WO2021048292A1 - Methods and compositions for treating melanoma - Google Patents

Abstract

The present invention relates to method and composition for treating melanoma. The inventors demonstrated that MITF loss favors the escape of melanoma cells from the immune system. This effect implies the secretion of Integrin subunit beta like 1 (ITGBL1) and the inhibition of NK cells. The inventors also showed that MITF repressed RUNX2, an activator of ITGBL1 transcription, and that VitaminD3, an inhibitor of RUNX2, sensitizes melanoma cells to death by immune cells. In particular, the present invention relates to a method for predicting whether a subject suffering from a melanoma is or is at risk of having resistant melanoma. The present invention also relates to methods for treating melanoma and resistant melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of an inhibitor of ITGBL1.

Description

METHODS AND COMPOSITIONS FOR TREATING MELANOMA

FIELD OF THE INVENTION:

The invention is in the field of oncology, more particularly the invention relates to methods and compositions for treating melanoma.

BACKGROUND OF THE INVENTION:

Despite recent therapeutic improvements, the prognosis for patients with metastatic melanoma is still very pejorative. Indeed, targeted therapies (TT) using BRAF inhibitors, now in combination with MEK inhibitors have shown dramatic results in terms of response rates. However, the initial enthusiasm was tempered by the observation of almost systematic recurrences, due to the acquisition of secondary resistances (Ugurel et al, 2017). Immuno- therapeutic approaches targeting negative check points for the immune response (ICT) showed promising results, especially anti-PDl (Nivolumab, Pembrolizumab), with which nearly 30% of sustained responses were observed (Carretero-Gonzalez et al, 2018). A recent clinical trial showed that the rate of progression-free survival between 2 and 4 years was around 40% in the nivolumab-plus-ipilimumab (anti-CTLA4) group, suggesting that 40% of patients might be cured by this treatment (Ugurel et al, 2017). Unfortunately, despite the undeniable progresses brought by these new therapies, most patients (50-60%) are resistant or develop resistance to such treatment, highlighting the need to find new therapeutic approaches.

Recently, compelling data dissecting the causes of resistances to TT and ICT have been gathered. Both, genetic (mutations, amplifications, translocations) and non-genetic (micro environment, transcriptional) factors are thought to contribute to acquisition of resistance. Indeed, mutations in NRAS, MEKs, and BRAF amplification or translocation (non-exhaustive list) are causative of some resistance to TT (Luke et al, 2017). Resistances to ICT involve mutations in JAK1/2 and B2M (MHC class I component) (Zaretsky et al., 2016). However, the main cause of resistance is non-genetic. It implies a phenotypic plasticity which results in rewiring of the transcriptional program allowing the adaptation of melanoma cells to stressful conditions imposed by the micro-environment (hypoxia, nutriment shortage) or by the treatment itself. Despite the diversity of non-genetic mechanisms of resistance, it seems that the loss of MITF, a de-differentiation process and the implementation of a pseudo-EMT (Epithelial- Mesenchymal transition) (Falletta et al., 2017; Holzel and Tuting, 2016) are central to the acquisition of the resistances to targeted (Richard et al., 2016) and immuno-therapies (Bu et al., 2016).

Focusing on immunotherapy, presence of pre-existing CD8 cells in the tumor and their active proliferation appear to be essential for an efficient anti-melanoma response (Tumeh et al, 2014). At the molecular level, pro-inflammatory cytokines produced by low MITF melanoma cells (Ohanna et al, 2011; Tirosh et al, 2016; Zhao et al, 2016) were reported to alter the immune microenvironment by increasing myeloid cells recruitment and thereby favoring melanoma cells proliferation, impairment of the immune responses and development of metastasis (Riesenberg et al, 2015).

A recent study identified a molecular signature predictive for the anti-PDl therapy response (IPRES signature gene) (Hugo et al, 2016). For instance, increased expression in tumors of several cytokines such as CCL2, CCL8, IL-10, VEGFA/C was associated to anti- PDl resistance. Along with this, AXL, PDGFRA and WNT5A, known markers of resistance to TT, were increased in tumors of non-responding patients. These markers were also upregulated in MITF low cells (Ahmed and Haass, 2018; Dai et al, 2013; Muller et al., 2014). As the IPRES signature overlaps with what is found in tumors resistant to targeted therapy and with that of MITF low cells, the inventors studied the role of MITF in the immune response towards melanoma cells. Here the inventors show that, in vitro , the loss of MITF favors the escape of melanoma cells from the immune system. This effect is largely mediated by a secreted protein Integrin subunit beta like 1 (ITGBL1), whose immune function is yet to be described. Upregulation of ITGBL1 decreases immune-mediated death of melanoma cells, while Vitamin D3, which inhibit RUNX2 and ITGBL1 expression, improved immune-mediated death of melanoma cells. There is a still for biomarker of the immune tolerance towards melanoma.

Surprisingly the results identify ITGBL1 as a new predictive marker of the immune response and a key mediator of the immune tolerance towards melanoma. Accordingly, targeting ITGBL1 in immunotherapy resistant patient could be of interest to alleviate resistance.

SUMMARY OF THE INVENTION:

The invention relates to methods for treating melanoma and therapy-resistant melanoma in a subject in need thereof comprising the step of administering said subject with a therapeutically effective amount of an inhibitor of ITGBL1. In particular, the present invention is defined by the claims.

DETAILED DESCRIPTION OF THE INVENTION: Resistances to immunotherapies remain still poorly understood. In melanomas, non- genetic resistances are associated with an increase in the mesenchymal phenotype, a de differentiation and the loss of the transcription factor MITF.

The inventors demonstrated that MITF loss favors the escape of melanoma cells from the immune system. This effect implies the secretion of Integrin subunit beta like 1 (ITGBL1). ITGBL1 inhibited immune cell activation and cytotoxicity against melanoma cells in vivo and in vitro. Moreover, NK cells activity is decrease by ITGBL1 decreasing melanoma growth. Mechanistically, the inventors showed that MITF repressed RUNX2, an activator of ITGBL1 transcription, and that VitaminD3, an inhibitor of RUNX2, sensitizes melanoma cells to death by immune cells. The data indicate that inhibition of ITGBL1, through vitamin D3 supplementation might improve response to immunotherapies.

Method for predicting the risk of sufferins from resistant melanoma

In a first aspect, the invention relates to a method for predicting whether a subject suffering from a melanoma has or is at risk of having resistant melanoma comprising the steps of i) quantifying the expression level of ITGBL1 in a biological sample obtained from the subject; ii) comparing the expression level quantified at step i) with a predetermined reference value and iii) concluding that the subj ect has or is at risk of suffering from resistant melanoma when the level determined at step i) is higher than the predetermined reference value.

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 cutaneous melanoma.

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

In a particular embodiment, the subject has or is susceptible to have resistant melanoma.

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-tumor 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), b- (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- m ethoxy-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 b- carboline derivative or a brassilexin derivative. In a particular embodiment, the IDO inhibitor is selected from 1 -methyl-tryptophan, b-(3- benzofuranyl)-alanine, 6-nitro-L-tryptophan, 3- Amino-naphtoic acid and b-[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-1, 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.

As used herein, the term "predicting the risk" refers to assessing the probability according to which the subject as referred to herein will develop resistant melanoma. As will be understood by those skilled in the art, such an assessment is usually not intended to be correct for 100% of the subjects to be investigated.

As used herein, the term “EGF-like protein family” is a conserved protein domain, which derives its name from the epidermal growth factor. It comprises about 30 to 40 amino- acid residues and has been found in a large number of mostly animal proteins

As used herein, the term “Integrin subunit beta like 1” (ITGBL1) refers to a beta integrin-related protein that is a member of the EGF-like protein family. The encoded protein contains integrin-like cysteine-rich repeats. Alternative splicing results in multiple transcript variants. The naturally occurring human ITGBL1 gene has a nucleotide sequence having the following number: GenelD: 9358 and the naturally occurring human ITGBL1 protein has an aminoacid sequence as shown in Genbank Accession numbers AAN16024.1 As used herein, the term “expression level” refers to the expression level of ITGBL1. Typically, the expression level of the ITGBL1 gene may be determined by any technology known by a person skilled in the art. In particular, each gene expression level may be measured at the genomic and/or nucleic and/or protein level. In a particular embodiment, the expression level of gene is determined by measuring the amount of nucleic acid transcripts of each gene. In another embodiment, the expression level is determined by measuring the amount of each gene corresponding protein. The amount of nucleic acid transcripts can be measured by any technology known by a man skilled in the art. In particular, the measure may be carried out directly on an extracted messenger RNA (mRNA) sample, or on retrotranscribed complementary DNA (cDNA) prepared from extracted mRNA by technologies well-known in the art. From the mRNA or cDNA sample, the amount of nucleic acid transcripts may be measured using any technology known by a man skilled in the art, including nucleic microarrays, quantitative PCR, microfluidic cards, and hybridization with a labelled probe. In a particular embodiment, the expression level is determined by using quantitative PCR. Quantitative, or real-time, PCR is a well-known and easily available technology for those skilled in the art and does not need a precise description. Methods for determining the quantity of mRNA are well known in the art. For example the nucleic acid contained in the biological sample 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) and/or amplification (e.g., RT-PCR). Preferably quantitative or semi-quantitative RT- PCR is preferred. Real-time quantitative or semi-quantitative RT-PCR is particularly advantageous. 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 do not need to 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. A wide variety of appropriate indicators are known in the art including, fluorescent, radioactive, enzymatic or other ligands (e. g. avidin/biotin). 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 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 method of the invention comprises the steps of providing total RNAs extracted from a biological sample 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 embodiment, the expression level is determined by DNA chip analysis. Such DNA chip or nucleic acid microarray consists of different nucleic acid probes that are chemically attached to a substrate, which can be a microchip, a glass slide or a microsphere-sized bead. A microchip may be constituted of polymers, plastics, resins, polysaccharides, silica or silica-based materials, carbon, metals, inorganic glasses, or nitrocellulose. Probes comprise nucleic acids such as cDNAs or oligonucleotides that may be about 10 to about 60 base pairs. To determine the expression level, a biological 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).

As used herein, the term “biological sample” refers to any sample obtained from a subject, such as a serum sample, a plasma sample, a urine sample, a blood sample, a lymph sample, or a tissue biopsy.

In a particular embodiment, biological sample for the determination of an expression level include samples such as a blood sample, a lymph sample, or a tissue biopsy. In a particular embodiment, the biological sample is a blood sample, more particularly, circulating tumor cells (CTCs). Detection of CTCs are available to the man skilled in the art (see e.g. the article by Long et al, Cancer Medecine, 2016, 5(6): 1022-1030 [9]).

Typically, the predetermined reference value is a threshold value or a cut-off value. Typically, a "threshold value" or "cut-off value" can be determined experimentally, empirically, or theoretically. A threshold value can also be arbitrarily selected based upon the existing experimental and/or clinical conditions, as would be recognized by a person of ordinary skilled in the art. For example, retrospective measurement of cell densities in properly banked historical subject samples may be used in establishing the predetermined reference value. The threshold value has to be determined in order to obtain the optimal sensitivity and specificity according to the function of the test and the benefit/risk balance (clinical consequences of false positive and false negative). Typically, the optimal sensitivity and specificity (and so the threshold value) can be determined using a Receiver Operating Characteristic (ROC) curve based on experimental data. For example, after quantifying the cell density in a group of reference, one can use algorithmic analysis for the statistic treatment of the measured densities in samples to be tested, and thus obtain a classification standard having significance for sample classification. The full name of ROC curve is receiver operator characteristic curve, which is also known as receiver operation characteristic curve. It is mainly used for clinical biochemical diagnostic tests. ROC curve is a comprehensive indicator that reflects the continuous variables of true positive rate (sensitivity) and false positive rate (1 -specificity). It reveals the relationship between sensitivity and specificity with the image composition method. A series of different cut-off values (thresholds or critical values, boundary values between normal and abnormal results of diagnostic test) are set as continuous variables to calculate a series of sensitivity and specificity values. Then sensitivity is used as the vertical coordinate and specificity is used as the horizontal coordinate to draw a curve. The higher the area under the curve (AUC), the higher the accuracy of diagnosis. On the ROC curve, the point closest to the far upper left of the coordinate diagram is a critical point having both high sensitivity and high specificity values. The AUC value of the ROC curve is between 1.0 and 0.5. When AUC>0.5, the diagnostic result gets better and better as AUC approaches 1. When AUC is between 0.5 and 0.7, the accuracy is low. When AUC is between 0.7 and 0.9, the accuracy is moderate. When AUC is higher than 0.9, the accuracy is quite high. This algorithmic method is preferably done with a computer. Existing software or systems in the art may be used for the drawing of the ROC curve, such as: MedCalc 9.2.0.1 medical statistical software, SPSS 9.0, ROCPOWER.SAS, DESIGNROC.FOR, MULTIREADER POWER S AS, CREATE-ROC.SAS, GB STAT VIO.O (Dynamic Microsystems, Inc. Silver Spring, Md., USA), etc.

In some embodiments, the predetermined reference value is determined by carrying out a method comprising the steps of a) providing a collection of tumor tissue samples from subject suffering from melanoma; b) providing, for each tumor tissue sample provided at step a), information relating to the actual clinical outcome for the corresponding subject c) providing a serial of arbitrary quantification values; d) quantifying the cell density for each tumor tissue sample contained in the collection provided at step a); e) classifying said tumor tissue samples in two groups for one specific arbitrary quantification value provided at step c), respectively: (i) a first group comprising tumor tissue samples that exhibit a quantification value for level that is lower than the said arbitrary quantification value contained in the said serial of quantification values; (ii) a second group comprising tumor tissue samples that exhibit a quantification value for said level that is higher than the said arbitrary quantification value contained in the said serial of quantification values; whereby two groups of tumor tissue samples are obtained for the said specific quantification value, wherein the tumor tissue samples of each group are separately enumerated; f) calculating the statistical significance between (i) the quantification value obtained at step e) and (ii) the actual clinical outcome of the subjects from which tumor tissue samples contained in the first and second groups defined at step f) derive; g) reiterating steps f) and g) until every arbitrary quantification value provided at step d) is tested; h) setting the said predetermined reference value as consisting of the arbitrary quantification value for which the highest statistical significance (most significant P-value obtained with a log-rank test, significance when P<0.05) has been calculated at step g).

For example the cell density has been assessed for 100 tumor tissue samples of 100 subjects. The 100 samples are ranked according to the cell density. Sample 1 has the highest density and sample 100 has the lowest density. 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 subject, 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 (log-rank test). The predetermined reference value is then selected such as the discrimination based on the criterion of the minimum P-value is the strongest. In other terms, the cell density corresponding to the boundary between both subsets for which the P-value is minimum is considered as the predetermined reference value. It should be noted that the predetermined reference value is not necessarily the median value of cell densities. Thus in some embodiments, the predetermined reference value thus allows discrimination between a poor and a good prognosis with respect to DFS and OS for a subject. Practically, high statistical significance values (e.g. low P values) are generally obtained for a range of successive arbitrary quantification values, and not only for a single arbitrary quantification value. Thus, in one alternative embodiment of the invention, instead of using a definite predetermined reference value, a range of values is provided. Therefore, a minimal statistical significance value (minimal threshold of significance, e.g. maximal threshold P value) is arbitrarily set and a range of a plurality of arbitrary quantification values for which the statistical significance value calculated at step g) is higher (more significant, e.g. lower P-value) are retained, so that a range of quantification values is provided. This range of quantification values includes a "cut-off value as described above. For example, according to this specific embodiment of a "cut-off value, the outcome can be determined by comparing the cell density with the range of values which are identified. In some embodiments, a cut-off value thus consists of a range of quantification values, e.g. centered on the quantification value for which the highest statistical significance value is found (e.g. generally the minimum P-value which is found).

The inventors all show that, ITGBL1 overexpressing cells decreased immune cells activity in vivo and NK cells are important to decrease melanoma tumors growth. Also, ITGBL1 overexpression decreased NK cytotoxic activity against melanoma. The inventors showed that NK cells have anti melanoma activity by killing melanoma cells and ITGBL1 decreased NK activation and cytotoxicity against melanoma.

As used herein “Natural killer cells” (NK cells) are a type of cytotoxic lymphocyte critical to the innate immune system.

As used herein “cytotoxicity” refers to the extent of the destructive or killing capacity of an agent, in particular it is meant to describe the character of the NK cell activity that limits the development of cancer cells. Cytotoxic potential can be expressed as the percent of target ceil death above background. NK cells are naturally endowed with a “cytolytic activity”: they are characterized by their ability to initiate an immediate and direct cytolytic response to virally infected or malignantly transformed cells. Method for treatins melanoma and resistant melanoma

ITGBL1 is regulated transcriptionally by RUNX2 that is repressed by MITF. The inventors have shown that Vitamin D3 may directly potentiate immune-mediated melanoma cell death by repressing RUNX2 and ITGBL1 expression.

Accordingly, in a second aspect, the 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 an inhibitor of ITGBL1.

In some embodiment, the present invention relates to an inhibitor of ITGBL1 for use in a method for treating melanoma in a subject in need thereof.

In a particular embodiment, the subject is identified as having melanoma resistance by performing the method as described above.

Accordingly, in a third 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 an inhibitor of ITGBL1.

In some embodiment, the present invention relates to a method for treating resistant melanoma in a subject in need thereof, wherein said method comprises the step of:

(i) Determining in a biological sample obtained from said subject the expression level of ITGBL1

(ii) Comparing the expression levels quantified at step i) with a predetermined reference value and

(iii) Concluding that the subject has or is at risk of suffering from resistant melanoma when the level determined at step i) is higher than the predetermined reference value.

(iv) And administering to said subject a therapeutically effective amount of an inhibitor of ITGBL1 if the expression level of ITGBL1 is higher than the predetermined reference value.

In some embodiment, the present invention relates to an inhibitor of ITGBL1 for use in a method for treating resistant melanoma in a subject in need thereof.

In a particular embodiment, the subject is identified as having melanoma resistant to a classical treatment.

In some embodiments, the subject is identified as having BRAF inhibitors resistance.

In some embodiments, the subject is identified as having MEK inhibitors resistance.

In some embodiments, the subject is identified as having NRAS inhibitors resistance. In some embodiments, the subject is identified as having immune checkpoint inhibitors resistance. Or resistance to all the possible combinations of therapies described above.

In some embodiments, the inhibitor of ITGBL1 may be combined with an immune checkpoint inhibitor. In some embodiment, the present invention relates to a method for treating melanoma and/or resistant melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of an inhibitor of ITGBL1 and of an immune checkpoint inhibitor. In some embodiment, the present invention relates to an inhibitor of ITGBL1 and an immune checkpoint inhibitor for use in a method for treating melanoma and/or resistant melanoma in a subject in need thereof.

As used herein, the term “Runt-related transcription factor 2” (RUNX2) is a protein that in humans is encoded by the RUNX2 gene. RUNX2 is a key transcription factor associated with osteoblast differentiation. RUNX2 plays a cell proliferation regulatory role in cell cycle entry and exit in osteoblasts, as well as endothelial cells.

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.]).

Mechanistically, the inventors showed that MITF repressed RUNX2, an activator of

ITGBL1 transcription, and that VitaminD3, an inhibitor of RUNX2, sensitizes melanoma cells to death by immune cells. The data indicate that inhibition of ITGBL1, through vitamin D3 supplementation might improve response to immunotherapies.

As used herein, the term “inhibitor of ITGBL1” refers to a natural or synthetic compound that has a biological effect to inhibit the expression and/or the activity of ITGBL1.

In one embodiment, the inhibitor of ITGBL1 is VitD3.

As used herein, the term “VitD3” also known as “la,25-Dihydroxyvitamin D3” or “calcitriol” has the formula C27H44O3 and the following structure in the art:

In one embodiment, the inhibitor of ITGBL1 is CADD522.

As used herein, the term “CADD522” has the following CAS Number: 199735-88-1 and the following structure in the art:

In a particular embodiment, the inhibitor of ITGBL1 is a peptide, petptidomimetic, small organic molecule, antibody, aptamers, siRNA or antisense oligonucleotide. The term “peptido mimetic” refers to a small protein-like chain designed to mimic a peptide. In a particular embodiment, the inhibitor of ITGBL1 is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity.

In a particular embodiment, the inhibitor of ITGBL1 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 macro molecules (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 some embodiments, the inhibitor of ITGBL1 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 Rabat et ak, 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 ah, 2006; Holliger & Hudson, 2005; Le Gall et ah, 2004; Reff & Heard, 2001 ; Reiter et ah, 1996; and Young et ah, 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 inhibitor of ITGBL1 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 inhibitor of ITGBL1 is an intrabody having specificity for ITGBL1. 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.

In some embodiment, the antibody can be any blocking antibody. In particular, the anti- ITGBL1 antibody is a blocking antibody. In particular, the blocking antibody (e.g. the antibody anti-ITGBL) is an antibody that blocks the interaction of ITGBL1 with its receptor.

In order to test the functionality of a putative ITGBL1 inhibitor a test is necessary. For that purpose, to identify ITGBL1 inhibitor an in vitro assay will be used. We will measure the IFNg produce by the PBMC, by ELISA, after activation with PMA/ionomycin or CD3/CD28 antibody cocktail in presence or not of the recombinant ITGBL1 and the potential inhibitor developed. Activated PBMC secrete a lot of IFNg that is blocked by rITGBLI. In presence of ITGBL1 inhibitor, level of IFNg is expected to rise compare to the condition with therITGBLI alone.

Acquired resistance to targeted therapies is currently a clinical challenge in the treatment of advanced metastatic melanoma. Therefore, inventors examined the impact of targeting ITGBL1 in melanoma cells resistant to BRAF inhibitors (BRAFi), MEK inbitors (MEKi). They have shown that melanoma treatment with pharmacological inhibitor of ITGBL1 can overcome resistance to drugs targeting oncogenic BRAF.

The resistance of cancer for the medication is caused by mutations in the genes which are involved in the proliferation, divisions or differentiation of cells or by phenotypic switch with a transcriptional profile favoring the resistance to treatment. In the context of the invention, the resistance of melanoma is caused by the mutations (single or double) in the following genes: BRAF, MEK or NRAS. The resistance can be also caused by a double-negative BRAF and NRAS mutation or phenotypic switch.

In a particular embodiment, the melanoma is resistant to a treatment with the inhibitors of BRAF mutations.

In some embodiments, the melanoma is resistant to a treatment.

In a further embodiment, the melanoma is resistant to a treatment with the inhibitors of BRAF. In a particular embodiment, the melanoma is resistant to a treatment with dabrafenib also known as tafmlar which is commercialized by Novartis. In a particular embodiment, the melanoma is resistant to a treatment with vemurafenib. Vemurafenib also known as PLX4032, RG7204 ou R05185426 and commercialized by Roche as Zelboraf. In a particular embodiment, the melanoma is resistant to a treatment with dacarbazine. Dacarbazine also known as imidazole carboxamide is commercialized as DTIC-Dome by Bayer.

In a further embodiment, the melanoma is resistant to a treatment with the inhibitors of MEK. The inhibitors of MEK are well known in the art. In a particular embodiment, the melanoma is resistant to a treatment with trametinib also known as mekinist which is commercialized by GSK. In a particular embodiment, the melanoma is resistant to a treatment with cobimetinib also known as cotellic commercialized by Genentech. In a particular embodiment, the melanoma is resistant to a treatment with Binimetinib also knowns as MEK 162, ARRY-162 is developed by Array Biopharma.

In a particular embodiment, the melanoma is resistant to a treatment with the inhibitors of NRAS. In a particular embodiment, the melanoma is resistant to a treatment with salirasib commercialized by Concordia Pharmaceuticals. The inhibitors of BRAF mutation or MEK are used to treat the melanoma with NRAS mutations. In a particular embodiment, the melanoma is resistant in which double-negative BRAF and NRAS mutant melanoma.

In a particular embodiment, the melanoma is resistant to a combined treatment. As used herein, the terms “combined treatment”, “combined therapy” or “therapy combination” refer to a treatment that uses more than one medication. The combined therapy may be dual therapy or bi-therapy. In the context of the invention, the melanoma is resistant to a combined treatment characterized by using an inhibitor of BRAF mutation and an inhibitor of MEK or an inhibitor of BRAF mutation and an inhibitor of NRAS as described above. For example, the combined treatment may be a combination of Vemurafenib (BRAFi) and Cobimetinib (MEKi), or a combination of Dabrafenib (BRAFi) and Trametinib (MEKi), or a combination of Cobimetinib Encorafenib (BRAFi) and Binimetinib(MEKi).

In a further embodiment, the melanoma is resistant to a treatment with an immune checkpoint inhibitor. The term “immune checkpoint inhibitor”, as used herein, is defined above.

In a particular embodiment, the melanoma is also resistant to a combined treatment characterized by using an inhibitor of BRAF mutation and an inhibitor of immune checkpoint.

In a particular embodiment, the melanoma is also resistant to a combined treatment characterized by using an inhibitor of BRAF mutation, an inhibitor of MEK and an inhibitor of immune checkpoint.

In a particular embodiment, the subject has or susceptible to have melanoma resistant to at least one of the treatments as described above. The subject having a melanoma resistant is identified by standard criteria. The standard criteria for resistance, for example, are Response Evaluation Criteria In Solid Tumors (RECIST) criteria, published by an international consortium including NCI.

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., an inhibitor of ITGBL1) into the subject, such as by 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.

By a "therapeutically effective amount" is meant a sufficient amount of inhibitor of ITGBL1 for use in a method for the treatment of melanoma at a reasonable benefit/risk ratio applicable to any medical treatment. It will be understood that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular subject will depend upon a variety of factors including the age, body weight, general health, sex and diet of the subject; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or coincidental with the specific polypeptide employed; and like factors well known in the medical arts. For example, it is well known within the skill of the art to start doses of the compound at levels lower than those required to achieve the desired therapeutic effect and to gradually increase the dosage until the desired effect is achieved. However, the daily dosage of the products may be varied over a wide range from 0.01 to 1,000 mg per adult per day. Typically, the compositions contain 0.01, 0.05, 0.1, 0.5, 1.0, 2.5, 5.0, 10.0, 15.0, 25.0, 50.0, 100, 250 and 500 mg of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. A medicament typically contains from about 0.01 mg to about 500 mg of the active ingredient, typically from 1 mg to about 100 mg of the active ingredient. An effective amount of the drug is ordinarily supplied at a dosage level from 0.0002 mg/kg to about 20 mg/kg of body weight per day, especially from about 0.001 mg/kg to 7 mg/kg of body weight per day.

The inhibitor of ITGBL1 as described above may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form pharmaceutical compositions. "Pharmaceutically" or "pharmaceutically acceptable" refer to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a mammal, especially a human, as appropriate. A pharmaceutically acceptable carrier or excipient refers to a non-toxic solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The pharmaceutical compositions of the present invention for oral, sublingual, subcutaneous, intramuscular, intravenous, transdermal, local or rectal administration, the active principle, alone or in combination with another active principle, can be administered in a unit administration form, as a mixture with conventional pharmaceutical supports, to animals and human beings. Suitable unit administration forms comprise oral-route forms such as tablets, gel capsules, powders, granules and oral suspensions or solutions, sublingual and buccal administration forms, aerosols, implants, subcutaneous, transdermal, topical, intraperitoneal, intramuscular, intravenous, subdermal, transdermal, intrathecal and intranasal administration forms and rectal administration forms. Typically, the pharmaceutical compositions contain vehicles which are pharmaceutically acceptable for a formulation 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 vacuum drying 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 intrap eritoneal 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

A further object of the present invention relates to a method of screening a drug suitable for the treatment of melanoma comprising i) providing a test compound and ii) determining the ability of said test compound to inhibit the expression and/or the activity of ITGBL1.

Any biological assay well known in the art could be suitable for determining the ability of the test compound to inhibit of the expression of ITGBL1. Such assay is briefly described above.

In particular, the effect triggered by the test compound is determined relative to that of the reference compound (VitD3), 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 expression of ITGBL1, 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 (such as VitD3), 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.

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: ITGBL1 secreted by melanoma cells inhibits melanoma cell death by PBMCs. (A) Overall survival of patients with high versus low ITGBL1 expression in male primary melanoma tumors from TCGA. P-value was calculated using the log-rank test. (B) ITGBL1 overexpressing SKMEL28 or parental with empty vector (P) cells were incubated for 48h with activated or quiescent PBMCs. Melanoma cell death was monitored with Incucyte.

Figure 2: Recombinant ITGBL1 inhibits PBMCs activity and Interferon pathway. (A) Resting or activated PBMCs were incubated for 48h in presence or absence of recombinant ITGBL1 (5ng/ml). PBMCs were subsequently added to 501 melanoma cells and cell death was analyzed with Incucyte. Quantification of melanoma cell death is displayed as the mean+/-SD of 3 independent experiments. (B) Melanoma cells isolated from patients were incubated with their autologous resting or activated PBMCs in presence or absence of rITGBLI . Quantification of melanoma cell death by Incucyte is shown as the mean+/-SD of 3 independent experiments.

Figure 3: MITF regulates RUNX 2, an ITGBL1 regulator, and is a potential pharmacological target to improve immune responses. (A) ITGBL1 (left panel) and MITF (right panel) expression analysis in high (2) and low (1) Runx2 expressing tumors from the TCGA melanoma cohort. (B) WM9 melanoma pretreated with 5mM VitD3 were incubated with resting or activated PBMs. Melanoma cell death was quantified using Incucyte. Results show as mean+/-SD of one representative experiment in triplicate.

Figure 4: ITGBL1 inhibits in vivo melanoma growth in a complete immune system model by decreasing immune cells activity. (A) 0.15 x 106 B16F10 cells overexpressing or not ITGBL1 were injected subcutaneously in C57BL/6J, and tumor weight was monitored after 12 days (mean tumor weight in g ±s.e.m.). Picture of representative tumors at 12 days is shown on the top (n=6 mice, *P<0.05). (B) mRNA from tumor overexpressing ITGBL1 (ITGBL1) or not (Ctl) were quantified (n=3) for IFNy and GZMB by QPCR and expressed as relative quantification in fold change+/-SD. (C) 0.15 x 106 BP cells overexpressing or not ITGBL1 were injected subcutaneously in C57BL/6J, and tumor growth was monitored every 3 days. Tumor volume is displayed (mean tumor volume in mm3 ±s.e.m.). (n=6 mice, *P<0.05). (D) mRNA from tumor overexpressing ITGBL1 (ITGBL1) or not (Ctl) were quantified for IFNy and GZMB by QPCR and expressed as relative quantification in fold change+/-SD (n=3).

Figure 5: ITGBL1 inhibits in vivo melanoma growth independently of T cells. (A) 0.15 x 106 B16F10 cells overexpressing or not ITGBL1 were injected subcutaneously in nude mice, and tumor growth was monitored every 3 days (mean tumor volume in mm3 ±s.e.m.). Picture of representative tumors at 15 days is shown on the top (n=6 mice, *P<0.05). (B) mRNA from tumor overexpressing ITGBL1 (ITGBL1) or not (Ctl) were quantified (n=3) for IFNY and GZMB by QPCR and expressed as relative quantification in fold change+/-SD. (C) 0.15 ^c 106 BP cells overexpressing or not ITGBL1 were injected subcutaneously in NSG mice, and tumor growth was monitored. Tumor volume is displayed (mean tumor volume in mm3 ±s.e.m.). (n=6 mice). Picture of representative tumors at 15 days is shown on the top.

Figure 6: ITGBL1 inhibits anti melanoma activity of NK cells. (A) NK-92 cells were activated (PMA/iono) for 1 hour in presence or absence of ITGBL1 (lOng/ml). Then, mRNA was extracted and analyzed by QPCR for IFNy expression. Results shown are mean+/-SD of 3 independent experiments (left panel). IFNy secretion in activated NK-92 cells incubated for 6h with or without ITGBL1. Results shown are mean+/-SD (right panel). (B) Red stained melanoma cells (501mel or Skmel-28) were cocultured with NK at different ration (NK 1/1 or NK 1/5) for 24hours. Cells were analyzed by FACS with dapi and melanoma death was quantified as % of melanoma positive dapi melanoma cells (mean+/-SD, n=3, *P<0.05, **P<0.001). (C) Red stained Skmel-28 cells, overexpressing ITGBL1 or not, were cocultured with NK at different ration (NKl/1 or NK 1/5) for 24hours. Cells were analyzed by FACS and % of melanoma positive for dapi was quantified and expressed as % of melanoma death (mean+/-SD, n=3, *P<0.05, **P<0.001).

Figure 7: NK cells decrease cytotoxic NK activity against melanoma independently of anti PD-1 treatment. (A) Red stained Skmel-28 cells, overexpressing ITGBL1 or not, were cocultured with NK at different ration (NKl/1 or NK 1/5) and treated with anti PD1 (5pg/ml) or its isotype control for 24 hours. Cells were analyzed by FACS with dapi and % of melanoma positive for dapi was quantified and expressed as % of melanoma death (mean+/-SD, n=3, *P<0.05, **P<0.001). (B) 0.15 x 106 BP cells overexpressing or not ITGBL1 were injected subcutaneously in C57BL/6J. When tumor reached 50mm3, mice were treated with anti PD1 and tumor growth was monitored. Tumor volume is displayed (mean tumor volume in mm3 ±s.e.m.). (n=6 mice, *P<0.05).

EXAMPLE 1:

Material & Methods

Experimental Models and Subject Details

Human study protocol was approved by the CPP ethics committees from Nice (DC- 2015*2531). Informed consent was obtained from all human subjects included in this study. All cells were obtained from metastasis specimen. Cl 1.33 cells were obtained from man aged 37 years and C.18.07 from man aged 27 years.

Antibodies and reagents

Antibodies against ERK2 and Hsp90 were from Santa Cruz Biotechnology. Anti FN 1 antibodies was from BD Biosciences (Franklin Lakes, NJ, USA). Anti RUNX2 was from Cell Signaling Technology (Boston, MA, USA). MITFm IFNy and Granzyme B antibodies were purchased from Abeam (Cambridge, MA, USA). ITGBL1 antibibody was from Abgent (San Diego, CA, USA). Secondary antibodies HRP was from Dako (Glostrup, Denmark) and alexa488 antibody and DAPI from Lifectech. BD cytofix/cytoperm plus kit was used for flow cytometry staining (San Jose, CA). Recombinant ITGBL1 was from My biosource (San Diego, CA). la,25-Dihydroxyvitamin D3 was purchased from Sigma- Aldrich (Lyon, France)

Cell culture

Cells (501Mel, WM3912, SKMEL-28 and WM-9) were grown in Dulbecco’s modified Eagle’s (DMEM) medium (Invitrogen, Carlsbad, CA, USA) supplemented with 7% FCS and penicillin/streptomycin (100 U/ml/50 pg/ml) at 37°C and 5% C02. Patient melanoma cells were (Cl 1.33 and C.18.07) were prepared as previously described (Cheli et ak, 2014) and grown in RPMI medium (Invitrogen, Carlsbad, CA, USA) supplemented with 7% FCS and penicillin/streptomycin (100 U/ml/50 pg/ml) at 37°C and 5% C02.

Peripheral Blood Mononuclear cells (PBMCs) preparation

PBMCs were obtained from healthy donors with informed consent following the Declaration of Helsinki according to the recommendations of an independent scientific review board. The project has been validated by The Etablissement Fran ais du Sang. Blood samples were collected using ethylene diamine tetra-acetic acid (EDTA)-containing tubes. Isolation of peripheral blood mononuclear cells (PBMCs) was performed by Ficoll gradient centrifugation (Lymphoprep®, Euromedex, France). Patient used for autologous experiment was undergoing anti PD-1 treatment.

Cells were transfected with siRNA against MITF as previously (Cheli et al., 2012) using lipofectamine RNAiMax as manufactured recommendations. RUNX2 or a scrambled sequence shRNA were purchased from Sigma Aldrich. Viral particles were produced as described previously (Ohanna et al, 2013). For infections, 8 ^c 104 cells were seeded in six-well plates for 24 h, infected with viral supernatant for 48h before processing. Cell infection with adenovirus expressing MITF (Vector Biolabs, Philadelphia, PA, USA) or the control adenovirus (empty vector) was carried out as described previously (Bonet et al, 2017).

Western blot

Cells were solubilized for 10 min at 4 °C in buffer containing 50 mM Tris pH 8, 150 mM NaCl, 1% Triton X-100 and EDTA-free protease inhibitor cocktail (Roche Applied Science). Thirty micrograms of protein were separated by electrophoresis on 10% polyacrylamide SDS- polyacrylamide gel electrophoresis and transferred onto a polyvinylidene difluoride membrane (Immobilon, Millipore, Molsheim, France). The membrane was saturated for 1 h at 25 °C in 10 mM Tris HC1, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% Tween 20, 3% bovine serum albumin (weight/volume) and 5% gelatin (weight/volume). Primary antibodies were incubated overnight at 4 °C. After three washes of 5 min in 10 mM Tris pH 7.4, 150 mM NaCl, 1% Triton X-100, the secondary antibody coupled with horse radish peroxidase (Dakopatts, Glostrup, Denmark) was then incubated for 1 h at room temperature. After three additional washes, proteins of interest were revealed by ECL (Amersham, Uppsala, Sweden) using ImageQuant LAS-4000 Fujifilm (GE healthcare, Pittsburgh, PA).

ELISA

Supernatant from stimulated PBMCs were collected and IFNy production was quantified by commercially available ELISA Kits (Peprotech, NJ, USA). Quantification was performed according to manufactured recommendations.

Supernatant from SKMel-28 control or overexpressing ITGBL1 was analyzed using Elisa kit from My biosource (San Diego, CA) after 48h in contact with cells according to manufacturer recommendations. Immunodepleted medium was obtained by adding 1 Opg of anti ITGBL1 antibody per ml of medium overnight at 4°C, then protein A/G immunomagnetic beads were for 1 h at 4°C and the tube was next placed on the magnetic separator to remove the immunomagnetic beads-antibody complex.

Flow cytometry PBMCs were seeded at 100 000 per well in presence of the different media in presence or absence of lOng/ml of PMA and ionomycin (PMA-iono) for 24h and treated for 12 h with monensin (Golgi Stop, BD Biosciences) according to manufacturer’s recommendation prior to staining. Briefly, cells were harvested, fixed and permeabilized using cytofix/cytoperm reagents. Granzyme B was diluted in staining buffer at 1/200 and incubated for 1 h on ice. Cells were washed 3 times and incubated with secondary antibody Alexa 488 labeled (1/500) for 1 h at 4 OC. Cells were washed and acquired on MACsQuant Cytometer (Miltenyi Biotech, Paris FRANCE).

Live imaging of melanocyte viability

Melanoma cells were plated in 96 well plates at 6000 cells per well. The day after, cells were stained with 0.5 mM CellTracker® Red CMPTX dye for 20 minutes at 370C according to manufacturer recommendations (Molecular Probes, USA) before adding either 100,000 cells per well of unstimulated PBMCs, the same number of activated PBMCs (pre-treated for 48h with lOng/mI of PMA (Sigma Aldrich) and lOng/mI of ionomycin (Sigma Aldrich)) or no PBMCs at all. Incucyte® green Cytotox Reagent (100 nM, Essen Bioscience, Michigan, USA) was added to all wells prior imaging and melanoma death was monitored in real-time using IncuCyte® Zoom live-cell imaging system (Essen Biosciences). Cells were maintained at 370C humidified environment and 5%C02 scanning every 2h at 10X magnification. Multiple images were collected per well and quantification of dead melanoma cells (yellow co-localised cells) was analysed using the integrated Zoom® software.

In experiments with conditioned media, PBMCs were preincubated in their respective media for 48h with PMA/ionomycin prior to be added to melanoma cells.

Vitamin D3 (5mM) was added to melanoma cells for 48h seeded in 96-well plates then media was replaced before adding PBMCs.

Bioinformatic analysis

We used the Skin Cutaneous Melanoma (SKCM) dataset from The Cancer Genome Atlas (TCGA). RNA-seq and clinical data were downloaded from the TCGA data portal (https://portal.gdc.cancer.gov). RNA-seq data were normalized using the Bioconductor package DESeq2 and log2 transformed. For each gene, we ranked patients by their expression levels and we classified the 25% highest and the 25% lowest in two groups. We next investigated the cellular components of the tumor microenvironment by inferring relative fractions of different immune cell types using CIBERSORT (Newman et al, 2015) from both groups of patients based on the ITGBL1 expression profile (https://cibersort.stanford.edu/). P- values were calculated by the Wilcoxon rank sum test. Survival curve from TCGA was obtained using R survival package. Overall survival was performed using primary male tumors data from TCGA. Significance was assessed using the log-rank test.

Results

MITF modulates immune system response through secreted factor

First, we tested if MITF affected melanoma cell death induced by PBMCs. Using melanoma cells isolated from lymph node metastasis (Cl 1.33) with low endogenous MITF level, we showed that forced MITF expression by adenoviral delivery increased significantly melanoma cell death induced by activated PBMCs (Data not shown). MITF expression is shown in western blot (Data not shown). Conversely, MITF silencing in 501MEL cells (high endogenous MITF level), using 2 different siRNA caused a 2-fold decrease in melanoma cells death induced by activated PBMCs (Data not shown). MITF silencing was verified by western blot (Data not shown).

These results indicate that modulation of MITF expression affects immune-mediated melanoma cells death. Next, we wondered if this effect was due to the inhibition of the intrinsic ability of melanoma cells to be killed by immune cells, or if melanoma cells through secretion of immunomodulating agents, damper the cytotoxic function of immune cells. First, we tested the effect of conditioned media (CM) obtained from 501MEL cells transfected with siCtl or siMITF. PBMCs were first incubated with these CM and then co-cultured with melanoma cells. In these conditions, we observed that CM from siMITF significantly decreased the cytotoxicity of activated PBMCs on melanoma cells (Data not shown) suggesting that melanoma cells may constitutively secrete negative immunomodulating agents (supported by decrease seen with siCtl as well) whose secretion and/or production is increased upon MITF silencing.

ITGBL1 secreted by melanoma cells decreases the cytotoxicity of immune cells

MITF low cells are known to have a pro-inflammatory secretory profile characterized by the production of numerous cytokines and immune regulators (Ohanna et ak, 2011; Riesenberg et ak, 2015; Wiedemann et ak, 2019). To identify key secreted factors that might impact the immune system we integrated the transcriptomic profile of melanoma cell lines (CCLE Broad, genes up regulated) expressing low MITF versus high MITF with the genes up regulated in non-responder to immune therapies (Hugo et ak, 2016). We identified 40 genes that are up regulated in both MITF Low cells and non-responders to immune therapy (Data not shown). Among these genes, 17 were described to encode secreted proteins that might affect the capacity of immune cells to kill melanoma cells (Data not shown). Among these proteins, ITGBL1 (integrin beta-1 like) was previously reported to be a bad prognosis factor for gastric, ovarian, lung and colorectal cancers (Gan et al., 2016; Liu et al, 2018; Qiu et al., 2018; Sun et al, 2016). Analysis of TCGA data showed that high expression of ITGBL1 in men is correlated to poor prognosis in melanoma and a decrease in overall survival (Figure 1A). First, we demonstrated that silencing MITF in 501MEL cells increased ITGBL1 expression (Data not shown) while MITF overexpression results in decreased ITGBL1 expression (Data not shown), confirming that ITGBL1 was negatively modulated by MITF. This was also observed in WM3912 cells (MITF expressing cells) (Data not shown). To further examine the role of ITGBL1, we stably overexpressed ITGBL1 in SKMEL28 cells which normally express low levels of ITGBL1. Increased expression can be observed in total cell extract (Data not shown) and in conditioned medium (Data not shown). When SKMEL28 cells overexpressing ITGBL1 were incubated with activated PBMCs, we observed reduced melanoma cell death compared to control cells (Figure IB). Note a weak basal anti-melanoma cytotoxic effect mediated by non- activated PBMCs. Next, we exposed PBMCs to the CM from parental and ITGBL1 expressing SKMEL28, and then studied their cytotoxic capacity towards naive 501MEL melanoma cells. We have shown an increase in melanoma cells death with activated PBMCs to that was inhibited by the CM from SKMEL28 cells expressing ITGBL1 (CM-ITGBL1) compared to CM from parental SKMEL28 (CM-Ctl) (Data not shown). Further, immunodepletion of ITGBL1 from the CM-ITGBL1 (CM- ITGBL1/IP) largely rescued the PBMCs cytotoxic effect and melanoma cell killing (Data not shown). The immunodepletion of ITGBL1 was verified by ELISA assay (Data not shown). These observations indicate that ITGBL1 secreted by melanoma cells is regulated by MITF and plays a key role in the regulation of immune-mediated melanoma cell death.

ITGBL1 secreted by melanoma cells impairs PBMCs activation

Next, we further investigated the effect of CM from melanoma cells on the immune cell functions. To do so, we evaluated by QPCR the effects of CM-Ctl or CM-ITGBL1 on the expression of IFNG, GZMB and B2M, the 3 genes known to play a key role in immune cell activation. Compared to resting PBMCs, PBMCs activated with PMA/ionomycin treatment induced a significant increase in expression of IFNG, GZMB and B2M in cells cultured with control media (CM-Ctl) (Data not shown). In PBMCs cultured in CM-ITGBL1, although the increase in IFNG, GZMB and B2M expression was also seen, this increase was statistically lower compared to the CM-Ctl. At the protein level, ELISA assay confirmed that CM-ITGBL1- induced increase in IFNy secretion evoked by PMA/ionomycin activation was significantly reduced compared to IFNy secretion induced by CM-Ctl activated PBMCs (Data not shown). In the same direction, FACS analysis showed that the expression of GZMB at the surface of immune cells was decreased in PBMCs incubated with CM-ITGBL1 compared to CM-Ctl (Data not shown). These observations indicated that ITGBL1 dampers the immune cell activation in vitro. To study the immune cells infiltrate in tumors expressing high versus low level of ITGBL1 and MITF, we analyzed TCGA data using Cibersort website (Stanford University). This analysis has shown that the most significant and common melanoma infiltrated immune cells was M2 macrophages. The percentage of M2 macrophages is increased in the ITGBL1 high tumors (Data not shown). Interestingly, Cibersort analysis of MITF low versus MITF high population showed that in contrary, the macrophage M2 population was reduced in MITF high tumors (Data not shown), which expressed low levels of ITGBL1. In agreement, incubation of PBMCs with CM-ITGBL1 increased CD206 and CD163 mRNA expression, M2 macrophages markers (Data not shown). Tumor M2 macrophages are generally associated with a poor prognosis and a reduced immune response. Finally, CFSE staining showed decreased proliferation in ITGBL1 stimulated PBMCs (Data not shown). Together these analyses further suggest that ITGBL1 contributes to impaired immune responses.

Recombinant ITGBL1 inhibits immune cells activation and cytotoxicity towards melanoma cells

To confirm our results obtained with conditioned media, we studied the direct effect of recombinant ITGBL1 protein (rITGBLI). Treatment of activated PBMCs with rITGBLI (5ng/ml) decreased their ability to kill 501MEL melanoma cells (Figure 2A). This was not observed in non-activated PBMCs. Furthermore, activation of PBMCs with PMA/ionomycin increased GZMB and IFNG mRNA expression that is abrogated by rITBLI (Data not shown). rITGLBl has no effect on GZMB and IFNG mRNA expression in resting PBMCs. At protein level, we also observed that rITGBLI induced a weak stimulation of STAT1 phosphorylation, a key event in lymphocyte activation. However, rITGBLI markedly impaired the activation of STAT1 induced by PMA/ionomycin, further supporting an inhibition of immune cell activation by rITGBLI (Data not shown). Finally, we tested the effect of rITGBLI in an autologous model. Using melanoma cells isolated from a patient and their cognate PBMCs (treated with anti-PDl), we observed increased melanoma cell death following PMA/ionomycin treatment of PBMCs and under these conditions, rITGBLI significantly impaired the ability of PBMCs to kill autologous melanoma cells (Figure 2B).

RUNX 2 regulates ITGBL1 and is a potential pharmacological target to decrease ITGBL1 and improve immune responses. Runt-related transcription factor 2 (RUNX2) has been shown to regulate ITGBL1 transcription in breast cancer cells (Li et al, 2015). First, analysis of the TCGA melanoma database revealed a significant increase in ITGBL1 expression in tumors with high RUNX2 expression, whereas MITF was decreased in these same patients (Figure 3A) suggesting that ITGBL1 is positively regulated by RUNX2 and negatively regulated by MITF. Next, the epistatic interaction between MITF and RUNX2 was demonstrated using siRNA approach to silence MITF (Data not shown) or adenoviral overexpression of MITF in WM3912 (Data not shown) or in 501MEL cells (Data not shown). In both cell lines, while inhibition of MITF lead to an increase in RUNX2, MITF overexpression lead to a dramatic decrease in RUNX2 expression (Data not shown). These observations were seen at both mRNA (Data not shown) and at protein level (Data not shown). RUNX2 shRNA efficiency was monitored by western blot, translated into decreased expression of RUNX2 (Data not shown). Next, we studied the effect of RUNX2 silencing on the cytotoxic activity of PBMCs. 501MEL melanoma cells were infected with control (shScr) or RUNX2 shRNA (sh#l, sh#2) expressing lentivirus and then incubated with PBMCs to monitor their ability to kill melanoma cells. No effect was observed in non-activated PBMCs. However, RUNX2 silencing increased melanoma cell death mediated by activated PBMCs compared to ShScr infected cells (Data not shown). Vit D3 has been shown to inhibit RUNX2 expression (Drissi et al., 2002). Therefore, we tested if melanoma cells treated with VitD3 could increase PBMCs cytotoxic activity. For this purpose, we pretreated WM9 melanoma cells with VitD3 (5mM) (Data not shown) and co-cultured them with PBMCs. Exposure of PBMCs to vitamin D3-treated melanoma cells triggered a stimulation of the cytotoxic activity of PBMCs equivalent to that achieved by PMA/ionomicyn treatment. Vitamin D3 -treated melanoma cells further increased the PBMCs activation evoked by PMA/ionomicyn treatment, thereby favoring the destruction of melanoma cells (Figure 3B). In agreement with previous observations, Cibersort analysis of RUNX2 low versus RUNX2 high population also showed that the macrophage M2 population was increased in RUNX2 high tumors (Data not shown), which expressed high level of ITGBL1. Our data thus suggest that VitD3 may directly potentiate immune-mediated melanoma cell death by repressing their RUNX2 and ITGBL1 expression.

EXAMPLE 2

Materials and methods

NK cells activity against melanoma Melanoma cells were stained with 0.5 mM CellTracker® Red CMPTX dye for 15 minutes at 370C according to manufacturer recommendations (Molecular Probes, USA) before to be plated in 48 well plates at density of 25000 cells per well. Five hours later, NK-92 cells were added at ratio of 1 melanoma cells for 1 NK cells (NK 1/1) or 1 melanoma cells for 5 NK cells (NK 1/5). Cells were then incubated for 24h at 370C in humidified environment and 5%C02. In case of treatment with anti PD1, NK cells were incubated 30mn with 5pg/ml of anti PD1 or its isotype control prior to be added to melanoma cells. 24hours later, cells were detached and analysed by flow cytometry for melanoma death. Briefly, cells were resuspended in 300m1 of PBS 0.5%BSA, 2mM EDTA. Dapi was added to final concentration of lpg/ml just before analyse using MACsQuant Cytometer (Miltenyi Biotech, Paris FRANCE).

In vivo experiments

The mice were maintained in a temperature-controlled facility (22° C) on a 12 - hour light/dark cycle and were given free access to standard laboratory chow (UAR, Epinay-S/Orge, France). B16F10 mouse melanoma cells or BP cells (derived from B-Raf/PTEN tumor mice) were injected subcutaneously (0.15X106) into the flank of C57BL/6J, nude mice or NSG (Janvier laboratory, Le Genest-Saint-Isle, France). Body weight and tumor volume were measured three times a week. Tumor size was measured with linear calipers and calculated using the formula: tumor width x tumor length2><0.5. When mice were treated with anti PD1, tumor were left growing to 50mm3 prior starting anti PD1 treatment. Mice were treated with 125pg/mice of anti PD1 or isotype control. Mice were sacrificed after 15 days maximum by cervical dislocation. Frozen tumors were crushed and resuspended in Trizol. RNA purification from tumor was performed using Direct-zol RNA miniprep kit according to manufacturer recommendation, then QPCR was performed as described previously (Cheli et al, 2011).

ELISA

Supernatant from stimulated NK-92 cells were collected and IFNy production was quantified by commercially available ELISA Kits (Peprotech, NJ, USA). Quantification was performed according to manufactured recommendations.

Results

We overexpressed ITGBL1 in B16F10 cells or BP cells. Overexpression was confirmed by QPCR (data not shown). Next, cells were injected subcutaneously in C57BL/6J and tumor growth was monitored. At the end of the experiment (12 days after injection), tumor weight of ITGBL1 overexpressing B16F10 cells was increased by two-fold compared to parental cells (Figure 4A). QPCR analysis showed a 2-3-fold decrease in IFNg and GZMB mRNA in the overexpressing ITGBL1 tumors (Figure 4B). To confirm these results, we injected BP overexpressing cells subcutaneously in C57BL/6J. Tumors from BP cells overexpressing ITGBL1 developed faster compared to BP control cells. Tumor volume at day fifteen presented a two-fold increase in ITGBL1 overexpressing tumor (Figure 4C). We next performed QPCR oflFNy and GZMB on tumor. ITGBL1 overexpressing tumors showed a decrease in IFNy and GZMB mRNA (Figure 4D). We concluded that overexpression of ITGBL1 in tumor grew faster than control tumor cells and decreased immune cells cytotoxicity. To confirm which immune cells are important for the phenotype, we performed the same experiment in nude mice that are lacking T cells. When injected subcutaneously, overexpressing ITGBL1 are still growing faster compare to control cells (Figure 5A). INFy and GZMB expression in tumor is also decreased in ITGBL1 overexpressing tumors (Figure 5B). So, ITGBL1 overexpressing cells inhibits immune cells independently of T cells. To confirm the importance of NK cells, we performed the same experiment in NSG mice that are lacking T and NK cells. Tumor growth of ITGBL1 overexpressing cells is the same than control cells (Figure 5C). Taking all together, ITGBL1 overexpressing cells decreased immune cells activity in vivo and NK cells are important to decrease melanoma tumors growth. Also, ITGBL1 overexpression decreased NK cytotoxic activity against melanoma.

To confirm this in vivo observation, we investigated if NK cells could be inhibited by ITGBL1 in vitro. When NK-92 cells (NK cell line) are stimulated with PMA and ionomycin, they increased IFNy mRNA expression and its secretion in the medium. However, when ITGBL1 is added to stimulated cells, IFNy mRNA is decreased by more than two-fold, like its protein secretion (Figure 6A). Next, we checked if NK-92 could have cytotoxicity on melanoma cells. We used two different cells lines (501mel and Skmel-28), in which, we added NK-92 at a ratio 1 melanoma cells for 1 NK cells or 1 melanoma cells for 5 NK cells. After 24 hours, we can observe melanoma death from 50% to 80% of death for the both cell lines dependent of the number of NK cells added (Figure 6B). Next, we quantified melanoma death of ITGBL1 overexpression cells or not. We can observe a two-fold decrease in melanoma death when ITGBL1 is overexpressed compared to control cells (Figure 6C). Taking together we showed that NK cells have anti melanoma activity by killing melanoma cells and ITGBL1 decreased NK activation and cytotoxicity against melanoma.

Finally, we tested if ITGBL1 could protect melanoma death independently of PD1 treatment. We quantified melanoma death with overexpression of ITGBL1 or not after pretreatment of NK-92 cells with anti PD1 or an isotype control (5pg/ml) NK-92 ratio from 1/1 or 1/5 was used as previously. When NK pretreated with anti PD1 were added on top of melanoma, we can observe a 10% increase of cell death in control or overexpressing ITGBL1 cells. However, we observed that ITGBL1 overexpression protected melanoma fromNK induce cell death compare to control with or without PD1 treatment (Figure 7A). ITGBL1 is protecting melanoma from NK induce cell death independently of PD1 treatment. We wanted to confirm this observation in vivo. We injected BP cells subcutaneously, and treated mice with anti PD1 when tumor reached 50mm3. Treatment with anti PD1 is effective by slowing down the tumor growth of control or overexpressed melanoma cells. However, ITGBL1 overexpressing tumor treated with anti PD1 are growing faster than control cells treated with PD1, but slower compare to ITGBL1 treated with isotype control (Figure 7B). In conclusion, ITGBL1 prevent tumor cells death independently of anti PD1 treatment.

Discussion

Although the recent implementation of targeted-and immune-therapies have shown marked improvements in the treatment of melanoma for many patients, the long-term outcome did not reach the initial expectations because of primary and acquired resistances developed in these patients. Even though melanoma cells are highly immunogenic, they secrete a large number of mediators that can reshape the immune environment, inhibit immune cell functions and therefore impair the response to immunotherapies.

As MITF was convincedly reported to control the secretory and inflammatory phenotype of melanoma cells, we studied the effect of MITF expression in melanoma cells on the activation and cytotoxic activity of immune cells. Using an in vitro immune cells activity assay, we have shown that loss of MITF impaired the cytotoxic effects of PMBC, while MITF forced expression did the opposite. These effects are largely mediated through secreted factors, as conditioned media from MITF depleted melanoma cells inhibited the cytotoxic activity of PBMCs.

In the search of secreted protein, expression of which is increased in MITF low melanoma cells and that may regulate the immune response, we identified ITGBL1. ITGBL1 is a secreted integrin-like protein and its role in the regulation of immune response has never been documented although ITGBL1 was increased in melanoma patients not responding to anti- PD1 immunotherapy. The key involvement of ITGBL1 in melanoma development was further supported by the association of high ITGBL1 expression with poor prognosis in male patients. As expected, MITF silencing increased ITGBL1 expression, while MITF forced expression inhibited its expression. SKMEL28 cells over-expressing ITGBL1 are less susceptible to the cytotoxic effect of PBMCs than the parental cells. The conditioned medium from ITGBL1 over- expressing cells inhibited the ability of PBMCs to kill melanoma cells and this effect is abrogated by ITGBL1 immuno-depletion demonstrating the pivotal role of ITGBL1 in the immunosuppressive effect of conditioned medium of melanoma cells.

This effect is due to the inhibition of the immune cell activation as shown by the inhibition of several activation markers, such as IFNy, granzyme B and B2M. Also, we observed a decrease in PBMCs proliferation (Data not shown) using CFSE staining. These effects of ITGBL1 on the immune system were supported by the analysis of the melanoma tumors from the TCGA with Cibersort that indicated an increase in M2 macrophage. This increase of M2 polarization was confirmed at mRNA level in PBMCs treated with either CM- ITGBL1 or the recombinant protein with the increase of CD206 and CD 163.

Then, the inhibition of immune activation and cytotoxic activity by recombinant ITGBL1 unambiguously demonstrate that ITGBL1 regulated the immune response. This effect might be due to the inhibition of the JAK/STAT pathway, a pivotal pathway in immune cell activation. Indeed, ITGBL1 blocked the phosphorylation of STAT1 induced by the activation with PMA/ionomycin, even though we observed a weak basal phosphorylation of STAT1 induces by ITGBL1. How ITGBL1 can affect the signalling pathway in immune cells is not known and the receptor of ITGBL1 remains to be identified. However, NIC cells have been reported to exhibit a strong anti-melanoma activity and responsive to the anti PD1 treatment. We identified, NK cells to be the main cells affected by ITGBL1 as shown by in vitro experiments, using NK-92 cells, and also as observed in incucyte experiments, were none stimulated cells ITGBL1 dampen melanoma death, reflect of the Innate immune activity. To confirm these observations in vivo , we used a syngenic animal model with complete immune system (C57BL/6J). ITGBL1 favours tumor growth and modifies the tumor microenvironment by decreasing immune cells cytotoxicity. In nude mice that lack T cells, we still observed ITGBL1 effect with the increase of tumor growth, and the decrease of cytotoxicity confirming the previous observations in C57BL/6j model. Also, we observed a decrease in IFNy and GZMB expression. In NSG mice, lacking immune system, no tumor growth difference was observed. Altogether, our observations indicate that ITGBL1 modulates NK activity in the tumor and plays a strong role in tumor rejection. Finally, we tested if this inhibition could be independent of PD1 treatment in vivo and in vitro. In all the case, overexpression of ITGBL1 protected melanoma cell death and favour tumor growth compare control cells. ITGBL1 protect melanoma cell death independently of PD1, and this could explain that patient resistant to anti PD1 have increase in ITGBL1 expression. It could be a new mechanism for immunotherapy resistance. ITGBL1 expression was reported to be regulated by RUNX2 and RUNX2 have recently been implicated in melanoma cells invasive potential, epithelial-mesenchymal transition, and in melanoma resistance to BRAF inhibitor (Boregowda et al., 2016). Analysis of TCGA cohort shown a correlation of RUNX2 with ITGBL1 and an inverse correlation with MITF. In vitro analysis demonstrated that MITF silencing increased RUNX2 expression while MITF forced expression did the opposite. As expected RUNX2 down regulation by 2 different shRNA resulted in ITGBL1 inhibition and made melanoma cells more sensitive to the cytotoxic activity of immune cells.

Taking advantage of the well described regulation of RUNX2 expression and function by Vitamin D3 (Boregowda et al., 2014; Timerman et al., 2017), we demonstrated that exposure of melanoma cells to Vitamin D3 (hormonal active form) decreased RUNX2 and ITGBL1, and rendered melanoma cell more susceptible to immune-mediated death. As melanoma cells expressed vitamin D receptor, CYP27B1 and CYP24A1, they are able to produce bioactive form of vitamin D3 and to convey cellular signals. Therefore, vitamin D3 might be a valuable mean to improve the immune response in melanoma patients, provided that vitamin D3 does not act on the immune system as a negative regulator. Several reports have already shown inhibitor effect of Vit D3 on the adaptative immune system (Mora et al., 2008) while a positive effect on the innate immune system (Medzhitov, 2007). However, doses used in experiments on the adaptative immune cells (Lemire et al., 1985) are much higher than the circulating hormone in blood. In conclusion, our data showing that ITGBL1 is a new immunomodulator that can be regulated by vitamin D3, the beneficial role of vitamin D in melanoma patient survival, and positive immunomodulator role of vitamin D prompted us to propose to evaluate the effect of Vit D3 supplementation during targeted- of immuno-therapy treatment in melanoma.

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Claims

CLAIMS:

1. A method for predicting whether a subject suffering from a melanoma has or is at risk of having resistant melanoma comprising the steps of i) quantifying the expression level of ITGBL1 in a biological sample obtained from the subject; ii) comparing the expression level quantified at step i) with a predetermined reference value and iii) concluding that the subject has or is at risk of suffering from resistant melanoma when the level determined at step i) is higher than the predetermined reference value.

2. A method for treating melanoma in a subject in need thereof comprising a step of administering said subject with a therapeutically effective amount of an inhibitor of ITGBL1.

3. 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 an inhibitor of ITGBL1.

4. A method for treating resistant melanoma in a subject in need thereof, wherein said method comprises the step of:

(i) Determining in a biological sample obtained from said subject the expression level of ITGBL1

(ii) Comparing the expression levels quantified at step i) with a predetermined reference value and

(iii) Concluding that the subject has or is at risk of suffering from resistant melanoma when the level determined at step i) is higher than the predetermined reference value.

(iv) And administering to said subject a therapeutically effective amount of an inhibitor of ITGBL1 if the expression level of ITGBL1 is higher than the predetermined reference value.

5. The method according to claims 1, 3 and 4, wherein the melanoma is resistant to a classical treatment.

6. The method according to claims 1, 3 and 4, wherein the melanoma is resistant to a treatment with the inhibitors of BRAF mutations.

7. The method according to claims 1, 3 and 4, wherein the melanoma is resistant to a treatment with the inhibitors of MEK.

8. The method according to claims 1, 3 and 4, wherein the melanoma is resistant to a treatment with the inhibitors of NRAS.

9. The method according to claims 1, 3 and 4, wherein the melanoma is resistant to a treatment with the immune checkpoint inhibitor.

10. The method according to claims 2 to 4 wherein the inhibitor of ITGBL1 is la, 25- Dihydroxy vitamin D3.

11. The method according to claims 2 to 4 wherein the inhibitor of ITGBL1 is CADD52.

12. 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 ITGBL1.