Combination of a SLC16a1 inhibitor and a SMAD3 inhibitor for treating cancer

Abstract

The present invention relates to the treatment of cancer. In this study, the inventors focused on evaluating the efficiency of SIS3 in cancer treatment concurrently with identifying genes driving resistance to SMAD3 inhibition, aiming to find efficient and long-lasting treatment options. Novel high-throughput methods such as CRISPR-screen and long-read RNA sequencing were used to identify and precisely characterise the genes and signalling pathways driving resistance to SMAD3 inhibition. They identified a cell surface protein SLC16A1 / MCT1 as a driver of resistance to SIS3. They demonstrated that loss of MCT1 by CRISPR-Cas9 KO or inhibition by a specific inhibitor AZD3965 results in higher sensitivity to SMAD3 inhibition. To study if the effect is common to other similar cancer types, they also overexpressed MCT1 in KRAS-mutated lung adenocarcinoma cells and observed higher resistance to the inhibitory effect of SIS3. Synergistic inhibition of SMAD3 and MCT1 resulted in efficient melanoma cell death in a dose-dependent manner, suggesting the use of combined inhibition to be a beneficial treatment option for cancer. Thus, the present invention relates to a combination of a SLC16A1 inhibitor and a SMAD3 inhibitor for use in the treatment of a cancer in a subject in need thereof.

Description

[0001] COMBINATION OF A SLC16A1 INHIBITOR AND A SMAD3 INHIBITOR FOR TREATING CANCER

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to a combination of a SLC16A1 inhibitor and a SMAD3 inhibitor for use in the treatment of a cancer in a subject in need thereof.

[0004] BACKGROUND OF THE INVENTION:

[0005] Despite advances in healthcare and medicine, cancer remains one of the leading causes of deaths worldwide. Major progress has been made in improving the efficiency of immune- and targeted therapies, which have become the first-line treatment options for many cancers. However, cancer cells developing resistance to treatment has emerged as one of the biggest challenges in the clinic. In addition to the complex tumour microenvironment and genetic factors, the cell plasticity and capacity to reprogram themselves can be affected by various non-genetic factors. Understanding these mechanisms presents a crucial step towards developing more efficient therapies (1).

[0006] Transforming growth-factor beta (TGF-P) signalling pathway has been identified to be highly active in several advanced cancers and correlates with poor prognosis. Moreover, TGF-P-induced epithelial-mesenchymal transition (EMT) has been shown to be closely related to cancer aggressiveness, more invasive phenotype and therapy resistance (2). Inhibiting TGF-P signalling pathway has proven to be a promising strategy for cancer treatment in pre-clinical studies and is being applied in numerous ongoing clinical trials. Of these, the most advanced options include using the small molecule inhibitors (SMI) to inhibit the kinase activity of TGF-P receptor. However, the clinical evidence is scarce and in several cases no effect has been observed for the overall survival, which could be explained by the pleiotropic downstream effects of the TGF-P receptor. Additional concerns with this approach include the adverse effects of long-term exposure and the stochiometric effects of the TGF-P receptor inhibition to the activity of its canonical downstream components (3). Strategies to fine-tune the inhibition of this signalling pathway have been suggested, focusing on more precise targeting of the downstream canonical or non-canonical signal transduction.

[0007] SMAD3 transcription factor is part of the canonical TGF-P pathway and facilitates most of the effects of TGF-P receptor activation (4). SMAD3 has been shown to be involved in modulating EMT and cancer progression and SMAD3 transcriptional signature has been associated with more aggressive and metastatic cancers (5). Furthermore, SMAD3 has been found to contribute to therapy resistance independently of TGF-P receptor activation (6). Thus, designing inhibitors specifically for SMAD3 have been suggested to be a valid strategy to surpass the non-canonical effects of TGF-P signalling and to address the TGF-P- independent effects of SMAD3 in therapy resistance.

[0008] SMAD3 inhibitors have been extensively used for in vitro studies showing promising results in reducing cancer cell growth, invasion and metastasis (7-9). Although no SMAD3 inhibitors have reached clinical trials, several of them have already been successfully used in preclinical models to reduce tumour growth. Of the currently developed SMAD3 inhibitors, a selective and potent inhibitor SIS3 has gained the most attention in research (10). Despite its low water-solubility, the bioavailability and effect on tumour growth has been successfully demonstrated in preclinical models (11-13). Additional efforts are being made to improve the inhibitor design (14).

[0009] SUMMARY OF THE INVENTION:

[0010] In this study, the inventors focused on evaluating the efficiency of SIS3 in cancer treatment concurrently with identifying genes driving resistance to SMAD3 inhibition, aiming to find efficient and long-lasting treatment options. Novel high-throughput methods such as CRISPR-screen and long-read RNA sequencing were used to identify and precisely characterise the genes and signalling pathways driving resistance to SMAD3 inhibition. They identified a cell surface protein SLC16A1 / MCT1 as a driver of resistance to SIS3. The SLC16 gene family has fourteen members. Four (SLC16A1, SLC16A3, SLC16A7, and SLC16A8) encode monocarboxylate transporters (MCT1, MCT4, MCT2, and MCT3, respectively) catalysing the proton-linked transport of monocarboxylates such as 1-lactate, pyruvate and ketone bodies across the plasma membrane (15). Of these, MCT1 has the highest affinity to lactate and has been demonstrated to affect tumour aggressiveness and metastasis by regulating cellular respiration in different cancers, including melanoma and lung adenocarcinoma (16,17).

[0011] They showed that activation of SLC16A1 promoter by CRISPR-SAM and overexpression of MCT1 protein increases cell viability in presence of SMAD3 inhibitor in BRAF-mutated melanoma cell lines. Contrarily, they demonstrated that loss of MCT1 by CRISPR-Cas9 KO or inhibition by a specific inhibitor AZD3965 results in higher sensitivity to SMAD3 inhibition. To study if the effect is common to other similar cancer types, they also overexpressed MCT1 in KRAS-mutated lung adenocarcinoma cells and observed higher resistance to the inhibitory effect of SIS3. Synergistic inhibition of SMAD3 and MCT1 resulted in efficient melanoma cell death in a dose-dependent manner, suggesting the use of combined inhibition to be a beneficial treatment option for cancer.

[0012] Thus, the present invention relates to a combination of a SLC16A1 inhibitor and a SMAD3 inhibitor for use in the treatment of a cancer in a subject in need thereof. Particularly, the invention is defined by its claims.

[0013] DETAILED DESCRIPTION OF THE INVENTION:

[0014] A first object of the present invention relates to a combination of a SLC16A1 inhibitor and a SMAD3 inhibitor for use in the treatment of a cancer in a subject in need thereof.

[0015] In another embodiment, the invention relates to i) a SLC16A1 inhibitor and ii) a SMAD3 inhibitor, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer in a subject in need thereof.

[0016] In another particular embodiment, the invention relates to i) a SLC16A1 inhibitor and ii) a SMAD3 inhibitor as a combined preparation for simultaneous use in the treatment of cancer in a subject in need thereof.

[0017] As used herein, the term “simultaneous use” denotes the use of the SLC16A1 inhibitor and the SMAD3 inhibitor compound of the invention occurring at the same time.

[0018] As used herein, the term “separate use” denotes the use of the SLC16A1 inhibitor and the SMAD3 inhibitor of the invention occurring not at the same time.

[0019] As used herein, the term “sequential use” denotes the use of the SLC16A1 inhibitor and the SMAD3 inhibitor of the invention occurring by following an order.

[0020] As used herein, the term “SLC16A1” also known as “MCT1” for “Monocarboxylate transporter 1” has its general meaning in the art and denotes a ubiquitous protein that in humans is encoded by the SLC16A1 gene. MCT1 is a proton coupled monocarboxylate transporter. Its Entrez reference number is 6566 its UniProt reference number is P53985.

[0021] As used herein, the term “SMAD3” for SMA- AND MAD-RELATED PROTEIN 3 has its general meaning in the art and denotes a member of the SMAD family of proteins. It acts as a mediator of the signals initiated by the transforming growth factor beta (TGF-P) superfamily of cytokines, which regulate cell proliferation, differentiation and death. [7] [8] Based on its essential role in TGF beta signalling pathway, SMAD3 has been related with tumor growth in cancer development. Its Entrez reference number is 4088 its UniProt reference number is P84022.

[0022] As used herein, the term “SLC16A1 inhibitor” or “SMAD3 inhibitor” denotes all molecules which inhibit the activity and / or the expression of SLC16A1 or SMAD3 (for example inhibition of the transporter activity for SLC16A1 or of transcription for SMAD3. These terms also denote inhibitors of the expression of the gene coding for protein MCT1 or SMAD3.

[0023] According to the invention, the cancer may be selected in the group consisting of adrenal cortical cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain and central nervous system cancer, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, non-Hodgkin's lymphoma, Kaposi's sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, liver cancer, lung cancer, mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, skin cancer, stomach cancer, testicular cancer, thymus cancer, thyroid cancer, vaginal cancer, vulvar cancer, and uterine cancer.

[0024] In a particular embodiment, the cancer is skin cancer and particularly a melanoma or a lung cancer.

[0025] In a particular embodiment, the cancer is a cancer expressing MCT1 or a cancer with a high level of MCT1.

[0026] 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. Particularly, the subject can suffer from a cancer and particularly a melanoma.

[0027] As used herein, the term "treatment" or "treat" refer to both prophylactic or preventive treatment as well as curative or disease modifying treatment, including treatment of subjects at risk of contracting the disease or suspected to have contracted the disease as well as subjects 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., disease manifestation, etc.]).

[0028] In one embodiment, the inhibitor of SLC16A1 or SMAD3 according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).

[0029] The term "small organic molecule" refers to a molecule (natural or not) of a size comparable to those organic molecules generally used in pharmaceuticals. The term excludes biological macromolecules (e. g., proteins, nucleic acids, etc.). Preferred small organic molecules range in size up to about 10000 Da, more preferably up to 5000 Da, more preferably up to 2000 Da and most preferably up to about 1000 Da.

[0030] According to the invention, SLC16A1 inhibitors are well known in the art ‘see for example Liu Tian et al Cancer Manag Res, 2023). Particularly, SLC16A1 inhibitors can be selected in the group consisting in but not limited to AZD3965 (see for example Silva Ana et al. Molecules. 2022), a-cyano-4-hydroxycinnamic acid(a-CHCA), BAY-8002 and 7ACC2. According to the invention, SMAD3 inhibitors can be selected in the group consisting in but not limited to SIS3 (see for example Jinnin, M. et al. Mol Pharmacol. 2006) or 16D (see for example Wu Nannan et al. Bioorganic & Medicinal Chemistry Letters. 2020). An exemplary chemical representation of SIS3 is depicted hereinafter:

[0031] An exemplary chemical representation of 16D is depicted hereinafter: In one embodiment, the inhibitor of SLC16A1 or SMAD3 is an antibody. Antibodies directed against SLC16A1 or SMAD3 can be raised according to known methods by administering the appropriate antigen or epitope to a host animal selected, e.g., from pigs, cows, horses, rabbits, goats, sheep, and mice, among others. Various adjuvants known in the art can be used to enhance antibody production. Although antibodies useful in practicing the invention can be polyclonal, monoclonal antibodies are preferred. Monoclonal antibodies against SLC16A1 or SMAD3 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 originally described by Kohler and Milstein (1975); the human B-cell hybridoma technique (Cote et al., 1983); and the EBV-hybridoma technique (Cole et al. 1985). Alternatively, techniques described for the production of single chain antibodies (see e.g., U.S. Pat. No. 4,946,778) can be adapted to produce anti-SLC16Al or anti-SMAD3 single chain antibodies. Compounds useful in practicing the present invention also include anti-SLC16Al or anti-SMAD 3 antibody fragments including but not limited to F(ab')2 fragments, which can be generated by pepsin digestion of an intact antibody molecule, and Fab fragments, which can be generated by reducing the disulfide bridges of the F(ab')2 fragments. Alternatively, Fab and / or scFv expression libraries can be constructed to allow rapid identification of fragments having the desired specificity to SLC16A1 or SMAD 3.

[0032] Humanized anti-SLC16Al or anti-SMAD3 antibodies and antibody fragments therefrom can also be prepared according to known techniques. "Humanized antibodies" are forms of non-human (e.g., rodent) chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (CDRs) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Methods for making humanized antibodies are described, for example, by Winter (U.S. Pat. No. 5,225,539) and Boss (Celltech, U.S. Pat. No. 4,816,397).

[0033] Then, for this invention, neutralizing antibodies of SLC16A1 or SMAD3 are selected.

[0034] In another embodiment, the antibody according to the invention is a single domain antibody against SLC16A1 or SMAD3. The term “single domain antibody” (sdAb) or "VHH" refers to the single heavy chain variable domain of antibodies of the type that can be found in Camelid mammals which are naturally devoid of light chains. Such VHH are also called “nanobody®”. According to the invention, sdAb can particularly be llama sdAb. The term “VHH” refers to the single heavy chain having 3 complementarity determining regions (CDRs): CDR1, CDR2 and CDR3. The term “complementarity determining region” or “CDR” refers to the hypervariable amino acid sequences which define the binding affinity and specificity of the VHH.

[0035] The VHH according to the invention can readily be prepared by an ordinarily skilled artisan using routine experimentation. The VHH variants and modified form thereof may be produced under any known technique in the art such as in-vitro maturation.

[0036] VHHs or sdAbs are usually generated by PCR cloning of the V-domain repertoire from blood, lymph node, or spleen cDNA obtained from immunized animals into a phage display vector, such as pHEN2. Antigen-specific VHHs are commonly selected by panning phage libraries on immobilized antigen, e.g., antigen coated onto the plastic surface of a test tube, biotinylated antigens immobilized on streptavidin beads, or membrane proteins expressed on the surface of cells. However, such VHHs often show lower affinities for their antigen than VHHs derived from animals that have received several immunizations. The high affinity of VHHs from immune libraries is attributed to the natural selection of variant VHHs during clonal expansion of B-cells in the lymphoid organs of immunized animals. The affinity of VHHs from non-immune libraries can often be improved by mimicking this strategy in vitro, i.e., by site directed mutagenesis of the CDR regions and further rounds of panning on immobilized antigen under conditions of increased stringency (higher temperature, high or low salt concentration, high or low pH, and low antigen concentrations). VHHs derived from camelid are readily expressed in and purified from the E. coli periplasm at much higher levels than the corresponding domains of conventional antibodies. VHHs generally display high solubility and stability and can also be readily produced in yeast, plant, and mammalian cells. For example, the “Hamers patents” describe methods and techniques for generating VHH against any desired target (see for example US 5,800,988; US 5,874, 541 and US 6,015,695). The “Hamers patents” more particularly describe production of VHHs in bacterial hosts such as E. coli (see for example US 6,765,087) and in lower eukaryotic hosts such as moulds (for example Aspergillus or Trichoderma) or in yeast (for example Saccharomyces, Kluyveromyces, Hansenula or Pichia) (see for example US 6,838,254).

[0037] In one embodiment, the compound according to the invention is an aptamer. Aptamers are a class of molecule that represents an alternative to antibodies in term of molecular recognition. Aptamers are oligonucleotide or oligopeptide sequences with the capacity to recognize virtually any class of target molecules with high affinity and specificity. Such ligands may be isolated through Systematic Evolution of Ligands by Exponential enrichment (SELEX) of a random sequence library, as described in Tuerk C. and Gold L., 1990. The random sequence library is obtainable by combinatorial chemical synthesis of DNA. In this library, each member is a linear oligomer, eventually chemically modified, of a unique sequence. Possible modifications, uses and advantages of this class of molecules have been reviewed in Jayasena S.D., 1999. Peptide aptamers consists of a conformationally constrained antibody variable region displayed by a platform protein, such as E. coli Thioredoxin A that are selected from combinatorial libraries by two hybrid methods (Colas et al., 1996).

[0038] Then, for this invention, neutralizing aptamers of SLC16A1 or SMAD3 are selected.

[0039] In one embodiment, the compound according to the invention is a polypeptide.

[0040] In a particular embodiment the polypeptide is an antagonist of SLC16A1 or SMAD3 and is capable to prevent the function of SLC16A1 or SMAD3. Particularly, the polypeptide can be a mutated SLC16A1 or SMAD3 or a similar protein without the function of SLC16A1 or SMAD3s.

[0041] In one embodiment, the polypeptide of the invention may be linked to a cellpenetrating peptide” to allow the penetration of the polypeptide in the cell.

[0042] The term “cell-penetrating peptides” are well known in the art and refers to cell permeable sequence or membranous penetrating sequence such as penetratin, TAT mitochondrial penetrating sequence and compounds (Bechara and Sagan, 2013; Jones and Sayers, 2012; Khafagy el and Morishita, 2012; Malhi and Murthy, 2012).

[0043] The polypeptides of the invention may be produced by any suitable means, as will be apparent to those of skill in the art. In order to produce sufficient amounts of polypeptide or functional equivalents thereof for use in accordance with the present invention, expression may conveniently be achieved by culturing under appropriate conditions recombinant host cells containing the polypeptide of the invention. Preferably, the polypeptide is produced by recombinant means, by expression from an encoding nucleic acid molecule. Systems for cloning and expression of a polypeptide in a variety of different host cells are well known.

[0044] When expressed in recombinant form, the polypeptide is preferably generated by expression from an encoding nucleic acid in a host cell. Any host cell may be used, depending upon the individual requirements of a particular system. Suitable host cells include bacteria mammalian cells, plant cells, yeast and baculovirus systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells. HeLa cells, baby hamster kidney cells and many others. Bacteria are also preferred hosts for the production of recombinant protein, due to the ease with which bacteria may be manipulated and grown. A common, preferred bacterial host is E coli.

[0045] In specific embodiments, it is contemplated that polypeptides used in the therapeutic methods of the present invention may be modified in order to improve their therapeutic efficacy. Such modification of therapeutic compounds may be used to decrease toxicity, increase circulatory time, or modify biodistribution. For example, the toxicity of potentially important therapeutic compounds can be decreased significantly by combination with a variety of drug carrier vehicles that modify biodistribution. In example adding dipeptides can improve the penetration of a circulating agent in the eye through the blood retinal barrier by using endogenous transporters.

[0046] A strategy for improving drug viability is the utilization of water-soluble polymers. Various water-soluble polymers have been shown to modify biodistribution, improve the mode of cellular uptake, change the permeability through physiological barriers; and modify the rate of clearance from the body. To achieve either a targeting or sustained-release effect, water-soluble polymers have been synthesized that contain drug moieties as terminal groups, as part of the backbone, or as pendent groups on the polymer chain.

[0047] Polyethylene glycol (PEG) has been widely used as a drug carrier, given its high degree of biocompatibility and ease of modification. Attachment to various drugs, proteins, and liposomes has been shown to improve residence time and decrease toxicity. PEG can be coupled to active agents through the hydroxyl groups at the ends of the chain and via other chemical methods; however, PEG itself is limited to at most two active agents per molecule. In a different approach, copolymers of PEG and amino acids were explored as novel biomaterials which would retain the biocompatibility properties of PEG, but which would have the added advantage of numerous attachment points per molecule (providing greater drug loading), and which could be synthetically designed to suit a variety of applications.

[0048] Those of skill in the art are aware of PEGylation techniques for the effective modification of drugs. For example, drug delivery polymers that consist of alternating polymers of PEG and tri -functional monomers such as lysine have been used by VectraMed (Plainsboro, N. J.). The PEG chains (typically 2000 daltons or less) are linked to the a- and e- amino groups of lysine through stable urethane linkages. Such copolymers retain the desirable properties of PEG, while providing reactive pendent groups (the carboxylic acid groups of lysine) at strictly controlled and predetermined intervals along the polymer chain. The reactive pendent groups can be used for derivatization, cross-linking, or conjugation with other molecules. These polymers are useful in producing stable, long-circulating pro-drugs by varying the molecular weight of the polymer, the molecular weight of the PEG segments, and the cleavable linkage between the drug and the polymer. The molecular weight of the PEG segments affects the spacing of the drug / linking group complex and the amount of drug per molecular weight of conjugate (smaller PEG segments provides greater drug loading). In general, increasing the overall molecular weight of the block co-polymer conjugate will increase the circulatory half-life of the conjugate. Nevertheless, the conjugate must either be readily degradable or have a molecular weight below the threshold-limiting glomular filtration (e.g., less than 60 kDa).

[0049] In addition, to the polymer backbone being important in maintaining circulatory halflife, and biodistribution, linkers may be used to maintain the therapeutic agent in a pro-drug form until released from the backbone polymer by a specific trigger, typically enzyme activity in the targeted tissue. For example, this type of tissue activated drug delivery is particularly useful where delivery to a specific site of biodistribution is required and the therapeutic agent is released at or near the site of pathology. Linking group libraries for use in activated drug delivery are known to those of skill in the art and may be based on enzyme kinetics, prevalence of active enzyme, and cleavage specificity of the selected disease-specific enzymes. Such linkers may be used in modifying the protein or fragment of the protein described herein for therapeutic delivery.

[0050] In another embodiment, SLC16A1 inhibitor or SMAD3 inhibitor according to the invention is an inhibitor of SLC16A1 or SMAD3 gene expression.

[0051] Small inhibitory RNAs (siRNAs) can also function as inhibitors of SLC16A1 or SMAD3 according to the invention for use in the present invention. SLC16A1 or SMAD3 according to the invention is an inhibitor of SLC16A1 gene expression can be reduced by contacting a subject or cell with a small double stranded RNA (dsRNA), or a vector or construct causing the production of a small double stranded RNA, such that SLC16A1 or SMAD3 inhibitor according to the invention is an inhibitor of SLC16A1 gene expression is specifically inhibited (i.e. RNA interference or RNAi). Methods for selecting an appropriate dsRNA or dsRNA-encoding vector are well known in the art for genes whose sequence is known (e.g. see for example Tuschl, T. et al. (1999); Elbashir, S. M. et al. (2001); Hannon, GJ. (2002); McManus, MT. et al. (2002); Brummelkamp, TR. et al. (2002); U.S. Pat. Nos. 6,573,099 and 6,506,559; and International Patent Publication Nos. WO 01 / 36646, WO 99 / 32619, and WO 01 / 68836). Short hairpin RNA or small hairpin RNA (shRNA) can also function as inhibitors of SLC16A1 or SMAD3 according to the invention for use in the present invention. shRNA is an artificial RNA molecule with a tight hairpin turn that can be used to silence target gene expression via RNA interference (RNAi). Expression of shRNA in cells is typically accomplished by delivery of plasmids or through viral or bacterial vectors. shRNA is an advantageous mediator of RNAi in that it has a relatively low rate of degradation and turnover. However, it requires use of an expression vector, which has the potential to cause side effects in medicinal applications.

[0052] MicroRNA (miRNA) can also function as inhibitors of SLC16A1 or SMAD3 expression for use in the present invention. MicroRNA (miRNA) are small, single-stranded, non-coding RNA molecules containing 21 to 23 nucleotides. Found in plants, animals and some viruses, miRNAs are involved in RNA silencing and post-transcriptional regulation of gene expression. miRNAs base-pair to complementary sequences in mRNA molecules, then silence said mRNA molecules.

[0053] Antisense oligonucleotides (ASOs) can also function as inhibitors of SLC16A1 or SMAD3 expression for use in the present invention. ASOs target messenger RNA (mRNA). ASOs are capable of altering mRNA expression through a variety of mechanisms, including ribonuclease H mediated decay of the pre-mRNA, direct steric blockage, and exon content modulation through splicing site binding on pre-mRNA. Several ASOs have been approved in the United States, the European Union, and elsewhere.

[0054] Ribozymes can also function as inhibitors of SLC16A1 or SMAD3 gene expression for use in the present invention. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage. Engineered hairpin or hammerhead motif ribozyme molecules that specifically and efficiently catalyze endonucleolytic cleavage of SLC16A1 or SMAD3 mRNA sequences are thereby useful within the scope of the present invention. Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which typically include the following sequences, GUA, GUU, and GUC. Once identified, short RNA sequences of between about 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site can be evaluated for predicted structural features, such as secondary structure, that can render the oligonucleotide sequence unsuitable. The suitability of candidate targets can also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using, e.g., ribonuclease protection assays.

[0055] Both antisense oligonucleotides and ribozymes useful as inhibitors of SLC16A1 or SMAD3 gene expression can be prepared by known methods. These include techniques for chemical synthesis such as, e.g., by solid phase phosphoramadite chemical synthesis. Alternatively, anti-sense RNA molecules can be generated by in vitro or in vivo transcription of DNA sequences encoding the RNA molecule. Such DNA sequences can be incorporated into a wide variety of vectors that incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Various modifications to the oligonucleotides of the invention can be introduced as a means of increasing intracellular stability and half-life. Possible modifications include but are not limited to the addition of flanking sequences of ribonucleotides or deoxyribonucleotides to the 5' and / or 3' ends of the molecule, or the use of phosphorothioate or 2'-O-methyl rather than phosphodiesterase linkages within the oligonucleotide backbone.

[0056] Antisense oligonucleotides siRNAs and ribozymes of the invention may be delivered in vivo alone or in association with a vector. In its broadest sense, a "vector" is any vehicle capable of facilitating the transfer of the antisense oligonucleotide siRNA or ribozyme nucleic acid to the cells and preferably cells expressing SLC16A1 or SMAD3. Preferably, the vector transports the nucleic acid to cells with reduced degradation relative to the extent of degradation that would result in the absence of the vector. In general, the vectors useful in the invention include, but are not limited to, plasmids, phagemids, viruses, other vehicles derived from viral or bacterial sources that have been manipulated by the insertion or incorporation of the antisense oligonucleotide siRNA or ribozyme nucleic acid sequences. Viral vectors are a preferred type of vector and include, but are not limited to nucleic acid sequences from the following viruses: retrovirus, such as moloney murine leukemia virus, harvey murine sarcoma virus, murine mammary tumor virus, and rouse sarcoma virus; adenovirus, adeno-associated virus; SV40-type viruses; polyoma viruses; Epstein-Barr viruses; papilloma viruses; herpes virus; vaccinia virus; polio virus; and RNA virus such as a retrovirus. One can readily employ other vectors not named but known to the art.

[0057] Preferred viral vectors are based on non-cytopathic eukaryotic viruses in which non- essential genes have been replaced with the gene of interest. Non-cytopathic viruses include retroviruses (e.g., lentivirus), the life cycle of which involves reverse transcription of genomic viral RNA into DNA with subsequent proviral integration into host cellular DNA. Retroviruses have been approved for human gene therapy trials. Most useful are those retroviruses that are replication-deficient (i.e., capable of directing synthesis of the desired proteins, but incapable of manufacturing an infectious particle). Such genetically altered retroviral expression vectors have general utility for the high-efficiency transduction of genes in vivo. Standard protocols for producing replication-deficient retroviruses (including the steps of incorporation of exogenous genetic material into a plasmid, transfection of a packaging cell lined with plasmid, production of recombinant retroviruses by the packaging cell line, collection of viral particles from tissue culture media, and infection of the target cells with viral particles) are provided in Kriegler, 1990 and in Murry, 1991).

[0058] Preferred viruses for certain applications are the adeno-viruses and adeno-associated viruses, which are double-stranded DNA viruses that have already been approved for human use in gene therapy. The adeno-associated virus can be engineered to be replication deficient and is capable of infecting a wide range of cell types and species. It further has advantages such as, heat and lipid solvent stability; high transduction frequencies in cells of diverse lineages, including hemopoietic cells; and lack of superinfection inhibition thus allowing multiple series of transductions. Reportedly, the adeno-associated virus can integrate into human cellular DNA in a site-specific manner, thereby minimizing the possibility of insertional mutagenesis and variability of inserted gene expression characteristic of retroviral infection. In addition, wild-type adeno-associated virus infections have been followed in tissue culture for greater than 100 passages in the absence of selective pressure, implying that the adeno-associated virus genomic integration is a relatively stable event. The adeno- associated virus can also function in an extrachromosomal fashion.

[0059] Other vectors include plasmid vectors. Plasmid vectors have been extensively described in the art and are well known to those of skill in the art. See e.g. Sambrook et al., 1989. In the last few years, plasmid vectors have been used as DNA vaccines for delivering antigen-encoding genes to cells in vivo. They are particularly advantageous for this because they do not have the same safety concerns as with many of the viral vectors. These plasmids, however, having a promoter compatible with the host cell, can express a peptide from a gene operatively encoded within the plasmid. Some commonly used plasmids include pBR322, pUC18, pUC19, pRC / CMV, SV40, and pBlueScript. Other plasmids are well known to those of ordinary skill in the art. Additionally, plasmids may be custom designed using restriction enzymes and ligation reactions to remove and add specific fragments of DNA. Plasmids may be delivered by a variety of parenteral, mucosal and topical routes. For example, the DNA plasmid can be injected by intramuscular, eye, intradermal, subcutaneous, or other routes. It may also be administered by intranasal sprays or drops, rectal suppository and orally. It may also be administered into the epidermis or a mucosal surface using a gene-gun. The plasmids may be given in an aqueous solution, dried onto gold particles or in association with another DNA delivery system including but not limited to liposomes, dendrimers, cochleate and mi croencap sul ati on .

[0060] In a particular embodiment, the antisense oligonucleotide, siRNA, shRNA or ribozyme nucleic acid sequence is under the control of a heterologous regulatory region, e.g., a heterologous promoter. The promoter may be specific for Muller glial cells, microglia cells, endothelial cells, pericyte cells and astrocytes For example, a specific expression in Muller glial cells may be obtained through the promoter of the glutamine synthetase gene is suitable. The promoter can also be, e.g., a viral promoter, such as CMV promoter or any synthetic promoters.

[0061] In a particular embodiment, an endonuclease can be used to abolish the expression of the gene, transcript or protein SLC16A1 or SMAD3.

[0062] Indeed, as an alternative to more conventional approaches, such as cDNA overexpression or downregulation by RNA interference, new technologies provide the means to manipulate the genome. Indeed, natural and engineered nuclease enzymes have attracted considerable attention in the recent years. The mechanism behind endonuclease-based genome inactivating generally requires a first step of DNA single or double strand break, which can then trigger two distinct cellular mechanisms for DNA repair, which can be exploited for DNA inactivating: the error prone non homologous end-joining (NHEJ) and the high-fidelity homology-directed repair (HDR).

[0063] In a particular embodiment, the endonuclease is CRISPR-cas. As used herein, the term “CRISPR-cas” has its general meaning in the art and refers to clustered regularly interspaced short palindromic repeats associated which are the segments of prokaryotic DNA containing short repetitions of base sequences.

[0064] In some embodiment, the endonuclease is CRISPR-cas9 which is from Streptococcus pyogenes. The CRISPR / Cas9 system has been described in US 8697359 Bl and US 2014 / 0068797. Originally an adaptive immune system in prokaryotes (Barrangou and Marraffini, 2014), CRISPR has been recently engineered into a new powerful tool for genome editing. It has already been successfully used to target important genes in many cell lines and organisms, including human (Mali et al., 2013, Science, Vol. 339 : 823-826), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), zebrafish (Hwang et al., 2013, PLoS One, Vol. 8:e68708.), C. elegans (Hai et al., 2014 Cell Res. doi: 10.1038 / cr.2014.11.), bacteria (Fabre et al., 2014, PLoS Negl. Trop. Dis., Vol. 8:e2671.), plants (Mali et al., 2013, Science, Vol. 339 : 823-826), Xenopus tropicalis (Guo et al., 2014, Development, Vol. 141 : 707- 714.), yeast (DiCarlo et al., 2013, Nucleic Acids Res., Vol. 41 : 4336-4343.), Drosophila (Gratz et al., 2014 Genetics, doi: 10.1534 / genetics. H3.160713), monkeys (Niu et al., 2014, Cell, Vol. 156 : 836-843.), rabbits (Yang et al., 2014, J. Mol. Cell Biol., Vol. 6 : 97-99.), pigs (Hai et al., 2014, Cell Res. doi: 10.1038 / cr.2014.11.), rats (Ma et al., 2014, Cell Res., Vol. 24 : 122-125.) and mice (Mashiko et al., 2014, Dev. Growth Differ. Vol. 56 : 122-129.). Several groups have now taken advantage of this method to introduce single point mutations (deletions or insertions) in a particular target gene, via a single gRNA. Using a pair of gRNA- directed Cas9 nucleases instead, it is also possible to induce large deletions or genomic rearrangements, such as inversions or translocations. A recent exciting development is the use of the dCas9 version of the CRISPR / Cas9 system to target protein domains for transcriptional regulation, epigenetic modification, and microscopic visualization of specific genome loci.

[0065] In some embodiment, the endonuclease is CRISPR-Cpfl which is the more recently characterized CRISPR from Provotella and Francisella 1 (Cpfl) in Zetsche et al. (“Cpfl is a Single RNA-guided Endonuclease of a Class 2 CRISPR-Cas System (2015); Cell; 163, 1-13).

[0066] In another embodiment, the invention relates to a method for treating a cancer comprising administering to a subject in need thereof a therapeutically effective amount of a SLC16A1 inhibitor and a SMAD3 inhibitor.

[0067] In order to test the functionality of a putative SLC16A1 inhibitor a test is necessary. For that purpose, to identify SLC16A1 inhibitor, intracellular lactate accumulation in cells is tested. SLC16A1 (MCT1) inhibitor will inhibit lactate transport and result in changes in lactate concentration intracellularly, extracellularly or both.

[0068] In order to test the functionality of a putative SMAD3 inhibitor a test is necessary. For that purpose, to identify SMAD3 inhibitor, loss of p-SMAD3 expression visualized by western blot is tested.

[0069] Therapeutic composition

[0070] Another object of the invention relates to a therapeutic composition comprising a SLC16A1 inhibitor and a SMAD3 inhibitor for use in the treatment of cancer in a subject in need thereof. Any therapeutic agent of the invention may be combined with pharmaceutically acceptable excipients, and optionally sustained-release matrices, such as biodegradable polymers, to form therapeutic compositions.

[0071] "Pharmaceutically" or "pharmaceutically acceptable" refers 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.

[0072] The form of the pharmaceutical compositions, the route of administration, the dosage and the regimen naturally depend upon the condition to be treated, the severity of the illness, the age, weight, and sex of the patient, etc.

[0073] The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

[0074] Preferably, 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.

[0075] The doses used for the administration can be adapted as a function of various parameters, and in particular as a function of the mode of administration used, of the relevant pathology, or alternatively of the desired duration of treatment.

[0076] In addition, other pharmaceutically acceptable forms include, e.g. tablets or other solids for oral administration; time release capsules; and any other form currently can be used.

[0077] Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent. The present invention also relates to a kit comprising an agonist, antagonist or inhibitor of the expression according to the invention and a further therapeutic active agent.

[0078] For example, anti-cancer agents may be added to the pharmaceutical composition as described below.

[0079] Anti-cancer agents may be Melphalan, Vincristine (Oncovin), Cyclophosphamide (Cytoxan), Etoposide (VP- 16), Doxorubicin (Adriamycin), Liposomal doxorubicin (Doxil) and Bendamustine (Treanda). Others anti-cancer agents may be for example cytarabine, anthracyclines, fludarabine, gemcitabine, capecitabine, methotrexate, taxol, taxotere, mercaptopurine, thioguanine, hydroxyurea, cyclophosphamide, ifosfamide, nitrosoureas, platinum complexes such as cisplatin, carboplatin and oxaliplatin, mitomycin, dacarbazine, procarbizine, etoposide, teniposide, campathecins, bleomycin, doxorubicin, idarubicin, daunorubicin, dactinomycin, plicamycin, mitoxantrone, L-asparaginase, doxorubicin, epimbicm, 5 -fluorouracil, taxanes such as docetaxel and paclitaxel, leucovorin, levamisole, irinotecan, estramustine, etoposide, nitrogen mustards, BCNU, nitrosoureas such as carmustme and lomustine, vinca alkaloids such as vinblastine, vincristine and vinorelbine, imatimb mesylate, hexamethyhnelamine, topotecan, kinase inhibitors, phosphatase inhibitors, ATPase inhibitors, tyrphostins, protease inhibitors, inhibitors herbimycm A, genistein, erbstatin, and lavendustin A. In one embodiment, additional anticancer agents may be selected from, but are not limited to, one or a combination of the following class of agents: alkylating agents, plant alkaloids, DNA topoisomerase inhibitors, anti-folates, pyrimidine analogs, purine analogs, DNA antimetabolites, taxanes, podophyllotoxin, hormonal therapies, retinoids, photosensitizers or photodynamic therapies, angiogenesis inhibitors, antimitotic agents, isoprenylation inhibitors, cell cycle inhibitors, actinomycins, bleomycins, MDR inhibitors and Ca2+ ATPase inhibitors.

[0080] Additional anti-cancer agents may be selected from, but are not limited to, cytokines, chemokines, growth factors, growth inhibitory factors, hormones, soluble receptors, decoy receptors, monoclonal or polyclonal antibodies, mono-specific, bi-specific or multi-specific antibodies, monobodies, polybodies.

[0081] Additional anti-cancer agent may be selected from, but are not limited to, growth or hematopoietic factors such as erythropoietin and thrombopoietin, and growth factor mimetics thereof.

[0082] In the present methods for treating cancer the further therapeutic active agent can be an antiemetic agent. Suitable antiemetic agents include, but are not limited to, metoclopromide, domperidone, prochlorperazine, promethazine, chlorpromazine, trimethobenzamide, ondansetron, granisetron, hydroxyzine, acethylleucine monoemanolamine, alizapride, azasetron, benzquinamide, bietanautine, bromopride, buclizine, clebopride, cyclizine, dunenhydrinate, diphenidol, dolasetron, meclizme, methallatal, metopimazine, nabilone, oxypemdyl, pipamazine, scopolamine, sulpiride, tetrahydrocannabinols, thiefhylperazine, thioproperazine and tropisetron. In a preferred embodiment, the antiemetic agent is granisetron or ondansetron. In another embodiment, the further therapeutic active agent can be an hematopoietic colony stimulating factor. Suitable hematopoietic colony stimulating factors include, but are not limited to, filgrastim, sargramostim, molgramostim and epoietin alpha.

[0083] In still another embodiment, the other therapeutic active agent can be an opioid or non-opioid analgesic agent. Suitable opioid analgesic agents include, but are not limited to, morphine, heroin, hydromorphone, hydrocodone, oxymorphone, oxycodone, metopon, apomorphine, nomioiphine, etoipbine, buprenorphine, mepeddine, lopermide, anileddine, ethoheptazine, piminidine, betaprodine, diphenoxylate, fentanil, sufentanil, alfentanil, remifentanil, levorphanol, dextromethorphan, phenazodne, pemazocine, cyclazocine, methadone, isomethadone and propoxyphene. Suitable non-opioid analgesic agents include, but are not limited to, aspirin, celecoxib, rofecoxib, diclofinac, diflusinal, etodolac, fenoprofen, flurbiprofen, ibuprofen, ketoprofen, indomethacin, ketorolac, meclofenamate, mefanamic acid, nabumetone, naproxen, piroxicam and sulindac.

[0084] In yet another embodiment, the further therapeutic active agent can be an anxiolytic agent. Suitable anxiolytic agents include, but are not limited to, buspirone, and benzodiazepines such as diazepam, lorazepam, oxazapam, chlorazepate, clonazepam, chlordiazepoxide and alprazolam.

[0085] In yet another embodiment, the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent.

[0086] Typically, the checkpoint blockade cancer immunotherapy agent is an agent which blocks an immunosuppressive receptor expressed by activated T lymphocytes, such as cytotoxic T lymphocyte-associated protein 4 (CTLA4) and programmed cell death 1 (PDCD1, best known as PD-1), or by NK cells, like various members of the killer cell immunoglobulin- like receptor (KIR) family, or an agent which blocks the principal ligands of these receptors, such as PD-1 ligand CD274 (best known as PD-L1 or B7-H1).

[0087] Typically, the checkpoint blockade cancer immunotherapy agent is an antibody.

[0088] In some embodiments, the checkpoint blockade cancer immunotherapy agent is an antibody selected from the group consisting of anti-CTLA4 antibodies, anti-PDl antibodies, anti-PDLl antibodies, anti-PDL2 antibodies, anti-TIM-3 antibodies, anti-LAG3 antibodies, anti -IDO 1 antibodies, anti-TIGIT antibodies, anti-B7H3 antibodies, anti-B7H4 antibodies, anti-BTLA antibodies, and anti-B7H6 antibodies. 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.

[0089] FIGURES:

[0090] Figure 1. SLC16A1 / MCT1 expression drives resistance to SMAD3 inhibitor in melanoma and lung cancer. A. Annotation of CRISPR-SAM cell lines; gene expression is induced in cell lines containing all three CRISPR-SAM components (corresponds to “3+”). B. Expression level of SLC16A1-AS1 and SLC16A1 measured by qPCR. sgRNA (sg7) targeting the shared promoter of SLC16A1-AS1 and SLC16A1 was introduced into 501Mel melanoma cell line using lentiviral vector to achieve stable overexpression by CRISPR-SAM. C. Protein level of SLC16A1 measured by Western blot. sgRNA (sg2) outside of the promoter was used for negative control. D. Dose-dependent effect of SMAD3 inhibition by SIS3 on the viability of 501Mel cells. Cells were seeded to 96w plates at low confluency and treated for 4 days. Viability was determined by methylene blue assay and is presented as measured absorbance of solubilized methylene blue ratios. E. Protein level of phospho-SMAD3 upon treatment with 17pM SIS3 for 48h. F. The effect of SLC16A1 overexpression by CRISPR-SAM (g7) on the resistance to SIS3. Cells were seeded on 6w plates at low confluency and treated with 17pM SIS3 for 2 weeks. Cell density is visualized by crystal violet staining and measured by the absorbance of solubilized crystal violet. G. The effect of SLC16A1 overexpression by CRISPR-SAM (g7) on the cell viability during SIS3 treatment for 2 weeks. Control cell population (2+, 90%) and MCT1 -overexpressing cell line (CRISPR-SAM g7, 10%) was mixed and treated with 17pM SIS3 for 2 weeks. H. Flow cytometry measurement of the mixed cell populations before (Day 1) and after (2 weeks) the treatment. I. Protein level of SLC16A1 measured by Western blot. sgRNA (sg7) targeting the promoter of SLC16A1 was introduced into A549 lung adenocarcinoma cell line using lentiviral vector to achieve stable overexpression by CRISPR-SAM. J. Dose-dependent effect of SMAD3 inhibition by SIS3 on the viability of A549 cells. K. The effect of SLC16A1 overexpression by CRISPR-SAM (g7) on the resistance to SIS3 in A549 cells. Cells were seeded on 6w plates at low confluency and treated with 20pM SIS3 for 2 weeks. Cell density is visualized by crystal violet staining and measured by the absorbance of solubilized crystal violet.

[0091] Figure 2. Combined inhibition of SMAD3 and MCT1 efficiently kills melanoma cells. A. The effect of MCT1 inhibitor AZD3965 on the viability of 501Mel cells. Cells were seeded on 6w plates at low confluency and treated with increasing concentration of AZD3965 for 6 days. Cell density is visualized by crystal violet staining. B. The measured absorbance of solubilized crystal violet. C. The combined effect of SMAD3 inhibitor SIS3 and MCT1 inhibitor AZD3965 on the viability of 501Mel cells. Cells were seeded on 6w plates at low confluency and treated with different combinations of SIS3 and AZD3965 for 2 weeks. Cell density is visualized by crystal violet staining. D. The measured absorbance of solubilized crystal violet. E-F. The combined effect of SIS3 and AZD3965 on the viability of 501Mel Naive (WT) cells. Cells were seeded on 96w plates at low confluency and treated with increasing concentrations of SIS3 and AZD3965 for 4 days. G-H. The combined effect of SIS3 and AZD3965 on the viability of 501Mel 3+ cells. Cells were seeded on 96w plates at low confluency and treated with increasing concentrations of SIS3 and AZD3965 for 4 days. I-J. The combined effect of SIS3 and AZD3965 on the viability of 501Mel Naive MCT1 KO cells. Cells were seeded on 96w plates at low confluency and treated with increasing concentrations of SIS3 and AZD3965 for 4 days.

[0092] Figure 3. CRISPR-screen identifies SLC16A1 / SLC16A1-AS1 promoter activation as resistance driver to SMAD3 inhibitor. A. Workflow of the CRISPR-screen (gene activation) targeting IncRNA genes to identify resistance drivers to SIS3. B. Volcano plot of sgRNA sequence count change after 2 weeks of SIS3 treatment compared to non-treated control cell library. Identification of NR 103743 (RefSeq gene ID) as the top candidate resistance driver to SIS3. C. Sequencing count number and genomic distribution of sgRNA target sites within SLC16A1 / SLC16A1-AS1 promoter after 2 weeks of SIS3 treatment compared to non-treated CRISPR-SAM cell library. D. Average count number of sgRNA(l- 3) and sgRNA(4-10) in non-treated and SIS3-treated cell library. E. Simultaneous induction of SLC16A1 / SLC16A1-AS1 gene expression 48h after transient transfection of 501Mel cells using sgRNAs within (sg5-9) or outside (sg2) of the promoter.

[0093] Figure 4. Resistance to SMAD3 inhibitor is caused by MCT1 protein. A. Stable overexpression of SLC16A1-AS1 IncRNA and MCT1 protein from ectopic vector introduced into 501Mel cell line using lentiviral vector. B. WB measurement of MCT1 protein in stable cell lines overexpressing SLC16A1-AS1 IncRNA and MCT1 protein. C. The effect of IncRNA or MCT1 overexpression on cell viability during SIS3 treatment. Cells were seeded on 6w plates at low confluency (10000 cells per well) and treated with 17pM SIS3 for 2 weeks. Cell density is visualized by crystal violet staining and measured by the absorbance of solubilized crystal violet. D. WB measurement of MCT1 protein levels in 501Mel WT cells and MCT1 KO cell line before and after rescued expression of MCT1 from ectopic vector. The effect of MCT1 KO and rescued protein expression on cell viability during 2 weeks of SIS3 treatment. E. WB measurement of MCT1 protein levels in SKMel28 WT and MCT1 KO cell line. The effect of MCT1 KO on cell viability during 2 weeks of SIS3 treatment.

[0094] Figure 5. MCT1 and SMAD3 may be markers of opposing gene regulatory programs. A. qPCR measurement of SMAD3 expression level in 501Mel cells 48h after transient transfection and SLC16A1 / SLC16A1-AS1 promoter activation by CRISPR-SAM using sgRNAs within (sg5-9) or outside (sg2) of the promoter. qPCR measurement of SMAD3 expression level in 501Mel and A549 cell lines with stable MCT1 overexpression by CRISPR-SAM using sg7. B. qPCR measurement of EMT marker genes’ expression level in 501Mel cells upon stable MCT1 overexpression by CRISPR-SAM using sg7. C. qPCR measurement of SLC16A1 expression level in 501Mel cells upon transient (48h) or stable SMAD3 overexpression by CRISPR-SAM using sgl. D. qPCR measurement of SLC16A1 expression levels in 501Mel SMAD3 KO cells. qPCR measurement of SLC16A1 expression level in 501Mel NT or sg7 cells after 2 weeks of SIS3 treatment. E. WB measurement of MCT1 protein level in 501Mel or A549 NT or sg7 cells after 2 weeks of SIS3 treatment. F. WB measurement of MCT1 protein level in 501Mel NT or sg7 cells after 24h of SIS3 treatment.

[0095] Figure 6. MCTl-dependent metabolic shift towards OXPHOS contributes to drug-resistance. A. Workflow of long-read direct RNA sequencing in 501Mel WT, MCTl(sg7)and MCT1(KO)cells. B. Pathway enrichment analysis of overexpressed genes in MCTl(sg7)cells compared to CTRL cells without drug treatment. C. Heatmap and hierarchical clustering of expression of genes related to respiratory electron transport chain in CTRL or MCTl(sg7)cells with or without 2 weeks of SIS3 treatment. Data is presented as log2 Z-score for each row. D. Heatmap and hierarchical clustering of expression of genes related to oxidative phosphorylation (OXPHOS), fatty acid P-oxidation (FAO) and glycolysis in CTRL or MCTl(sg7)cells with or without 2 weeks of SIS3 treatment.

[0096] EXAMPLE 1:

[0097] Material & Methods

[0098] Medium, Chemicals, and Antibodies.

[0099] RPML1640 and DMEM culture medium, fetal bovine serum (FBS) and penicillinstreptomycin antibiotics were purchased from Gibco (Grand Island, NY, USA). SIS3 (HY- 13013) and AZD3965 (HY-12750) was purchased from MedchemExpress (Monmouth Junction, NJ, USA). Antibodies for MCT1 (#85680) and p-SMAD3 (C25A9) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody for a-Tubulin (T6199) was purchased from Sigma-Aldrich (Munich, Germany). Goat anti-rabbit IgG-HRP antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human MCT1 / SLC16A1 PE-conjugated Antibody and Mouse IgG2A PE-conjugated Antibody were purchased from Bio-Techne (Minneapolis, MN, USA). Puromycin, Blasticidin, Hygromycin and Zeocin selection antibiotics were purchased from InvivoGen (San Diego, CA, USA).

[0100] Cell lines.

[0101] 501Mel, A549 and HEK293 cell lines were obtained from ATCC. Cells were maintained at 37°C and 5% CO2 in humidified air. 501Melmelanoma cell lines were grown in RPMI-1640 culture medium supplemented with 10% FBS and 1% penicillin-streptomycin. A549 and HEK293 cell lines were grown in DMEM culture medium supplemented with 10% FBS and 1% penicillin-streptomycin.

[0102] Generation of lentiviral cell lines.

[0103] Lentivirus was produced in HEK293 cells using pPAX2 (Addgene #12259) and pMD2.G (Addgene #12259) vectors according to previously established protocols (D.Trono lab). For CRISPR-SAM, 501Mel and A549 cells were infected to stably express dCAS-VP64 (Addgene #61425) and MS2-P65-HSF1 (Addgene #61426). Cells were infected with lentivirus overnight in presence of 5p / ml polybrene and selected during 5-10 days using Blasticidin (2-4pg / ml) and Hygromycin (200-400pg / ml).To activate target gene expression, sgRNA sequences were cloned into lenti sgRNA(MS2)_zeo backbone (Addgene #61427). For CRISPR-Cas9 KO, sgRNA sequences were cloned into lentiCRISPR v2 backbone (Addgene #52961). Infected CRISPR-SAM cell lines were selected using Zeocin (500pg / ml) during 7- 10 days. Infected KO cell lines were selected using Puromycin (2.5pg / ml) during 3 days followed by clonal selection.

[0104] Isolation of RNA and mRNA measurement by qPCR.

[0105] NucleoSpin RNA, Mini kit for RNA purification (Macherey-Nagel, Duren, Germany) was used for RNA extraction. Reverse transcription was performed using High-Capacity cDNA Reverse Transcription Kit (Life Technologies, Carlsbad, CA, United States) followed by quantitative PCR using Power SYBR™ Green PCR Master Mix (Life Technologies). PCR cycling conditions were as follows: 95oC for 10 min, 40 cycles of 95oC for 10 sec and 60oC for 1 min. Results were analyzed with QuantStudio 5 Real-Time PCR Systems (Life Technologies). PCR reactions were performed in three replicates. The results were analyzed with Design & Analysis 2 (DA2) software (Life Technologies). Relative expression level of the target gene (defined as fold change from control Ct values) was calculated using the 2- AACt method.

[0106] Western blot.

[0107] Cells were lysed with RIPA Lysis and Extraction Buffer (Life Technologies) containing Pierce™ Protease Inhibitor Mini Tablets, EDTA-free (Life Technologies) and resolved on NuPAGE 4 to 12% Bis-Tris 1.5mm gels (Life Technologies). Following electrophoresis, proteins were transferred to nitrocellulose membrane using Trans-Blot Turbo Transfer System (Biorad, Hercules, CA, USA) and blocked with TBST (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20) containing 4% BSA for Ih. The membrane was incubated overnight at 4oC with primary antibody. Goat anti-rabbit secondary IgG-HRP, ECL Select™ Western Blotting Detection Reagent (Amersham, Buckinghamshire, United Kingdom) were used prior to detecting the signal with G:Box Chemi XX6 imager (Syngene).

[0108] Methylene blue assay.

[0109] Cells were seeded on 96-well plates at low confluency (2000-5000 cells / well). 24h later, media containing SIS3 or AZD3965 was added. 4 days later, cells were washed with PBS and fixed using 100% ethanol. Fixed cells were dried and incubated with methylene blue staining solution for 30 minutes. The plates were rinsed in water and dried. Methylene blue was solubilized using 0. IN HC1 during 30 minutes and absorbance was measured using Tecan Infinite200 Pro (Tecan, Mannedorf, Switzerland).

[0110] Crystal violet assay.

[0111] Cells were seeded on 6-well plates at low confluency (10000 cells / well). 24h later, media containing SIS3 or AZD3965 was added. Treatment was performed during 6 days (AZD3965) or 2 weeks (SIS3 or SIS3+AZD3965). Media change was performed after every 2 days. At the end of treatment, cells were washed once with PBS and incubated with 0.1% Crystal violet staining solution for 10 minutes. Cells were washed with PBS 3 times and dried. Crystal violet was solubilized using 0.1N HC1 during 30 minutes and absorbance was measured using Tecan Infinite200 Pro (Tecan).

[0112] Flow cytometry.

[0113] Cells were harvested and resuspended in cold PBS containing 2% FBS, saturated for 5 minutes on ice and labeled with PE-conjugated MCT1 or IgG2A during 30 minutes. Cells were then washed and resuspended using cold PBS (2% FBS) and analyzed using Novocyte cytometer (Agilent Technologies, Santa Clara, CA, United States). Viable single cells were gated for detection of MCT1. Results

[0114] SLC16A1 / MCT1 expression drives resistance to SMAD3 inhibitor in melanoma and lung cancer.

[0115] To identify genes that drive resistance to SMAD3 inhibitor using melanoma as a model, a CRISPR activation screen was performed. Gene expression in cells was induced by the CRISPR-SAM (synergistic activation mediator) method. Lentiviral vector was used to introduce all three components of the CRISPR-SAM complex into the cells (Figure 1A). A CRISPR-SAM cell library was generated to target the promoters of 10504 IncRNA genes and treated with SMAD3 inhibitor SIS3 for 2 weeks. SIS3 concentration was selected based on the performed viability assay using 501Mel cells, aiming to achieve significant selective pressure (Figure IB). A decrease in the phospho-SMAD3 levels was observed upon treatment with 17pM SIS3 (Figure 1C). Genomic DNA sequencing of SIS3-treated cell library revealed a high enrichment of single guide RNA (sgRNA) sequences targeting the promotor of SLC16A1-AS1 IncRNA gene compared to non-treated cells.

[0116] To validate the result, a cell line was generated to stably activate this promoter by CRISPR-SAM. For this, sgRNA 7 (sg7) was selected to transduce previously engineered 501Mel 2+ melanoma cells and maintain constant assembly of the CRISPR-SAM complex in the promoter. qPCR measurements showed increased expression levels of SLC16A1-AS1 IncRNA and SLC16A1 protein-coding gene from the shared promoter (Figure ID). Western blot measurement confirmed the increase in SLC16A1 (also known as MCT1) protein levels using sg7 (3+ cells), compared to control sgRNA (sg2) outside of the promoter (Figure IE). 2-week treatment with SIS3 resulted in higher cell density of 3+ (g7) cells compared to control cells, indicating increased resistance to the inhibitor (Figure IF). For further validation, a mixed cell population consisting of 10% 3+ (g7) cells and 90% WT cells was treated with SIS3 for 2 weeks (Figure 1G). A significant SIS3 -dependent increase in the population of 3+ (g7) cells was measured by flow cytometry, demonstrating better survival in presence of drug (Figure 1H).

[0117] To evaluate the observed effect on resistance to SIS3 in other cancer subtypes, A549 lung adenocarcinoma cells were engineered to stably activate SLC16A1-AS1 / SLC16A1 promoter by CRISPR-SAM using the same sgRNA (g7). The expression of MCT1 protein in these cells was confirmed by Western blot (Figure II). The sensitivity of these cells to SIS3 was determined by the viability assay (Figure II). Treatment with a high concentration of SIS3 resulted in increased cell density in 3+ (g7) cell line compared to control cells, suggesting a conserved effect is different tumours (Figure IK). Combined inhibition of SMAD3 and MCT1 efficiently kills melanoma cells.

[0118] As the CRISPR-SAM proximity-based upregulation of MCT1 protein in addition to the IncRNA from the targeted promoter was observed, we aimed to study its effect on melanoma cell growth. Using a specific inhibitor of MCT1 protein AZD3965, we observed a dose-dependent decrease of cell density after 6-day treatment (Figure 2A). Cells overexpressing MCT1 by CRISPR-SAM were significantly more resistant to AZD3965 at low concentrations (lOnM), indicating the presence of functional protein and its importance to cell growth. The inhibitory effect of AZD3965 was restored to the same level as WT cells at higher concentration (50nM) (Figure 2B). No additional effect was observed by higher concentration (lOOnM), suggesting that MCT1 inhibition alone may not be sufficient to provide desired treatment outcomes.

[0119] To study the effect of MCT1 and SMAD3 inhibition co-inhibition, melanoma cells were treated with inhibitors for 2 weeks. Simultaneous treatment with SIS3 (17pM) and AZD3965 (lOnM or lOOnM) resulted in efficient cell death of 501Mel cells (Figure 2C). Cells overexpressing MCT1 by CRISPR-SAM elicited higher resistance to SIS3 alone and in combination with AZD3965 (lOnM), but also cease to survive at higher AZD3965 concentrations (lOOnM) when combined with SIS3 (Figure 2D).

[0120] To precise in more detail the synergistic effect of SMAD3 and MCT1 co-inhibition, 501Mel cells were treated during 4 days with different combinations of the inhibitors. Cell viability analysis demonstrated a dose-dependent inhibitory effect of SIS3 and AZD3965, whereas the most potent growth arrest was achieved by the simultaneous use of both inhibitors (Figure 2E-F). Similar synergistic effect was also detected in cell line with high MCT1 expression (3+ g7) (Figure 2G-H). Correspondingly, higher levels of MCT1 in cells reflect higher resistance to the inhibitors in various combinations. Contrarily, MCT1 KO cells were most responsive to the co-treatment of SIS3 and AZD3965 (Figure 2I-J). The observed effect of AZD3965 on cell death in MCT1 KO cells might be due to its lower affinity for MCT4, which could compensate for the loss of MCT1.

[0121] Conclusion:

[0122] Overall, these data demonstrate that combined treatment of SMAD3 and MCT1 inhibition may prove to be an efficient treatment option for cancer, also suggesting altogether that the efficacy of treatment could be potentially predicted by the expression profile of MCT1 in different cancer subtypes.

[0123] EXAMPLE 2: Material & Methods

[0124] Medium, Chemicals, and Antibodies

[0125] RPMI-1640 and DMEM culture medium, fetal bovine serum (FBS) and penicillinstreptomycin antibiotics were purchased from Gibco (Grand Island, NY, USA). SIS3 (HY- 13013) and AZD3965 (HY-12750) were purchased from MedChemExpress (Monmouth Junction, NJ, USA). Antibodies for MCT1 (#85680), SMAD3 (#9523), p-SMAD3 (#9520) and MYC (#5605) were purchased from Cell Signaling Technology (Beverly, MA, USA). Antibody for a-Tubulin (T6199) was purchased from Sigma-Aldrich (Munich, Germany). Goat anti-rabbit IgG-HRP antibody was obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Human MCT1 / SLC16A1 PE-conjugated Antibody and Mouse IgG2A PE- conjugated Antibody were purchased from Bio-Techne (Minneapolis, MN, USA). Puromycin, Blasticidin, Hygromycin and Zeocin selection antibiotics were purchased from InvivoGen (San Diego, CA, USA).

[0126] Cell lines

[0127] 501Mel, SKMel28, M229, M238, A549 and HEK293 cell lines were obtained from ATCC. Cells were maintained at 37°C and 5% CO2 in humidified air. 501Mel, SKMel28, M229 and M238 melanoma cell lines were grown in RPMI-1640 culture medium supplemented with 10% FBS and 1% penicillin-streptomycin. A549 and HEK293 cell lines were grown in DMEM culture medium supplemented with 10% FBS and 1% penicillinstreptomycin. All cell lines were regularly subjected for mycoplasma testing.

[0128] Generation of lentiviral cell lines

[0129] Lentivirus was produced in HEK293 cells using pPAX2 (Addgene #12259) and pMD2.G (Addgene #12259) vectors according to previously established protocols (D.Trono lab). For CRISPR-SAM, 501Mel, M229, M238 and A549 cells were infected to stably express dCas9-VP64 (Addgene #61425) and MS2-P65-HSF1 (Addgene #61426). Cells were incubated with lentivirus overnight in presence of 5pg / ml polybrene and selected during 5-10 days using Blasticidin (2-4pg / ml) or Hygromycin (200-400pg / ml). In order to induce target gene expression, individual sgRNA sequences were cloned into lenti sgRNA(MS2)_zeo backbone (Addgene #61427). For CRISPR-Cas9 KO, individual sgRNA sequences were cloned into lentiCRISPR v2 backbone (Addgene #52961). Infected cell lines with stable target gene overexpression by CRISPR-SAM were selected using Zeocin (500pg / ml) during 7-10 days. Infected KO cell lines were selected using Puromycin (2.5pg / ml) during 3 days followed by clonal selection. For stable ectopic overexpression of SLC16A1-AS1 or S C16A l, an ectopic lentiviral vector was used (VectorBuilder). Infected cells were selected over 5-10 days using Puromycin (SI.C16A l-ASl) or Hygromycin (SLC16A1). All lentiviral infections were performed on cells at 70% confluency.

[0130] Transient transfection of cell lines

[0131] For transient transfection, cells were seeded on 6-well plates. Next morning, media was replaced to remove antibiotics. 2-3h later, cells were transfected at 70% confluency using Lipofectamine2000 (Life Technologies) and with 5pg / well CRISPR-SAM plasmid vectors containing individual sgRNA sequences. 6h after transfection, media was changed. 48h after transfection, cells were frozen or lysed immediately for RNA or protein extraction.

[0132] CRISPR-SAM gene activation screen

[0133] Human CRISPR IncRNA Activation Pooled Library (SAM 3 -Plasmid System) was used for transcriptional activation of IncRNA genes (Addgene Pooled Library #1000000106). Plasmid library was amplified and used to infect 501Mel CTRL cell line expressing dCas9- VP64 and MS2-P65-HSF1 CRISPR-SAM components according to previously established protocol68. Shortly, plasmid library was amplified in Lucigen E cloni. 10G Elite electrocompetent bacteria (Cat. #60051-2, LGC Biosearch technologies, Hoddeston, UK) and purified using NucleoBond Xtra Maxi kit (Macherey-Nagel, Duren, Germany). HEK293 cells were transfected to produce lentiviral library and engineered 501Mel CTRL cells were infected with MOI<0.2. Infected pooled cell library was treated with 500pg / ml Zeocin for 2 weeks for cell selection. After amplification, 150M cells were collected for each of 3 replicates of non-treated control condition and frozen. Remaining cells were seeded in 3 replicates (150M cells for each replicate) and treated with 17pM SIS3 over 2 weeks. Cells were split with 2 / 5 ratio after every 2-3 days at 90% confluency. Frozen pellets of untreated and SIS3-treated cells were subjected to genomic DNA extraction and sequencing library preparation according to previously established protocols68. Genomic DNA was sequenced using NovaSeq 6000 sequencing system (Illumina) within Institut Curie CRISPR’it platform (Paris, France). sgRNA enrichment analysis, including ranking of positively or negatively enriched sgRNAs based on fold change, p-values and false discovery rates were computed using MAGeCK software. Ranking of genes based on fold change and uniformity of targeting sgRNAs was computed using MAGeCK software.

[0134] Direct long-read RNA sequencing

[0135] 501Mel CTRL, MCTl(sg7)and MCT1(KO)cells were treated or not with 17pM SIS3 over 2 weeks. RNA was extracted from 100M cells from at least 3 replicates of each condition using NucleoSpin RNA kit (Macherey-Nagel). Library preparation was performed according to manufacturer’s protocol (Oxford Nanopore Technologies Ltd, Oxford, UK). Sequencing was done using MinlON Flow Cells with GridlON device (Oxford Nanopore Technologies Ltd). Read basecalling was done using MinKNOW software (Oxford Nanopore Technologies Ltd).

[0136] Pathway enrichment analysis

[0137] Gene Set Enrichment Analysis (GSEA) or GProfiler software were used on Reactome pathway database for pathway enrichment analysis of differentially regulated or enriched genes from RNA sequencing.

[0138] Isolation of RNA and mRNA measurement by qPCR

[0139] NucleoSpin RNA, Mini kit for RNA purification (Macherey -Nagel) was used for RNA extraction. Reverse transcription was performed using High-Capacity cDNA Reverse Transcription Kit (Life Technologies) followed by quantitative PCR using Power SYBR™ Green PCR Master Mix (Life Technologies). PCR cycling conditions were as follows: 95°C for 10 min, 40 cycles of 95°C for 10 sec and 60°C for 1 min. Results were analyzed with QuantStudio 5 Real-Time PCR Systems (Life Technologies). PCR reactions were performed in three replicates. The results were analyzed with Design & Analysis 2 (DA2) software (Life Technologies). Relative expression level of the target gene (defined as fold change from control Ct values) was calculated using the 2-AACt method.

[0140] Western blot

[0141] Cells were lysed with RIPA Lysis and Extraction Buffer (Life Technologies) containing Pierce™ Protease Inhibitor Mini Tablets, EDTA-free (Life Technologies) and resolved on NuPAGE 4 to 12% Bis-Tris 1.5mm gels (Life Technologies). Following electrophoresis, proteins were transferred to nitrocellulose membrane using Trans-Blot Turbo Transfer System (Biorad, Hercules, CA, USA) and blocked with TBST (20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 0.1% Tween 20) containing 4% BSA for Ih. The membrane was incubated overnight at 4°C with primary antibody. Goat anti -rabbit secondary IgG-HRP, ECL Select™ Western Blotting Detection Reagent (Amersham, Buckinghamshire, United Kingdom) were used prior to detecting the signal with G:Box Chemi XX6 imager (Syngene). NE-PER Nuclear and Cytoplasmic Extraction Kit was used for extraction of separate cytoplasmic and nuclear protein fractions (Life Technologies).

[0142] Methylene blue assay

[0143] Cells were seeded on 96-well plates at low confluency (2000-5000 cells / well). 24h later, media containing SIS3 or AZD3965 was added. 4 days later, cells were washed with PBS and fixed using 100% ethanol. Fixed cells were dried and incubated with methylene blue staining solution for 30 minutes. The plates were rinsed in water and dried. Methylene blue was solubilized using 0. IN HC1 during 30 minutes and absorbance was measured using Tecan Infinite200 Pro (Tecan, Mannedorf, Switzerland).

[0144] Crystal violet assay

[0145] Cells were seeded on 6-well plates at low confluency (10000-50000 cells / well). Media containing inhibitor was added 24h later. Media change was performed after every 2-3 days. At the end of treatment, cells were washed once with PBS and incubated with 0.1% Crystal violet staining solution for 10 minutes. Cells were washed with PBS 3 times and dried. Crystal violet was solubilized using 0.1N HC1 during 30 minutes and absorbance was measured using Tecan Infinite200 Pro (Tecan).

[0146] Flow cytometry

[0147] Cells were harvested and resuspended in cold PBS containing 2% FBS, saturated for 5 minutes on ice and labeled with PE-conjugated MCT1 or IgG2A during 30 minutes. Cells were then washed and resuspended using cold PBS (2% FBS) and analyzed using Novocyte cytometer (Agilent Technologies, Santa Clara, CA, United States). Viable single cells were gated for detection of MCT1.

[0148] Statistical Analysis

[0149] Data were obtained from at least three independent experiments. Student t-test was used when comparing mean differences of two experimental groups. For multiple comparisons between treatment groups, one-way ANOVA was used, followed by Tukey’s post-hoc test. The level of statistical significance was established at p value of < 0.05. Data are expressed as means ± SEM.

[0150] Results

[0151] CRISP R-screen identifies SLC16A1 / SLC16A1-AS1 promoter activation as resistance driver to SMAD3 inhibitor

[0152] To identify genes that drive resistance to SMAD3 inhibitor we performed a CRISPR activation screen (Figure 3A). We used the CRISPR-SAM (synergistic activation mediator) method to induce gene expression. A lentiviral vector was used to engineer BRAFV600E- mutated 501Mel melanoma cell line to stably express the CRISPR-SAM complex components. The expression of dCas9-VP64 and MS2-p65-HSFl was validated by qPCR as well as by the induction of SMAD3 gene expression after introducing SMAD3 promotertargeting sgRNA into the cells (data not shown). To reduce heterogeneity, we selected a clone. CRISPR-SAM mediated gene activation was confirmed in these cells (data not shown). A CRISPR-SAM cell library was generated to target the promoters of 10504 long non-coding RNA (IncRNA) genes (each promoter targeted by 10 different sgRNAs) and treated with SMAD3 inhibitor SIS3. A viability assay on 501Mel cells was performed to determine and select the IC70 SIS3 concentration (17pM) to achieve significant cell selection pressure (data not shown). This concentration is sufficient to significantly decrease SMAD3 phosphorylation and activity (data not shown). The cell library was treated with SIS3 for 2 weeks and genomic DNA (gDNA) sequencing was performed to determine the abundance of sgRNA sequences. Distinct sgRNA enrichment profiles were observed in drug-treated compared to non-treated cells (data not shown).

[0153] We identified the highest enrichment among sgRNA sequences targeting the SLC16A1 / SLC16A1-AS1 promoter (RefSeq identifier NR 103743) (Figure 3B, data not shown). From the 10 sgRNA target sequences, we found 7 (sgRNAs 4-10) to be highly enriched in SIS3-treated cells compared to non-treated cell library, whereas no change was detected for the sgRNAs 1-3 (Figure 3C). To exclude the possibility of any initial amplification bias, we sequenced the used gDNA plasmid library and observed no significant fluctuation within the 10 sgRNA target sequences of this promoter (data not shown). The enriched sgRNA (sg4-10) target sequences are located between 2 transcription start sites (TSS) of nearby genes, while the non-affected sgRNA (sgl-3) target sequences are located within the coding region of SLC16A1 (Figure 3C, data not shown). Furthermore, we observed an increase in the average sg(4-10) count number compared to sg(l-3) after SIS3 treatment, but a decrease in the non-treated cell library (Figure 3D). These data suggest that the activation of this promoter contributes to increased cell survival specifically as a response to drug treatment and could require expression of the protein-coding SLC16A1 gene (encoding for MCT1 - Monocarboxylate Transporter 1). Thus, we hypothesized that CRISPR-SAM complex assembly is hindered within the SLC16A1 coding region. Accordingly, transient CRISPR-SAM gene activation using individual sgRNAs resulted in no change with sg2, whereas sgRNAs targeting the shared promoter (sg5, sg7, sg8, sg9) induced simultaneous expression of nearby SLC16A1-AS1 IncRNA and SLC16A1 proteincoding gene (Figure 3E).

[0154] SLC16A1 / SLC16A1-AS1 promoter activation drives resistance to SMAD3 inhibitor in melanoma and lung adenocarcinoma

[0155] To validate SLC16A1 / SLC16A1-AS1 promoter activation as a SIS3 resistance driver, we created a cell line with stable promoter activation by CRISPR-SAM. We used lentiviral vector to introduce into the CTRL cells individual sgRNA sg7 (and sg2 for control) to allow the assembly of CRISPR-SAM complex and stable endogenous expression of the nearby genes (Figure 1A). We confirmed by qPCR the concurrent expression of SLC16A1 and SLC16A1-AS1 in MCTl(sg7), but not in MCTl(sg2)501Mel melanoma cell line (Figure ID). Measurement of protein levels by WB showed a significant increase of MCT1 in MCTl(sg7), but not in MCTl(sg2)cell line compared to control (Figure IE). To evaluate the response to drug, we seeded the cells at low density and treated with SIS3 (17pM) for 2 weeks. We observed a significant increase in cell density using sg7, but not sg2, showing that adequate promoter activation and increased gene expression supports cell survival (Figure IF). To confirm the role of MCT1 in SIS3 resistance, we mixed together MCTl(sg7)and CTRL cells with 1 :9 ratio (Figure 1G). Flow cytometry analysis confirmed the MCTl-positive population to be close to 10% at day 1 after seeding (data not shown). After 2 weeks of culture in presence of SIS3 we detected a clear enrichment of cells overexpressing MCT1, consisting up to half the population (Figure 1H). The proportional increase in MCTl- positive cells was not likely due to drug-induced MCT1 expression, indicated by decreased relative proliferation of MCTl(sg7)cells compared to CTRL cells without SIS3 (Figure 1H). Similar results were obtained with a starting population containing 1% of MCTl(sg7)cells (data not shown), verifying that SLC16A1 / SLC16A1-AS1 promoter activation stimulates resistance to SMAD3 inhibitor.

[0156] Next, we aimed to evaluate this effect in other cancer cell lines. We engineered BRAFV600E-mutated M238 and M229 melanoma cell lines18to express CRISPR-SAM components and allow transcriptional activation of target genes. The expression of SAM components was confirmed by qPCR in both cell lines, as well as the induced transcription of SLC16A1 and SLC16A1-AS1 by transient transfection using a plasmid expressing the sg7 for CRISPR-SAM assembly (data not shown). Simultaneous induction of these genes was also observed in cell lines with stable activation of this promoter, as measured by qPCR and WB of MCT1 protein (data not shown). Treatment with SIS3 at IC70 concentration for 2 weeks increased cell density compared to CTRL cells, demonstrating that increased drugresistance in response to this promoter activation is not confined to a single melanoma cell line (data not shown). Additionally, we applied this approach to A549 lung adenocarcinoma cells and observed very similar effects (Figure II, 1J, IK; data not shown). Furthermore, we examined this effect in B16 mouse melanoma cells (data not shown). For promoter activation by CRISPR-SAM we selected 2 sgRNAs (sgl and sg2) based on sequence similarities to human SLC16A1 / SLC16A1-AS1 promoter. Using 2 different sgRNAs, we demonstrate that transcriptional activation of this promoter is followed by upregulation of both Slcl6al and Slcl6al-asl expression in B16 cells (data not shown). We determined the IC70 concentration of SIS3 to be lOpM by a viability assay. We found that promoter activation by both sgRNAs increased resistance to SIS3, as measured by increased cell density after 1 week of drug treatment (data not shown). Taken together, we validated the CRISPR-screen top candidate to promote resistance to the growth inhibitory effects of SIS3. Additionally, we show that this effect is not tissue or species specific, but more broad and likely conserved.

[0157] Resistance to SMAD3 inhibitor is caused by MCT1 protein

[0158] To determine if the increased drug-resistance is mediated by SLC16A1-AS1 IncRNA or SLC16A1 protein-coding gene, we used ectopic vectors to overexpress both of these genes separately. We decided to use 501Mel cell line as a model due to previously observed low SLC16A1-AS1 expression and relatively low MCT1 protein levels. Using a lentiviral delivery to generate cell lines with stable overexpression we verified increased expression levels compared to WT cells for both of these genes by qPCR (Figure 4A). Overexpression of SLC16A1-AS1 did not have an effect on transcriptional activation of SLC16A1 and vice versa (Figure 4A). WB measurement showed that SLC16A1 overexpression also resulted in increased MCT1 protein level, whereas SLC16A1-AS1 IncRNA overexpression does not increase the amount of MCT1 protein (Figure 4B). We treated these cell lines with SIS3 for 2 weeks and found that IncRNA overexpression had no effect on cell growth (Figure 4C). However, SLC16A1 overexpression significantly increased cell survival, implying that drugresistance is driven by MCT1 protein (Figure 4C). No difference in the growth speed of these cells was observed in non-treated control condition, suggesting that the effect is specific to drug treatment (data not shown). To confirm this, we created a CRISPR-Cas9 mediated knock-out (KO) of MCT1 protein in 501Mel cell line. We corroborated the KO of MCT1 by WB (Figure 4D). KO of MCT1 drastically increased drug-sensitivity and resulted in reduced growth of the cells (Figure 4D). We rescued MCT1 protein levels in KO cells from a lentiviral-delivered cDNA vector and detected a restored phenotypic response to drug-resistance, as demonstrated by recovery of cell survival (Figure 4D). Without SIS3 treatment, modulation of MCT1 protein level had little or no effect on the proliferation rate of the cells, showing that MCTl-elicited impact on cell growth during drug treatment is a specific response to SIS3 (data not shown). In addition, we used CRISPR-Cas9 to deplete MCT1 protein in SKMel28 melanoma cell line. Similarly, we measured a decrease in the growth of these cells compared to WT cells in presence of SIS3 (Figure 4E), while MCT1 KO did not affect proliferation without the drug (data not shown). Collectively, these results confirm that MCT1 protein causes resistance to SMAD3 inhibitor and suggest that SLC16A1-AS1 IncRNA, at least in trans, does not contribute to this effect.

[0159] MCT1 and SMAD3 are markers of opposing gene regulatory programs

[0160] To gain insight into how MCT1 overexpression causes resistance to SMAD3 inhibitor, we first studied if MCT1 is involved in regulation of SMAD3 expression and signaling. Transient overexpression of MCT1 by CRISPR-SAM using several sgRNAs did not change SMAD3 expression level in 501Mel cells (Figure 5 A). Stable overexpression of MCT1 by CRISPR-SAM (sg7) in 501Mel, M229, M238 or A549 cell lines had no effect on SMAD3 mRNA levels, indicating lack of direct transcriptional regulation (Figure 5A, data not shown). MCT1 overexpression had no effect on the expression level of known SMAD3 target genes, indicating that MCT1 does not modulate SMAD3 transcriptional activity (Figure 5B). Contrarily, we aimed to study if SMAD3 regulates SLC16A1 expression. Transient or stable SMAD3 overexpression by CRISPR-SAM had no effect on transcriptional activation of SLC16A1 promoter, as indicated by unchanged expression levels of SLC16A1 and SLC16A1-AS1 (Figure 5C, data not shown). Loss of SMAD3 by CRISPR- Cas9 mediated KO resulted in minor upregulation of SLC16A1-AS1 IncRNA levels, but had no effect on SLC16A1 mRNA levels (Figure 5D, data not shown). Inhibition of SMAD3 activity by SIS3 in CTRL or MCT1 -overexpressing cells after 2 weeks of treatment did not affect the expression levels of SLC16A1 or SLC16A1-AS1, suggesting that SMAD3 does not regulate SLC16A1 transcriptionally (Figure 5D, data not shown). Additionally, long-term SIS3 treatment did not change MCT1 protein levels in 501Mel or A549 cells with low basal SLC16A1 expression level (Figure 5E). However, we found a significant increase in MCT1 protein levels after 2 weeks of inhibitor treatment in MCT1 -overexpressing cells (Figure 5E). We did not observe this effect with short-term 24h SIS3 treatment, suggesting that SMAD3 inhibition does not directly modulate translation activity or protein stability of MCT1 (Figure 5F). Thus, the increase in MCT1 protein after 2 weeks is likely the result of positive selection of cells with high MCT1 expression within the polyclonal cell population.

[0161] Due to seemingly favored selection of MCT1 -conferred signaling in presence of SMAD3 inhibition, but no direct transcriptional regulation between them, we hypothesized that MCT1 and SMAD3 might be markers of different gene regulatory programs. Corroborating this, we found a significant reverse correlation between the expression level of SLC16A1 and SMAD3 in melanoma cell lines (data not shown). We also evaluated gene expression levels in different melanoma subtypes, classified by the differentiation stage dependent transcriptional signature18. We noticed lower levels of SLC16A1 in neural-crest like melanoma cells, a subtype with high SMAD3 expression level (data not shown). On the contrary, SLC16A1 expression levels are high in melanocytic and transitory cells, which exhibit low SMAD3 expression (data not shown). To confirm the observation, we measured and compared SLC16A1 mRNA levels in transitory M229 and neural-crest like M238 cells and observed a stage-dependent decrease compared to melanocytic SKMel28 cell line, opposite to the SMAD3 expression and transcriptional signature as previously determined (data not shown)6,18. Thus, MCT1 and SMAD3 likely are markers of opposing gene regulatory programs. Taken together, MCT1 -conferred resistance to SIS3 does not result from direct regulation of SMAD3 signaling, but is likely due to systemic reprogramming that has favorable survival advantage in presence of SMAD3 inhibition and drug-induced stress.

[0162] MCT1 promotes a metabolic shift towards OXPHOS

[0163] To deeper understand the mechanisms of resistance we characterized the systemic changes induced by SIS3 in 501Mel CTRL, MCTl(sg7)and MCT1 KO cells. We profiled RNA expression using direct long-read nanopore sequencing to precisely capture transcript abundance at isoform level (Figure 6A). We identified cell cycle regulation and respiratory electron transport to be the most affected pathways among downregulated genes in SIS3- treated cells, indicating reduced metabolic activity and energy production (data not shown). Contrarily, analysis of genes upregulated by endogenous MCT1 overexpression showed enrichment in pathways related to the tricarboxylic acid cycle (TCA), respiratory electron transport chain (ETC), ATP synthesis and mitochondrial biogenesis (Figure 6B). SIS3 treatment resulted in downregulation of most common genes related to the electron transport chain, whereas MCT1 overexpression caused upregulation of these genes (Figure 6C, data not shown). Moreover, MCT1 overexpression results in elevated expression of genes promoting mitochondrial biogenesis, including the mitochondrial biogenesis master regulator gene PPARGC1A, suggesting that MCT1 positively affects energy production which may compensate the effects of SIS3 (data not shown). We observed PPARGC1A to inversely correlate with SMAD3 in different melanoma subtypes, suggesting that MCT1 and SMAD3 expressing cells might rely on different metabolic programs (data not shown). Next, we evaluated the effects of SMAD3 inhibition and MCT1 overexpression on the gene expression profile of major metabolic pathways involved in energy production - oxidative phosphorylation (OXPHOS), fatty acid P-oxidation (FAO) and glycolysis. We found that MCT1 overexpression induced upregulation of most relevant genes in closely related OXPHOS and FAO metabolic pathways, whereas most genes were downregulated by SIS3 treatment (Figure 6D). Differential gene expression profile was also observed in the glycolytic pathway, suggesting that MCT1 has a significant impact on modulating energy balance and promoting OXPHOS and FAO (Figure 6D). Accordingly, we found several key genes within these pathways to be lowly expressed in cells with high SMAD3 levels, but having relatively high expression in melanocytic and transitory subtypes (data not shown). Increased expression of CTNNB1 and MYC in these subtypes reflect higher WNT / p-catenin pathway activity, which has been shown to promote OXPHOS and FAO19. Opposingly, significantly reduced expression of glucose transporter gene SLC2A1 within these cells, but increased expression in parallel to SMAD3 corroborates that distinct stages of melanoma differentiation are likely accompanied by different metabolic regulation and SMAD3 expressing cells may rely more on glycolytic pathway (data not shown). These data suggest that MCT1 functions as a metabolic switch by promoting mitochondrial biogenesis and aerobic respiration, which are likely to contribute to increased survival and resistance to SIS3 drug-treatment.

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[0165] Throughout this application, various references describe the state of the art to which this invention pertains. The disclosures of these references are hereby incorporated by reference into the present disclosure.

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Claims

CLAIMS:

1. A combination of a SLC16A1 inhibitor and a SMAD3 inhibitor for use in the treatment of a cancer in a subject in need thereof.

2. A i) SLC16A1 inhibitor and ii) a SMAD3 inhibitor, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer in a subject in need thereof.

3. A combination for use according to the claim 1 or a combined preparation for use according to the claim 2 wherein the cancer is a skin cancer or a lung cancer.

4. A combination or a combined preparation for use according to the claim 3 wherein the skin cancer is a melanoma.

5. A combination for use according to the claim 1 or a combined preparation for use according to the claim 2 wherein the cancer is a cancer expressing MCT1 or a cancer with a high level of MCT1.

6. A combination for use according to the claim 1 or a combined preparation for use according to the claim 2 wherein the SLC16A1 inhibitor can be selected in the group consisting in but not limited to AZD3965, BAY-8002 and 7ACC2.

7. A combination for use according to the claim 1 or a combined preparation for use according to the claim 2 wherein the SMAD3 inhibitor can be selected in the group consisting in but not limited to SIS3 or 16D.

8. A method for treating a cancer comprising administering to a subject in need thereof a therapeutically effective amount of a SLC16A1 inhibitor and a SMAD3 inhibitor.

9. A therapeutic composition comprising a SLC16A1 inhibitor and a SMAD3 inhibitor for use in the treatment of cancer in a subject in need thereof.

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