compared to 0MM1.3 control cell lines (Ctll, Ctl2 and Ctl3) (Data not shown). The validation was performed in additional models showing enhanced colony-forming ability of LKB 1 -KO in human OMM2.5 metastatic uveal melanoma cells and in human 92.1 primary uveal melanoma cells (Data not shown). Next, we showed in two different LKB 1 -KO clones that adding back the LKB1 wild-type form but not a kinase dead mutant, both expressed at the same level, alleviated the overproliferative phenotype mediated by LKB1 loss (Data not shown), thereby indicating that LKB1 kinase activity is required for rescuing the effects. To determine the role of LKB1 in tumorigenesis, we established xenograft tumors via subcutaneous inoculation of LKB1 wild-type (Ctl) and LKB1-KO 0MM1.3 cells into the left flanks of nude mice. LKB1-K0 cells displayed a significant increase of tumor growth compared to LKB1 -wild-type cells (Data not shown). These results indicate that LKB1 is critically required in constraining proliferation and tumorigenesis of uveal melanoma cells.

LKB1 is a well-known tumor suppressor that is inactivated by mutation in several cancers such as non-small cell lung cancer and cervical carcinomas (Cancer Genome Atlas Research Network, 2014; Wingo et al., 2009). Some loss-of-function mutations have been reported in cutaneous melanomas (Guldberg et al., 1999; Rowan et al., 1999), but not in uveal melanomas. LKB1 expression in primary uveal melanomas (TCGA cohort) is not associated with patient survival or metastasis development, likely because the expression of LKB1 is very homogeneous, and that its activity rather than its expression is important as a kinase. In human metastatic uveal melanoma (Karlsson et al., 2020), LKB1 is also rather homogeneously expressed, and at same extend in liver or skin metastasis (Data not shown). However, immunohistochemistry analyses of human uveal melanoma skin metastases showed some intratumoral heterogeneity with high or low LKB1 expression areas (Data not shown). LKB1 is found to be expressed mainly in the cytoplasm with, in some region, a reinforcement of the labelling at the membrane that was reported to be associated with LKB1 activation (Dogliotti et al., 2017). As LKB1 loss favored metastatic uveal melanoma cells proliferation, the low or negative LKB1 areas might mark the active regions within the tumors.

LKB1 regulates calcium metabolism and SLC8A1 expression in metastatic uveal melanoma cells

The role of LKB1 in uveal melanoma has never been studied. To gain insights into the mechanism by which LKB1 restricts metastatic uveal melanoma cell growth, we profiled the transcriptomes of Ctl and LKB1-KO cells. We identified genes that were specifically upregulated (n=198) and downregulated (n=72) in LKB1-KO compared to Ctl cells (Data not shown). Gene Set Enrichment Analysis (GSEA) of the datasets uncovered 4 gene sets out of the 30 related to calcium (Ca2+) in LKB1-KO cells (Data not shown). Together, these observations indicate that the Ca2+ metabolism might play a critical role in LKB1 mediated effect in uveal melanoma cells. We next used FURA-2-AM staining and F340/F380 ratio for qualitative description of changes in the cytosolic Ca2+ concentration. Remarkably, LKB1-KO was associated with enhanced intracellular Ca2+ level (Data not shown). Moreover, when reducing extracellular free calcium concentration with EGTA, 0MM1.3 Ctll cell number was strongly reduced, demonstrating that metastatic uveal melanoma cells struggle in proliferating when external calcium concentration is very low (Data not shown). Importantly, EGTA displayed stronger effect in LKB1 -KO cells, suggesting that LKB1 loss rendered 0MM1.3 cells more addict to Ca2+ than the control cells. BAPTA-AM, a well-known membrane permeable chelator of intracellular calcium also strongly impaired the proliferative ability of LKB1-KO cells (Data not shown . Collectively, these data indicate that the proliferative effect triggered by LKB1 loss might highly rely on Ca2+ metabolism. Searching for the genes up-regulated in LKB1-KO cells and involved in Ca2+ metabolism, our attention has been drawn by the sodium (Na+)/calcium (Ca2+) exchanger SLC8A1 (solute carrier family 8 member A) belonging to the NCX family. Indeed, among the calcium exchanger/transporter regulators comprising members of the ORAI, TRPC, TRPV, CACNA, MICU, NCX families (3), only the sodium (Na+)/calcium (Ca2+) exchanger SLC8A1 (solute carrier family 8 member A) belonging to the NCX family, was statistically significantly deregulated in LKB1-KO cells (Data not shown .

Increased SLC8A1 expression in LKB1-KO cells, that was confirmed by RT-qPCR, was impaired upon re-introduction of a wild-type but not a kinase dead version of LKB1, demonstrating the specificity of the effect (Data not shown}. Importantly, expression of SLC8A1 is associated with worse prognosis and survival in uveal melanoma (Figure 1}. Moreover, SLC8A1 expression negatively correlates with BAP1 expression, which loss in uveal melanomas is associated with the metastatic risk (Data not shown}. It is worth noting that SLC8A2 level is weak, not affected by LKB1 loss nor significant of the prognostic (Data not shown} and SLC8A3 is not expressed.

SLC8A1 plays a predominant role in metastatic uveal melanoma cell proliferation

To investigate the role of SLC8A1 in uveal melanoma biology, we first analysed its expression in human metastatic uveal samples. Given the lack of high quality SLC8A1 antibody for immunochemistry, its expression in human skin metastasis was evaluated by RNAscope® fluorescence in situ hybridization assay in two different patients combined with immunofluorescence staining of CD44 to detect the membrane contour. The staining was heterogeneous showing cells with high and low SLC8A1 level (Data not shown}. Positive and negative control staining are shown (Data not shown}. In agreement with the expression pattern in primary uveal melanomas, data showed that SLC8A1 expression is heterogeneous compared to the homogeneous LKB1 expression in the cohort of metastatic uveal melanomas (Karlsson et al., 2020). Moreover, SLC8A1 level is higher in liver metastases than in skin metastases (Data not shown}. Altogether, these observations suggest that LKB 1 function is reduced in the liver and/or favors a liver tropism. We next evaluated the role of SLC8A1 in LKB1-KO mediated proliferative effect. Two different siRNA, which efficiently reduced SLC8A1 at both mRNA and protein level (Data not shown), significantly impaired the enhanced colony formation ability induced by LKB1 loss (Data not shown). The effect of SLC8A1 knock-down by siRNA on reducing metastatic uveal melanoma cell formation capacity was confirmed in a different clone (Data not shown). These results indicate that the upregulation of SLC8A1 expression downstream of LKB1 controls the proliferative capacity of metastatic uveal melanoma cells.

SIK2 controls SLC8A1 expression and metastatic uveal melanoma cell proliferation driven by LKB1 loss.

Next, we asked how LKB1 suppression enhanced SLC8A1 expression. Interestingly, our CRISPR/Cas9 screen also disclosed enrichment for sgRNAs targeting SIK2. SIK2 is one of the several kinases activated by LKB 1. As performed for LKB 1 , the functional impact of SIK2 loss was validated by introducing individual sgRNAs. SIK2 inhibition in pooled 0MM1.3 cells was confirmed by immunoblot (Data not shown) and resulted in an increased colony-forming capacity (Data not shown). While SIK2 was detected as a doublet, both sgRNAs and siRNA indicated that SIK2 corresponds to the upper band and confirmed the efficacy of both approaches to inhibit SIK2 (Data not shown). Next, clonal cell lines were derived to generate 0MM1.3 SIK2-KO cell lines (SIK2-KO1, SIK2-KO2 and SIK2-KO3) in which knockout was confirmed by immunoblot (Data not shown). SIK2 deficiency resulted in an increased colonyforming capacity (Data not shown) and growth rate (Data not shown). The validation was performed in additional models showing enhanced colony-forming ability upon SIK2 suppression in human metastatic OMM2.5 cells and in human 92.1 primary cells (Data not shown). The overproliferative phenotype caused by SIK2 loss was rescued when SIK2-WT was reintroduced into SIK2-KO clonal cell lines (SIK2-KO1 and SIK2-KO2) compared to noninfected cells or introduction of an empty vector (Data not shown). Then, we asked whether SIK2 phosphorylation by LKB1 was involved in constraining proliferation. Our data showed that LKB 1 -KO dramatically reduced SIK2 phosphorylation to an extent similar to that of SIK2- Q(Data not shown). Adding back a constitutively active form of SIK2 (SIK2 T175D), but not a kinase dead form (SIK2 K49M), alleviated the overproliferative phenotype mediated by LKB1 suppression in two different clones (LKB 1 -KOI and LKB1-KO2), indicating that SIK2 kinase activity is required for LKB1 function in constraining uveal melanoma cell proliferation (Data not shown). Then, we assessed the effect of SIK2 loss on SLC8A1 expression level. Our data showed that SLC8A1 expression was enhanced in SIK2-KO cells while it returned to the basal level upon adding back SIK2 WT (Data not shown). Likewise, the increase in SLC8A1 mediated by LKB1 loss was dramatically reduced by SIK2 WT forced expression (Data not shown). These observations were confirmed in additional SIK2-KO and LKB1-KO clones (Data not shown). Finally, we also showed that SIK2-KO cells treated with SLC8A1 siRNA proved to be less proliferative compared to cells treated with control siRNA (Data not shown). These results demonstrate that SIK2 is critical for controlling SLC8A1 expression and for constraining proliferation downstream of LKB1 in uveal melanoma cells.

LKB1 and SIK2 suppression are associated with enhanced mitochondrial Ca2+ and ROS level.

Mitochondria has been reported to act as a spatial Ca2+ buffer in many cells. We therefore measured mitochondrial Ca2+ (mtCa2+) level using the sensitive dye Rhod-2 AM. Data revealed more Ca2+ taken up by mitochondria in LKB1-KO and SIK2-KO cells compared to the control cells (Data not shown). High mtCa2+ level stimulates respiratory chain activity leading to higher amounts of mitochondrial reactive oxygen species (mtROS) (Gbrlach et al., 2015) which can activate intracellular signaling pathways and contribute to proliferation and survival in many cancers (Sabharwal and Schumacker, 2014). To determine mtROS, we used dihydrorhodamine 123 based assays. Enhanced mtROS level was detected in LKB1-KO and SIK2-KO uveal melanoma cells compared to the control cells (Data not shown). We next asked whether mtROS could be exploited to inhibit the growth of uveal melanoma cells. We evaluated the effect of a mitochondria-targeted antioxidant, mitoquinol (MitoQ), which has been used in clinical trials to selectively deactivates mitochondrial superoxide. The SLC8A1 inhibitor KB- R7943 or MitoQ alone did not affect WT, or LKB1 and SIK2-deficient cell survival, while the combination of these 2 inhibitors promoted a massive apoptosis to a much higher extent in LKB1-KO and SIK2-KO cells (Data not shown).

Clinical implications of the LKB1-SIK2-SLC8A1 cascade.

We evaluate the impact of SLC8A1 and mitochondrial ROS on tumor growth in vivo. Subcutaneous injections of control and LKB1-KO cells were performed in athymic nude mice, then when the tumors reached 100 mm3, they were treated with KB-R7943, MitoQ or their combination and vehicle. Note that 0MM1.3 control cells did not grow as xenografts within the time frame used in this experiment. A strong reduction in tumor volume, size and weight was observed in LKB 1 -KO cells treated with the combination of KB-R7943 and MitoQ (Figure 2A and Figure 3), suggesting that Ca2+ metabolism and mtROS are critical for uveal melanoma progression. The treatment did not affect body weight (Data not shown . Collectively, these results indicate the selectivity and potency of SLC8A1 and mitochondrial ROS inhibition toward LKB 1 -deficient UM cells.

Interestingly, from the RNA-seq analysis we developed a eleven-genes signature associated with LKB1 depletion (Data not shown), that will be useful to identify tumors with LKB1 decreased activity, regardless of its expression level. High LKB1 loss signature score is associated with a reduced survival (Figure 2B) and with a high sensitivity and specificity to identify patients that will develop metastasis and eventually die as shown by the ROC curve (Figure 2C). ROC analyses indicated that this signature performed as well, or even slightly better than the gene expression profiling (GEP) signature used to predict the risk of metastasis in patients with uveal melanoma (Onken et al., 2004). In another ROC analyses, we demonstrate the progression-free survival of the LKB1 loss signature in another cohort of patients (Laurent et al data) (Figure 2D). We also shows that the LKB1 loss signature (which does not include LKB1) is more strongly associated with tumors with a low level of LKB 1 compared to tumors with a higher level of LKB 1 (Figure 2E).

Therefore, the LKB1 loss gene signature might serve as a prognosis stratification tool to predict survival outcomes of uveal melanoma patients. Since we have shown in vitro and in xenograft model that LKB1 loss increases uveal melanoma cells sensitivity to the combination KB-R7943 with MitoQ, this signature therefore predicts the response to the treatment.

Discussion

In this work, using a kinome-wide CRISPR screen, we disclose new kinases, LKB1 and SIK2, whose loss is critically required for uveal melanoma cell proliferation. Importantly, lack of LKB1 expression is detected in human metastasis of uveal melanoma tissues. These data demonstrate the critical pathological role of deficiency in the LKB1-SIK2 module and suggest that their inhibition contributes to the deadliest phase in the malignant progression of uveal melanomas. Indeed, while metastases are responsible for most cancer deaths, it is not the metastatic process by itself that is lethal, but proliferation of isolated metastatic cells into overt, clinically detectable metastatic lesions which in turn results in organ failure and patient mortality. Although genetic alterations in LKB1 have not been reported in uveal melanomas, nongenetic inactivation could decrease its expression and/or activity. How LKB1 function is regulated in human metastatic uveal melanomas remains to be determined. We found that LKB1 enhances intracellular Ca2+ level and expression of SLC8A1 a unique calcium transport system that plays a major role in the regulation of the intracellular Ca2+ concentration.

While generally SLC8A transport Ca2+ ions out of the cell in exchange for sodium ions and are considered one of the most important cellular mechanisms for removing Ca2+, in pathological settings such as cancers, they can work in a reverse mode, promoting Ca2+ influx (Chovancova et al., 2020). Our data suggest that SLC8A1 functions in a reverse mode in uveal melanoma given that LKB1 loss increases intracellular Ca2+ concentration. Likewise, in cutaneous melanoma cells, SLC8A1 has been shown to operate in the reverse mode (Rodrigues et al., 2019; Sennoune et al., 2015).

How LKB1 and SIK2 loss impair SLC8A1 level and/or activity has to be determined. There are few reports indicating that SLC8A1 activity is regulated by different factors, such as pH, ATP, phosphatidylinositol 4, 5 -bisphosphate (PIP2), and post-translational modifications including phosphorylation or palmitoylation (Morad et al., 2011; Plain et al., 2017; Schulze et al., 2003). These factors might perturb the structure of the transporter or the macromolecular complex in which it is functioning and impact on its membrane anchorage.

We detected more mitochondrial Ca2+ level in LKB1 and SIK2 deficient cells. Ca2+ stimulates mitochondrial oxidative metabolism which plays an important role in regulating ATP production, to meet the cells’ energy demands, and ROS generation. Alterations in both mitochondrial Ca2+ and ROS are hallmarks of tumors.

These results provide the basis for some potential anti-cancer therapeutic strategies targeting cellular metabolism. LKB1 is a master kinase that activates several kinases of the AMPK subfamily. A well-known substrate of LKB1 is AMPK, which is likely to mediate most, if not all, of the tumor suppressor effects of LKB1 including cell survival and proliferation (Shackelford and Shaw, 2009), suggesting that LKB1 itself or AMPK could be a therapeutic target. In this regard, although small molecules activating do not currently exist, activators of AMPK could potentially be used to activate LKB 1 function.

However, we found that AMPK knockdown by siRNA reduced the growth of uveal melanoma cell growth (data not shown). This is reminiscent of a previous study showing similar effect of AMPK inhibition (Chua et al., 2022).

Our data also show that LKB1 or SIK2-deficient uveal melanoma cells are more sensitive and vulnerable to disruption of Ca2+ homeostasis and oxidative stress. This metabolic vulnerability provides a therapeutic strategy to treat these metastatic uveal melanoma subtypes. In fact, here we find that the combination of the SLC8A1 inhibitor, KB-R7943, and the mitochondria- targeted ROS scavenger MitoQ, the only mitochondrial antioxidant which has safely been used in clinical trials (Rossman et al., 2018; Smith and Murphy, 2010), have an enhanced cell death efficacy in LKB1 and SIK2 -negative uveal melanoma cells.

In summary, our data demonstrate that LKB 1 or SIK2 deficiency creates an hypersensitivity to SLC8A1 inhibition and ROS blockers, revealing promising therapeutic perspectives for a subset of metastatic uveal melanomas.

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Claims

CLAIMS:

1. A method for treating melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of a combination of SLC8A1 inhibitor and mitochondria-targeted antioxidant.

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

3. The method according to claims 1 and 2 wherein the melanoma is uveal melanoma resistant.

4. The method according to claims 1 to 3 wherein the SLC8A1 inhibitor is KB-R7943.

5. The method according to claims 1 to 3 wherein the mitochondria-targeted antioxidant is mitoquinol (MitoQ).

6. The method according to claims 1 to 5 wherein the combination of SLC8A1 inhibitor and mitochondria-targeted antioxidant is administered by topical or intravitreal administration.

7. i) SLC8A1 inhibitor, ii) mitochondria-targeted antioxidant and iii) a classical treatment, as a combined preparation for use in the treatment of melanoma.

8. The combined preparation for use according to claim 7 wherein the melanoma is uveal melanoma.

9. The combined preparation for use according to claims 7 to 8 wherein the SLC8A1 inhibitor is KB-R7943.

10. The combined preparation for use according to claims 7 to 8 wherein the mitochondria- targeted antioxidant is mitoquinol (MitoQ).

11. A pharmaceutical composition comprising an SLC8A1 inhibitor and a mitochondria- targeted antioxidant for use in the treatment of melanoma and/or uveal melanoma.

12. The pharmaceutical composition for use according to claim 11 comprising i) SLC8A1 inhibitor, ii) mitochondria-targeted antioxidant and iii) a classical treatment, as a combined preparation for use in the treatment of melanoma.

13. The pharmaceutical composition for use according to claims 11 to 12 wherein the SLC8A1 inhibitor is KB-R7943.

14. The pharmaceutical composition for use according to claims 11 to 12 wherein the mitochondria-targeted antioxidant is mitoquinol (MitoQ).

15. A method of screening a drug suitable for the treatment of melanoma comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression and/or the activity of SLC8A1.

16. A method for predicting the survival time of a patient suffering from uveal melanoma comprising i) determining in a tumor sample obtained from the patient the loss gene expression level of LKB1 ii) comparing the expression level determined at step i) with a predetermined reference value and iii) providing a good prognosis when the loss expression level of LKB1 determined at step i) is lower than the predetermined reference value, or providing a bad prognosis when the loss expression level of LKB1 determined at step i) is higher than the predetermined reference value.

17. A method for determining whether a patient suffering from uveal melanoma will respond to a combination treatment of SLC8A1 inhibitor and mitochondria-targeted antioxidant of comprising i) determining in a tumor sample obtained from the patient the loss gene expression level of LKB1 ii) comparing the loss expression level determined at step i) with a predetermined reference value and iii) concluding that the patient will significantly respond to the treatment when the expression level of LKB1 determined at step i) is higher than the predetermined reference value.