Combination of VDAC2 modulators and BH3 mimetics for treating cancer

Abstract

The present invention relates to the field of the treatment of cancer. In this study, the inventors found that VDAC2 is heterogeneously expressed in MM cells. VDAC2 protein expression correlated with BAK but not with BAX protein levels. Transient silencing of VDAC2, but not VDAC1 or VDAC3, sensitized MM cells to intrinsic mitochondrial apoptosis signals, alongside with the induction of pre-activated BAK and the increase of global, MCL1 and BCL2 mitochondrial priming. They also found a that a VDAC2 compound, efesevin recapitulated the sensitization effect of VDAC2 knock-down on BH3 mimetics apoptotic response. This novel VDAC2 modulator sensitized MM cells to BH3 mimetics targeting MCL1 (S63845) or BCL2 (Venetoclax) without modifying BAK or BAX protein expression. The efficiency of the VDAC2 modulator was directly correlated with the levels of VDAC2 protein. To better understand the VDAC2 / BAK interplay, The inventors generated VDAC2 KO myeloma cells. VDAC2 KO cells exhibited an important decrease of BAK protein expression while BAX remained unchanged. Accordingly, VDAC2 KO cells completely lost their mitochondrial priming. Interestingly, they also found that BAK KO myeloma cells displayed decreased levels of VDAC2. The reciprocal regulation between VDAC2 and BAK was dependent on both the proteasome and lysosome degradation pathways. Thus, the present invention relates to a combination of a VDAC2 modulator and a BH3- mimetics compound for use in the treatment of a cancer in a subject in need thereof.

Description

[0001] COMBINATION OF VDAC2 MODULATORS AND BH3 MIMETICS FOR TREATING CANCER

[0002] FIELD OF THE INVENTION:

[0003] The present invention relates to combination of a VDAC2 modulator and a BH3- mimetic compounds for use in the treatment of a cancer in a subject in need thereof.

[0004] BACKGROUND OF THE INVENTION:

[0005] Evasion of apoptosis is a hallmark of cancer cells. The BCL2 family has emerged as a promising therapeutic target in cancer due to its ability to tightly regulate apoptosis. The BCL2 family encompasses anti-apoptotic proteins (MCL1, BCL2 and BCLXL) and two sets of pro- apoptotic proteins, the BH3-only (e.g. BIM, BMF, NOXA) and the pore-forming effectors BAK and BAX (1). They regulate themselves through protein-protein interactions, the balance between the two main groups determines cell fate. Triggering of the apoptotic pathway involves the activation and oligomerization of the pro-apoptotic effectors BAX and BAK, which ultimately causes a breach in the mitochondrial outer membrane (MOM) (2). Complex mechanisms are involved in the regulation of these effector proteins. Beyond BAX / BAK sequestration by their anti-apoptotic BCL2 counterparts, non-BCL2 family proteins are emerging as partners of these effectors and are considered as novel regulators of apoptosis (3). One of them is VDAC2, which belongs to the family of voltage dependent anion channels along with VDAC1 and VDAC3. VDACs are the most abundant proteins in the MOM, where they allow the passage of ions and metabolites (4,5). Although the three forms can substitute for each other in the metabolite and ion flux (6), they also have nonredundant roles. Thus, VDAC2 draw particular attention as only VDAC2 deletion was embryonic lethal in mice (7), indicating a unique and fundamental role in apoptosis regulation. Nevertheless, it is not clear how this mechanistically occurs with contrasting evidence for the role of VDAC2 in regulating BAX and BAK effectors. Notably, VDAC2 was reported to be necessary for efficient BAX dependent apoptosis (8), while conversely inhibited BAK apoptotic function (7). These contrasting arguments prompted us to study the implication of VDAC2 in the regulation of apoptotic function of multiple myeloma cells.

[0006] Multiple Myeloma (MM) is a B-cell hemopathy accounting for 10% of hematologic malignancies and is the second most common hematologic cancer. The median age at diagnosis is 66-70 years. MM cells overexpress the anti-apoptotic BCL2 members, which can be selectively targeted with the novel agents termed BH3 -mimetics. BH3-only proteins as well as BAX and BAK protein effectors are also readily detected in MM patients’ cells (9). The inventors recently demonstrated that in MM cells harboring TP53 wild type, BAX was critical for optimal cell response to MCL1 BH3 mimetic (10). However, abnormal p53 expression is also found in MM, whether the apoptotic response would rely on BAK effector and which mechanisms are implicated in this setting remains to be clarified.

[0007] SUMMARY OF THE INVENTION:

[0008] In the present study, the inventors found that VDAC2 is heterogeneously expressed in MM cells. VDAC2 protein expression correlated with BAK but not with BAX protein levels. Transient silencing of VDAC2, but not VDAC1 or VDAC3, sensitized MM cells to intrinsic mitochondrial apoptosis signals, alongside with the induction of pre-activated BAK and the increase of global, MCL1 and BCL2 mitochondrial priming. They also found a VDAC2 compound that recapitulated the sensitization effect of VDAC2 knock-down on BH3 mimetics apoptotic response. This novel VDAC2 modulator sensitized MM cells to BH3 mimetics targeting MCL1 (S63845) or BCL2 (Venetoclax) without modifying BAK or BAX protein expression. The efficiency of the VDAC2 modulator was directly correlated with the levels of VDAC2 protein. To better understand the VDAC2 / BAK interplay, The inventors generated VDAC2 KO myeloma cells. VDAC2 KO cells exhibited an important decrease of BAK protein expression while BAX remained unchanged. Accordingly, VDAC2 KO cells completely lost their mitochondrial priming. Interestingly, they also found that BAK KO myeloma cells displayed decreased levels of VDAC2. The reciprocal regulation between VDAC2 and BAK was dependent on both the proteasome and lysosome degradation pathways.

[0009] Thus, the present invention relates to a combination of a VDAC2 modulator and a BH3- mimetic compound for use in the treatment of a cancer in a subject in need thereof.

[0010] Particularly, the invention is defined by its claims.

[0011] DETAILED DESCRIPTION OF THE INVENTION:

[0012] A first object of the present invention relates to a combination of a VDAC2 modulator and a BH3 -mimetic compound for use in the treatment of a cancer in a subject in need thereof.

[0013] In another embodiment, the invention relates to i) a VDAC2 modulator and ii) a BH3- mimetic compound, as a combined preparation for simultaneous, separate or sequential use in the treatment of cancer in a subject in need thereof. As used herein, the term “simultaneous use” denotes the use of the modulator and the BH3 -mimetic compound of the invention occurring at the same time.

[0014] As used herein, the term “separate use” denotes the use of the modulator and the BH3- mimetic compound of the invention occurring not at the same time.

[0015] As used herein, the term “sequential use” denotes the use of the modulator and the BH3- mimetic compound of the invention occurring by following an order.

[0016] In another particular embodiment, the invention relates to a VDAC2 modulator and ii) a BH3 -mimetic compound as a combined preparation for simultaneous use in the treatment of cancer.

[0017] In a particular embodiment, the invention relates to a VDAC2 modulator and ii) a BH3- mimetic compound as a combined preparation wherein the modulator is initially administrated alone and then the modulator and the BH3 mimetic compound are administered together.

[0018] As used herein, the term “VDAC2” for “Voltage-dependent anion- selective channel protein 2” has its general meaning in the art and denotes a protein that in humans is encoded by the VDAC2 gene on chromosome 10. This protein is a voltage-dependent anion channel and shares high structural homology with the other VDAC isoforms. VDACs are generally involved in the regulation of cell metabolism, mitochondrial apoptosis, and spermatogenesis. Additionally, VDAC2 plays a critical role in cardiac function by influencing cellular calcium signalling. (Shankar TS et al Nat Comm 2021). Its Entrez accession number is 7417 and its UniProt accession number is P45880.

[0019] As used herein, the term “BH3 -mimetic compound” denotes a compound which directly activates the apoptotic machinery in malignant cells. This compound binds to and inhibits specific pro-survival BCL2 family proteins, thereby mimicking their interaction with the BH3 domains of proapoptotic BCL2 family proteins. BH3-mimetic compounds are well known in the art (see for example Diepstraten S.T. et al, Nature Review 2022 or Kalonil D. et al, Apoptosis 2023). They are inhibitors of anti-apoptotic molecules like BCL2, BCLXL or MCL1 (herein the anti-apoptotic BCL2 family protein members), once bound to the anti-apoptotic BCL2 member they trigger the release of the BCL2 pro-apoptotic proteins and in turn cell death. The released pro-apoptotic proteins are apoptosis effectors like BAX and BAK and proapoptotic BH3-only molecules considered as direct activators (BID, BIM and PUMA) or apoptosis sensitizers like, BAD, NOXA and HRK (see for example Townsend Paul A. J Exp Clin Cancer Res 2021).

[0020] As used herein, the term “VDAC2 modulator” denotes all molecules which modulate the activity of VDAC2 on the regulation of the apoptotic response. The VDAC2 modulator Efsevin binds into a VDAC2 pocket formed by the channel wall and the pore-lining helix facilitating the closure of VDAC2 channel, state that enhances calcium flux (Wilting F et al. Br J Pharmacol 2020). The inventors show that efsevin significantly sensitized multiple myeloma cells (notably) to BH3 mimetics targeting MCL1 or targeting BCL2. Accordingly, efsevin increased the mitochondrial global priming and the specific dependence on MCL1 and BCL2 without modifying the protein levels of BAK or BAX apoptotic effectors in these cells.

[0021] 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, glioblastoma, astrocytoma, breast cancer, Castleman disease, cervical cancer, colorectal cancer, endometrial cancer, esophagus cancer, gallbladder cancer, gastrointestinal carcinoid tumors, Hodgkin's disease, haematological cancer, non-Hodgkin's lymphoma like Mantle cell 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, multiple myeloma and uterine cancer.

[0022] In a particular embodiment, the cancer is a multiple myeloma or a multiple myeloma resistant.

[0023] In a particular embodiment, the cancer is a non-Hodgkin's lymphoma like Mantle cell lymphoma.

[0024] In a particular embodiment, the cancer is a cancer with a hight level of VDAC2 protein, particularly a multiple myeloma with a hight level of VDAC2 protein. Particularly, the level of expression of the VDAC2 protein is determined by Western blot.

[0025] In a particular embodiment, the cancer can have or not a p53 mutation. 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 suffers from a cancer and particularly a multiple myeloma.

[0026] 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.]).

[0027] In one embodiment, the VDAC2 modulator and the BH3-mimetc according to the invention may be a low molecular weight compound, e. g. a small organic molecule (natural or not).

[0028] 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.

[0029] According to the invention, VDAC2 modulator can be efsevin (described in Shimizu H et al eLife 2015, Wilting, F. et al. Br J Pharmacol 2020). In a particular embodiment, the VDAC2 modulator is a derivative of efsevin as described in the patent application W02016100379 or WO2023062451.

[0030] According to the invention, the BH3 -mimetic compound can be a BH3 -mimetic drug and can be an inhibitor of BCL2, Bcl-xL or MCL1 and can be selected in the group consisting in ABT-737, Navitoclax, Gossypol, Obatoclax, Venetoclax, APG1387, AT -406, A1210477, AMG-176, AZD-5991, S63845, MIMI, VU661013, Abbvie compound 4 or Amgen compound 3.

[0031] Particularly, the BH3-mimetic drug is an inhibitor of BCL2 like Venetoclax or and inhibitor of MCL1 like S63845.

[0032] 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 VDAC2 modulator and a BH3 -mimetic compound.

[0033] In order to test the functionality of a putative VDAC2 modulator a test is needed to evaluate the capacity of a said putative VDAC2 modulator to sensitize cancer cells to an apoptotic signal. For that purpose, to identify VDAC2 modulator a cell death assay can be performed in vitro on cells treated with the combination of the putative VDAC2 modulator with a particular therapeutic agent. After treatment, cells can be stained with Annexin V and analyzed by flow cytometry. Alternatively, a viability assay of the combination can be also performed using a Luminiscent CellTitre Gio assay.

[0034] In order to test the functionality of a putative BH3-mimetic compound a test is nedeed. For that purpose, to identify a BH3 -mimetic compound a cell death assay can be performed in vitro on cells treated with the combination of the putative VDAC2 modulator with a particular BH3 mimetic. After treatment, cells can be stained with Annexin V and analyzed by flow cytometry. Alternatively, a viability assay of the combination can be also performed using a Luminiscent CellTitre Gio assay.

[0035] Therapeutic composition

[0036] Another object of the invention relates to a therapeutic composition comprising a VDAC2 modulator and a BH3 -mimetic compound for use in the treatment of cancer in a subject in need thereof.

[0037] 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.

[0038] "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.

[0039] 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.

[0040] The pharmaceutical compositions of the invention can be formulated for a topical, oral, intranasal, parenteral, intraocular, intravenous, intramuscular or subcutaneous administration and the like.

[0041] 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.

[0042] 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.

[0043] 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. Pharmaceutical compositions of the present invention may comprise a further therapeutic active agent. The present invention also relates to a kit comprising a modulator or BH3 mimetic according to the invention and a further therapeutic active agent.

[0044] For example, anti-cancer agents may be added to the pharmaceutical composition as described below.

[0045] Anti-cancer agents may be Melphalan, Vincristine (Oncovin), Cyclophosphamide (Cytoxan), Etoposide (VP- 16), Doxorubicin (Adriamycin), Liposomal doxorubicin (Doxil) and Bendamustine (Treanda).

[0046] 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, hexamethyhnel amine, 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] In still another embodiment, the other therapeutic active agent can be an opioid or nonopioid 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.

[0052] 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.

[0053] In yet another embodiment, the further therapeutic active agent can be a checkpoint blockade cancer immunotherapy agent.

[0054] 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).

[0055] Typically, the checkpoint blockade cancer immunotherapy agent is an antibody. 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.

[0056] 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.

[0057] FIGURES:

[0058] Figure 1. A) HMCLs were treated with Efsevin (lOpM) and S63845 combination during 24h or pre-treated with Efsevin (lOpM) during 72h or 96h followed by the combination treatment with Efsevin and S63845 for the next 24h. Cell death was assessed by Annexin V staining using flow cytometry. LD50 values corresponded to the mean ±SD of 3 independent experiments. Statistical analysis was performed using the ordinary one-way ANOVA test with multiple comparisons. B) HMCLs were ordered according to VDAC2 relative protein levels normalized to ACTIN obtained from figure 3D (left panel). Analysis of the correlation between VDAC2 relative protein levels and the ratio of LD50 S63845 / LD50 S63845 + Efsevin (lOpM) combination at 24h, 72h or 120h is shown in the right panel. Statistical analysis was performed by Spearman test, p and r values are indicated. C) HMCLs were treated with Efsevin (lOpM) and Venetoclax combination during 24h or pre-treated with Efsevin (lOpM) during 72h or 96h followed by the combination treatment with Efsevin and Venetoclax for the next 24h. Cell death was assessed as in A). Values represent the mean and ±SD of at least 3 independent experiments. Statistical analysis was performed using ordinary one-way ANOVA test with multiple comparisons and unpaired t-test, respectively. D) Death response of primary MM samples (n=20) to Efsevin (15pM), S63845 (25nM) or their combination during 24h. Cell death was assessed by the loss of CD138 staining using flow cytometry 20,21,24. Left panel represents all primary samples analyzed, middle panel samples with > 50% of cell death S63845 alone and right panel samples with <50% of cell death with S63845 alone. Statistical analysis was performed using Wilcoxon test. E) A Mantle Cell Lymphoma, JEKO, was treated with the combination of Efsevin and S63845 as in A). Cell death was measured as in A).

[0059] Figure 2. A) Identification of the differentially expressed proteins between VDAC2 KO clones vs CTR clones. Proteomic analysis of KMS12PE and KMM1 VDAC2 KO clones (3 clones for each cell line) compared to their respective control clones (3 clones for each cell line). Differentially expressed proteins were selected according the Log-fold change between 0.5 and -0.5 and p val <0.05. Left panel represents the number of differentially expressed proteins for each cell line. Two downregulated proteins were shared between KMS12PE and KMM1 (right panel). B) Western blotting analysis of VDAC2, BAK and BAX protein expression in CTR and VDAC2KO clones on KMS12PE (CT=3, VDAC2 KO=4) and KMM1 (CT=3, VDAC2 KO=3) cells. A representative western blot is shown in the top panel. Protein expression levels were quantified and normalized to ACTIN; results corresponded to the mean ± SD and are representative of at least 4 experiments (bottom panel). C) RT-qPCR analysis of VDAC2 and BAK1 genes was performed on cells analyzed in B). Values corresponded to the mean ± SD and are representative of at least 4 experiments. Statistical analysis was performed using Mann-Whitney test. D) BH3 profiling was performed on KMS12PE and KMM1 VDAC2 KO clones. Cytochrome c release was measured by flow cytometry after challenging cells with BH3-derived peptides or Venetoclax. BIM peptide measures global mitochondrial priming, MSI peptide and Venetoclax measure MCL1 and BCL2 dependence, respectively. PUMA 2A is an inert peptide. Values corresponded to the percentage of cytochrome c loss (mean ± SD) of 3 independent experiments. Statistical analysis was performed using the 2-way Anova test. E) KMM1 BAX KO and BAX KO were treated with Efsevin (lOpM) and S63845 combination during 24h or pre-treated with Efsevin (lOpM) during 72h or 96h followed by the combination treatment with Efsevin and S63845 for the next 24h. Cell death was assessed by Annexin V staining. LD50 values corresponded to the mean ±SD of at least 3 independent experiments. Statistical analysis was performed using the ordinary one-way ANOVA test with multiple comparisons. F) BH3 profiling was performed on KMS12PE and KMM1 cells pre-treated with Efsevin (lOpM) during 72h. Cytochrome c release was measured by flow cytometry after challenging cells with BH3-derived peptides. BIM peptide measures global mitochondrial priming, MSI peptide and Venetoclax measure MCL1 and BCL2 dependence, respectively. PUMA 2A is an inert peptide. Values corresponded to the percentage of cytochrome c loss (mean ± SD) of 3 independent experiments. G) VDAC2, BAK and BAX protein expression in KMM1 and KMS12PE cell lines treated with lOpM of Efsevin for the indicated times. A representative western blot from 3 experiments is shown.

[0060] Figure 3. A) KMM1 CTR2 and VDAC2KO6 cells were treated with 5nM Bortezomib (Bz), 50nM BafilomycinAl (Baf) or the combination of both for the indicated times. The expression of the indicated proteins was assessed by western blotting. NOXA and pEIF2 alpha protein expression were included as controls of Bortezomib and bafilomycinAl treatment, respectively. B) KMM1 CTR and BAK-KO cell lysates were analyzed by western blotting. A representative western blot from 3 experiments is shown. C) KMM1 CTR4 and BAK -KO 11 cells were treated with Bortezomib (5nM), BafilomycinAl (50nM) or the combination of both for the indicated times. The expression of the indicated proteins was assessed by western blotting. NOXA and pEIF2alpha protein expression were included as controls of Bortezomib and Bafilomycin Al treatment, respectively. D) VDAC2, BAK and BAX protein levels were analyzed by western blotting in 20 HMCLs (NAN12 was excluded). LP1 cell lysate was included as an internal control. VDAC2, BAK and BAX relative protein levels were quantified and normalized to ACTIN. Results represent the mean of at least 3 independent experiments. Correlation of VDAC2 vs BAK and VDAC2 vs BAX relative protein levels were assessed by Spearman test, p and r values are indicated.

[0061] Figure 4. The VDAC2 modulator Efsevin sensitized Myeloma cells to BH3-mimetics targeting MCL1 or BCL2. Human Myeloma Cell Lines (HMCL) were treated with EF (10 pM) for 72h and then treated with the combination of EF and S63845 (A) or EF and Venetoclax (C) for the next 24 hours. Cell death was assessed by Annexin V staining using flow cytometry. LD50 values corresponded to the mean ± SD of 3 independent experiments. Statistical analysis was performed using paired t test. Cells were pre-treated 72 hours with EF and then with the combination of EF and S63845 (B) or Venetoclax (D) for the next 24 hours, as indicated. BLISS scores are indicated in each graph and were calculated using https: / / synergyfinder.fimm.fi / . Results represent the mean ± SD of 3 independent experiments. (E) The VDAC2 modulator Efsevin sensitizes myeloma cells to cell death induced by BH3 mimetics targeting MCL1 independent of p53 status. NCI-H929NI#1 (TP53+ / +) and NCI- H929 TP53KO#\ (TP53~ / _) clones, previously generated in our laboratory, were pre-treated with EF (1 OpM) for 96 hours and then with the combination of EF with S63845 for the next 24h. Cell death was assessed by Annexin V staining. LD50 values corresponded to the mean ± SD of 3 independent experiments. Statistical analysis was performed using paired t test.

[0062] Figure 5. The combination of Efsevin and the BH3 mimetic targeting MCL1 was effective in tumor cells from Myeloma patients. (A) The graph represents cell death response of myeloma cells from patient samples (n=27) to EF (15 pM), S63845 (25 nM), or their combination after 24 hours of treatment. (B) The graph represents the difference between observed cell death (25 nM S63845 combined with 15 pM EF) minus expected cell death (sum of cell death induced by 25 nM S63845 and by 15 pM EF) (right). Cell death was assessed after 24 hours by flow cytometry. Statistical analysis was performed using the Wilcoxon rank sum test.

[0063] Figure 6. Efsevin sensitizes Myeloma cell lines to the BH3 mimetic targeting BCLXL (Al 155463). Human Myeloma Cell Lines (HMCL) were treated with EF (10 pM) for 72h and then treated with the combination of EF and Al 155463 for the next 24 hours. Cell death was assessed by Annexin V staining using flow cytometry. LD50 values corresponded to the mean ± SD of 3 independent experiments. Statistical analysis was performed using paired t test.

[0064] Table 1. HMCLs characteristics and LD50 values for S63845 or Venetoclax in combination with Efsevin

[0065] A)

[0066] B)

[0067] HMCLs were previously described18. A) HMCLs were treated with Efsevin (lO M) and S63845 combination during 24h or pre-treated with Efsevin ( I O M) during 72h or 96h followed by the combination treatment with Efsevin and S63845 for the next 24h. B) HMCLs were treated with Efsevin (lO M) and Venetoclax combination during 24h or pre-treated with Efsevin (lO M) during 72h or 96h followed by the combination treatment with Efsevin and Venetoclax for the next 24h. Cell death was assessed by Annexin V staining. LD50 values corresponded to the mean of at least 4 independent experiments. Table 2. Cell death induced by S63845, Efsevin and their combination in MM primary cells

[0068]

[0069] Mononuclear cells isolated from bone marrow (BM) or peripheral blood (PB) from myeloma patients (MYRACLE cohort, NTC03807128) (2) were cultured 24 hours with S63845 (25nM), Efsevin (15mM) or their combination, and myeloma cell death was assessed by flow cytometry. Dell7p, lq21 gain, t(4,14) and t(ll;14) were determined by FISH (threshold >50% of cells). D: Diagnosis. P: Progression; R: Relapse; x:not performed.

[0070] Table 3A. HMCLs’ characteristics and LDso values of S63845 + / - Efsevin

[0071] Table 3B. HMCLs’ characteristics and LDso values of Venetoclax + / - Efsevin Human Myeloma Cell Lines (HMCLs) were pre-treated or not during 72 hours with Efsevin (lO M), and then treated 24 hours with A) S63845 or B) Venetoclax (Ven). Cell death was assessed by Annexin V staining. LD50 values corresponded to the mean of at least 4 independent experiments. Wild type (wt)

[0072] EXAMPLES:

[0073] Material & Methods

[0074] Human myeloma cell lines and primary myeloma cells.

[0075] Human Myeloma Cell Lines (HMCLs, n= 20) were characterized as previously describedl8. MDN, BCN, XG5, XG6, XG7, XG10, NAN11 and NAN12 were derived in our laboratory and are cultured with RPMH640 with 5% FCS and 3ng / ml recombinant IL-6. The KMM1, KMS12PE and KMS11 cell lines were kindly provided by Dr. Otsuki (Kawasaki Medical School, Kurashiki, Japan); KARPAS-620 (K620) by Dr. Karpas (Cambridge Clinical School, Cambridge UK); ANBL6 by Dr. Jelinek (Rochester, USA); MM IS by Dr. S. Rosen (Northwestern University, Chicago, USA); AM01, LP1, L363, NCI-H929 (NCI), U266, OPM2 HMCLs were from DSMZ (Braunsweig, Germany) and JIM3 was provided by Dr. I. MacLennan (Birmingham Medical School, Birmingham, United Kingdom). After informed consent, MM bone marrow / blood samples were collected at University Hospital of Nantes. Department of Hematology, (MYRACLE study; NTC03807128)19.

[0076] Reagents and antibodies.

[0077] Venetoclax (ABT-199) and Bortezomib were from Selleck Chemicals (Houston, TX, USA); S63845 from Chemietek (Indianapolis, USA) and BafilomycinAl (BFA) from Sigma- Aldrich. Efsevin was kindly provided by Dr. Ohyun Kwon and Tony Moreno, (Department of Chemistry and Biochemistry, University of California, Los Angeles) and previously characterized 16. Anti-VDAC2 (PA5-28106) was purchased from Thermo Fisher. Anti-BAK (12105S), anti-BAX (2772S), anti-BCL2 (Dako, M0887), anti-BCLXL (2764S), anti-PUMA (12450S), anti-NOXA (14766), anti-TUBULIN (3873S) and anti-pEIF2alpha (9721S) from Cell Signaling. BCLXL (sc-271121), MCL1 (sc-12756) and VDAC1 (sc-8828) from Santa Cruz. ACTIN (MAB1501) and BIM (Abl7003) from Millipore.

[0078] Cell death assays.

[0079] Cell death in HMCLs and CRISPR-Cas9 gene editing cell lines was assessed using AnnexinV-FITC staining (Beckman Coulter). Fluorescence acquisition in flow cytometry and analysis were performed using a FACs Accuri (Becton Dickinson) and FlowJo Software respectively. For primary myeloma cells, mononuclear Cells (MNC) were isolated from blood samples by Ficoll-Hypaque density gradient centrifugation and immediately cultured in RPMI- 1640 media with 5% FCS. Plasma cells were identified using CD138 staining (anti-CD138-PE, Beckman Coulter). MNC were incubated 24h with S63845 in the presence or absence of Efsevin (15pM) and an untreated condition was included as a control. Cell death response was measured by the loss of CD138 expression, as previously described20,21. Specific cell death was expressed as the percentage relative to the untreated control condition. Fluorescence acquisition in flow cytometry and analysis were performed using a FACs Cantoll (Becton Dickinson) and Flow Jo software.

[0080] CRISPR-CAS9 genome editing.

[0081] The generation of a CRISPR-Cas9 gene editing tool was employed to edit the KMM1 and KMS12PE cell lines to create either a VDAC2, a BAK1 or a BAX knockout (KO) (VDAC2KO, BAKKO, BAXKO named respectively). For VDAC2, Leucine L26 (TTG) was modified to Stop codon (TAG). For BAK1, Serine S3 (TCG) was modified to Stop codon (TAG). For BAX the Arginine R109 (CGG) was modified in Stop codon (TAG). 0.5 x 106 cells were electroporated with 36 pmols SpCas9 Nuclease V3, 44 pmols CRISPR-Cas9 tracRNA ATTO 550, 44 pmols Alt-R CRISPR-Cas9 crRNA XT, 44 pmols Alt-R Cas9 Electroporation Enhancer and 36 pmols Ultramer DNA Oligo (IDT-Integrated DNA Technologies). Electroporation was performed using Amaxa Transfection System (Lonza). Cells were then cultured and further cloned in 96-well plates (limiting dilution 0.3 cells per well). After cloning, generated clones were analyzed by DNA extraction, PCR amplification with VDAC2, BAK1 or BAX primers and validated by Sanger Sequencing. Finally, protein lysates were collected from KO cells to assess VDAC2, BAK and BAX protein levels by Western Blot. For KMM1 cell line, 3 clones VDAC2-KO, 3 clones BAK -KO, 3 clones BAX-KO and 3 control clones (CTR) were generated. For KMS12PE cell line, 4 clones VDAC2-KO and 3 control clones (CTR) were generated. Control clones (CTR) were obtained using the same electroporation procedure, but without the addition of target crRNA.

[0082] Transient transfection.

[0083] KMM1 cell line was transfected with lOOpmol siRNA using RNAimaxTM Reagent (Life Technologies) according to the manufacturer’s instructions. KMS12PE cell line was transfected by nucleofection using Amaxa Transfection System (Lonza) using the SF Solution and CA 137 program. Transfected cells were treated 24h with BH3-mimetics after 48h or 24h of transfection, respectively. siRNAs scramble (D-001810-10-20), VDAC1 (L-019764-00- 0005), VDAC2 (L-019766-00-0005) and VDAC3 (L-020850-00-0005) were from Dharmacon.

[0084] Intracellular BH3 profiling. BH3 profiling assay was performed in HMCLs cell lines as previously described, using BH3 derived peptides. BIM and BMF peptides measure global priming, NOXA (MSI) peptide for MCL1 dependence and PUMA (PUMA2A) inert peptide as a negative control. Venetoclax for BCL2 dependence and DMSO as control condition. Briefly, cells were permeabilized in DTEB buffer containing 0.002% of digitonin and exposed to peptides and Venetoclax for 1 hour at 27°C, before fixation with 2% formaldehyde at room temperature for 15 min. After addition of neutralizing buffer (Tris / Glycine buffer) for 5 min, cells were stained with anticytochrome c antibody Alexa 647 (BD Biosciences) in 0.1%Saponin / l%BSA / PBS overnight at 4°C. Cytochrome c release was analyzed by flow cytometry using a FACs Cantoll (Becton Dickinson) and the quantification of cytochrome c loss induced by each peptide was calculated by gating the cytochrome c positive population.

[0085] BAX and BAK activation.

[0086] The detection of BAK or BAX activation was performed as previously described21 . Briefly, 1x106 cells were fixed and permeabilized using the FOXP3 transcription factor staining buffer set (Thermo Fischer Scientific, 00-5523-00) following the manufacturer’s recommendations. Cells were then incubated with the following antibodies: BAX (clone 6A7, , sc-23959, Santa Cruz), BAK (clone AB-1 (TC-100), Calbiochem), mouse IgGl or IgG2a isotypic controls (Miltenyl, 130-106-545) for 30 min. After washing, cells were incubated with the Alexa fluor 647 antibody for 30 min, washed once in PBS and resuspended in PBS-1% formaldehyde. The flow cytometry acquisition and analysis were performed on a Facs Canto (Becton Dickinson) and FlowJo software, respectively.

[0087] Immunoblotting.

[0088] Cells pellets were lysed 40 minutes on ice in 0.5% NP40 containing lysis buffer for immunoblotting. Lysates were then centrifugated 30 minutes at 10 000 rpm at 4°C and supernatants were collected. 50pg total protein lysates per lane were separated by SDS-PAGE using precast gels (Bio-Rad or Thermo Fisher Scientific) and then electro-transferred using trans-blot turbo transfer system (Bio-rad). Membranes were then blocked in 5% skim milk- TBS-Tween. Chemiluminescence was detected using ECL detection reagent (Pierce, Rockford, IL, USA) in a ChemiDoc Imaging System (Bio-Rad) and protein expression levels were quantified using ImageLab and Image J software.

[0089] Quantitative real-time PCR.

[0090] Total RNA was isolated using the NucleoSpin RNA II kit (Macherey-Nagel) following the manufacturer’s recommendations. Two micrograms of the total RNA were reverse transcribed using the Maloney murine leukemia virus reverse transcriptase (Invitrogen) and random hexamers (Amersham Bioscience). Quantitative PCR was performed using the TaqMan Universal PCR Master Mix (Applied Biosystems) and the MX4000 instrument (Stratagene) as previously described22. The probes VDAC1 (Hs04978484_ml), VDAC2 (Hs00762994_sl), VDAC3 (Hs01091534_gl), BAK1 (Hs00832876_gl), BAX (Hs00180269_ml), MCL1 (HS00172036_ml), BCL2 (Hs00608023_ml), BCL2L1 (Hs00236329_ml), BCL2L11 (Hs00708019_sl) and RPL37a (HsOl 102345 ml) TaqMan gene expression assays were from Applied Biosystems. The thermal cycling parameters used were 50°C for 2 min and 95°C for 10 min for optimal Amperase UNG activity. Then 40 cycles at 95°C for 30 s and 60°C for 1 min. Amplification of RPL37a was conducted for each sample as a housekeeping gene control. The relative expression (fold change) of VDAC1, VDAC2, VDAC3, BAK1, BAX, MCL1, BCL2, BCL2L1 and BCL2L11 mRNAs was calculated using Pfaffl method23.

[0091] RNA-seq analysis.

[0092] Genomic profiling of HMCLs, clones or purified myeloma cells was assessed by RNA sequencing (RNA-seq; 3Z-digital gene expression sequencing DGE-seq) as previously described24.

[0093] Statistical analysis.

[0094] Statistical analyses were conducted using the Mann-Whitney, ANOVA, or paired Student t tests. Correlation was assessed by the Spearman correlation method, (r) and p values are indicated.

[0095] Results

[0096] VDAC2, but not VDAC1 or VDAC3, is an important regulator of the intrinsic mitochondrial apoptosis in myeloma cells.

[0097] Previous studies suggested that VDAC2 can regulate apoptosis with conflicting evidence for its implication in regulating BAX and BAK effectors (7,8,11). Additionally, VDAC1 has been reported as a mediator of mitochondria-induced apoptosis through its interaction with BAX, BCLXL or BCL2 (12,13,14,15). In light of these arguable results, we examined the implication of the three VDACs in MM apoptotic response.

[0098] We first analyzed the expression of VDACs in a collection of human MM cell lines (HMCL) by DGEseq. The VDAC family members (1, 2 and 3) were equivalently expressed among the cell lines analyzed, although VDAC2 mRNA levels were lower than VDAC1 and VDAC3 mRNA (data not shown). We next studied the role of each VDAC in cell death induction by BH3 mimetics targeting MCL1 (S63845) or BCL2 (Venetoclax). To this aim, we performed the transient silencing of the three VDACs in 2 HMCL expressing similar levels of the three VDAC family members and belonging to 2 different MM subgroups. KMS12PE harbors a t(l 1 ; 14) translocation and KMM1 a t(6; 14) translocation. Cell death induced by the MCL1 BH3 mimetic was significantly increased upon VDAC2 knock-down in both cells lines whereas the silencing of neither VDAC1 nor VDAC3 induced the same effect (data not shown). Indeed, we observed 4.4 and 3 fold decrease on S63845 LD50 in KMS12PE and KMM1 cells, respectively (data not shown). The increased cell death induced by Venetoclax observed in KMS12PE cells upon VDAC2 silencing is in agreement with the sensitivity of t(l l;14) MM cells to this BCL2 inhibitor (data not shown). Indeed, the knock-down of VDAC2 induced 13.4 fold decrease of Venetoclax LD50 in this cell line (data not shown). Of note, no effect of the silencing of any of the VDACs was observed on cell death induced by recombinant TRAIL, suggesting their lack of effect on the regulation of the extrinsic apoptotic pathway (data not shown). These results support the notion that VDAC2 rather than VDAC1 or VDAC3, modulates the intrinsic mitochondrial apoptotic pathway in MM.

[0099] We next explored whether the silencing of VDAC2 could modify the level of mitochondrial apoptotic priming (proximity to the threshold at which a cell commits to cell death) as well as the dependence on specific pro-survival MCL1 or BCL2 proteins. Thus, using the BH3 profiling assay, we demonstrated that cell death increase observed upon VDAC2 siRNA knock-down was associated with an enhanced global mitochondrial priming, which was measured by the release of cytochrome c induced by Bim BH3 peptides in both cell lines (data not shown). Interestingly, the specific priming or dependence was also significantly increased in VDAC2 knocked-down cells. Indeed, higher cytochrome c release was observed upon MSI peptide (targeting MCL1) in KM12PE and KMM1 cells, as well as upon Venetoclax challenging (targeting BCL2) in KMS12PE cells (data not shown). These results suggested that VDAC2 regulates apoptosis upstream mitochondrial outer membrane permeabilization (MOMP). Consistent with these findings, the silencing of VDAC2 induced BAK conformation change in both cell lines, as assessed by the exposure of its al helix, which is an early marker of BAK activation (data not shown). In contrast, there was no induction of BAX conformational changes upon VDAC2 knocked-down, as no significant exposure of the Bax N-terminal region was observed in this condition (data not shown). Together these results indicate a crucial role of VDAC2 in the regulation of intrinsic apoptosis pathway in MM, which could be supported by the control of mitochondrial priming and the early steps of BAK activation. Efsevin, a novel VDAC2 modulator, sensitizes MM cells to BH3 mimetics targeting MCL1 or BCL2

[0100] Our abovementioned results prompted us to look for a VDAC2 compound that could recapitulate the sensitization effect of VDAC2 knock-down on BH3 mimetics apoptotic response. Recently, Efsevin was identified through library screening for chemicals that were capable to reverse the arrhythmia phenotype observed in the tremblor zebrafish model (16). This activity was shown to be dependent on VDAC2, accordingly Efsevin was found bound to VDAC2 (17). Thus, we hypothesized that Efsevin could modulate the apoptotic response to BH3 mimetics S63845 (MCL1 specific) or Venetoclax (BCL2 specific) through VDAC2 targeting. To this aim, we pre-treated HMCLs with Efsevin during 72h or 96h followed by the combination treatment with Efsevin and S63845 for the next 24h. The combination effect was also assessed at 24h of combination treatment without pre-incubation (Table 1A). Efsevin itself did not induced apoptosis in HMCLs at different time points or at different concentrations (5-20microM) (data not shown). Strikingly, as shown in figure 1A, the combination of Efsevin with S63845 was efficient to significantly sensitize 4 out of 6 HMCLs tested. This effect was readily observed from 24h of combination treatment in 3 / 4 HMCLs, while longer treatment times were effective for 4 / 4 HMCL tested. Furthermore, the effect of Efsevin was irrespective of the MM molecular subgroup and the TP53 status of the HMCLs tested (Table 1A and IB). Accordingly, a similar sensitized effect of Efsevin on S63845 apoptosis induction was observed in NCLH929 TP53KO compared to NCLH929 wt, a comparable S63845 LD50 fold decrease (LD50 S63845 / LD50 S63845+Efsevin) was observed in both cell lines, 1.6 and 1.8, respectively (data not shown). Noteworthy, the efficacy of Efsevin in MCL1 BH3 mimetic apoptosis sensitization seemed to be related to the level of expression of VDAC2 protein (Figure IB left panel). Accordingly, Efsevin did not sensitize XG7 and L363 HMCLs, which expressed the lowest VDAC2 protein levels. Indeed, we demonstrated a direct correlation between the fold decrease of LD50 S63845 / LD50 S63845+Efsevin and the protein expression level of VDAC2 at 72h (p=0.0028; r=l) and 120h (p=0.0028; r=l) (Figure IB). Additionally, we also investigated the effect of Efsevin on Venetoclax apoptosis induction. Similar to the results obtained on S63845 apoptosis response, longer Efsevin treatment demonstrated more efficient effect of the combination in KMS12PE cells. We observed 6.83 and 3.1 Venetoclax LD50 fold decrease (LD50 Venetoclax / LD50 Venetoclax+Efsevin) at 96h and 120h, respectively (Figure 1C, left panel). Further analysis on another HMCLs harboring t(l l;14) translocation, MDN, confirmed the significant effect of Efsevin on Venetoclax sensitization (Figure 1C, right panel). To further confirm the effect of Efsevin on cell death sensitization, we assessed death response to S63845, Efsevin and their combination in myeloma cells from 20 consecutive patients at diagnosis, progression or relapse (Table 2). In agreement with the results obtained in HMCLs, the combination was more effective than S63845 alone (p=0.0001) (Figure ID, left panel). Interestingly, a more efficient effect of the combination was observed in the group of S63845 poor responder samples (cell death less than 50%, p=0.0093) (middle panel) than those with cell death more than 50% (p=0. 0078) (Figure ID right panel).

[0101] Importantly, we also demonstrated that Efsevin sensitized JEKO cells, a Mantle Cell lymphoma cell line to S63845 apoptosis induction. Figure IE showed a 1.5, 1.8 and 2.7 fold decrease of S63845 LD50 (LD50 S63845 / LD50 S63845+Efsevin) at 24h, 96h and 120h of combination treatment, respectively. Together these findings uncover a novel and unexpected effect of Efsevin as an apoptotic modulator, probably due through VDAC2 targeting.

[0102] The regulation of mitochondrial apoptosis by VDAC2 is essentially dependent on BAK To investigate the functional link between VDAC2 and the sensitization response to mitochondrial apoptosis, we generated myeloma VDAC2 KO cells by inserting a stop codon to the proximity of the ATG initiator codon. This strategy, known as KI Crispr-CAS9, is based on specific cut followed by homologous recombination with the DNA matrix bearing the stop codon. This approach allows the generation of identic clones, since the stop codon will always be at the same position. In addition, it ensures the deletion of all isoforms. Thus, VDAC2 KO clones were selected for each cell line (4 for KMS12PE and 3 for KMM1) and their protein expression profiling was analyzed in comparison to control clones (CTR). We used a label- free LC-MS / MS proteomics approach to identify proteins differentially expressed in VDAC2- KO stablished clones from KMS12PE and KMM1 cell lines. The comparison between VDAC2-KO vs CTR clones identified 307 and 165 proteins differentially expressed in KMS12PE and KMM1, respectively (Figure 2A). Only 2 proteins were commonly identified in both cells lines, as expected one of them being VDAC2. Strikingly, the other protein significantly decreased was the multidomain effector BAK (Figure 2A). Protein levels of VDAC1 and VDAC3 remained unchanged in VDAC2-KO cells derived from both cell lines (data not shown). Additionally, DGEseq analysis of VDAC2-KO vs CTR cells identified 17 and 50 genes differentially expressed in KMS12PE and KMM1 VDAC2-KO, respectively, but none, excepting VDAC2 itself, was shared between both cell lines (data not shown). Proteins differentially expressed between the 2 groups along with the other multidomain effector protein BAX were selected for verification using immunoblot analysis (Figure 2B, upper panel). In agreement with the proteomic results, only BAK was significantly decreased in VDAC2-KO cells (Figure 2B, upper and lower panel), BAX and all the other members of the BCL2 family remained constant (Figure 2B). The decreased BAK protein remained exclusively localized at the mitochondria (data not shown). In addition, qPCR analysis confirmed the diminution of VDAC2 mRNA while BAK and BAX mRNA remained constant in VDAC2-KO cells (Figure 2C). These results demonstrated that VDAC2 regulates BAK expression exclusively at the protein level.

[0103] To assess the mitochondrial impact of VDAC2 loss accompanied by the decrease of BAK, we performed BH3 profiling. Interestingly, VDAC2-KO cells lost their global mitochondrial priming, as well as the specific dependence to MCL1 and BCL2. Indeed, cytochrome c release induced by BIM, MSI peptides or Venetoclax was completely abolished in both VDAC2-KO cell lines (Figure 2D). Together these results support the notion that VDAC2 controls the mitochondrial apoptotic threshold, whether this effect is mainly through the protein effector BAK remains to be determined. To address this question, we generated KMM1 BAK -KO and BAX-KO. Then, we treated these cells with S63845 alone or in combination of Efsevin. Interestingly, Efsevin failed to sensitize myeloma BAK-KO cells to S63485 while it still significantly increased cell death in BAX-KO cells, further supporting a selective role for VDAC2 in inhibiting exclusively BAK and not BAX in MM cells (Figure 2E). It is worth noting, that while Efsevin induced significant sensitization to BH3 mimetics in cells displaying considerable levels of VDAC2 (Figure 1A) it also significantly heightened the global mitochondrial priming, as observed in KMS12PE and KMM1 challenged with the Bim peptide (Figure 2F). The MCL1 and BCL2 dependences were also increased by Efsevin in these cells without modifying the protein levels of BAK, BAX or VDAC2 (Figure 2F and 2G). Moreover, the anti-apoptotic BCL2 members MCL1, BCL2 and BCLXL remained constant upon Efsevin treatment at the indicated time points in both cell lines (data not shown). Together these data not only highlight the regulation of mitochondrial apoptosis by VDAC2 but also indicate that this function relies on its interplay with BAK.

[0104] Reciprocal protection between VDAC2 and BAK at the MOM

[0105] The role of VDAC2 in the stabilization of BAK has been already proposed (7), yet the mechanism behind the strong decrease in BAK protein levels in VDAC2-KO cells has never been documented before. This singularity observed in our human myeloma model prompted us to investigate if the main protein degradation pathways were implicated in the decrease of BAK protein in the absence of VDAC2. As shown in Figure 3 A, proteasome inhibition by Bortezomib or the blockage of lysosomal degradation by Bafilomycin were not enough to recover BAK protein levels. Interestingly, the concomitant inhibition of both pathways rescued BAK protein. To go deeper into the VDAC2 / BAK interplay, we analyzed VDAC2 protein expression in cells singly deficient for BAK. Strikingly, BAK-KO clones exhibited decreased levels of VDAC2 (Figure 3B). Furthermore, we also identified the implication of the proteasome and the lysosomal protein degradation pathways in the decrease of VDAC2 in BAK-KO cells (Figure 3C). Although to a lesser extent than VDAC2 KO cells, the impact of BAK knock-out in VDAC2 protein levels, illustrates for the first time the reciprocal regulation between both mitochondrial proteins. Accordingly, analyzing a collection of HMCLs, we demonstrated that VDAC2 protein levels were directly correlated with BAK protein expression (p=0.0212; r=0.5113), whereas no correlation was observed with BAX protein levels (p=0.8403; r=0.04812) (Figure 3D).

[0106] Together these results support the notion that VDAC2 and BAK finely regulated each other and shed light on the preferential role of VDAC2 in regulating BAK instead of BAX in MM cells

[0107] Efsevin sensitizes myeloma cells to cell death induced by BH3 mimetics

[0108] To confirm the impact of Efsevin on mitochondrial priming, we combined Efsevin with S63845 or Venetoclax in 9 Human Myeloma Cell Lines. LD50 values were determined after 96 hours incubation with Efsevin, with S63845 or Venetoclax added for the last 24 hours. Figure 4A shows that Efsevin significantly sensitized 4 out of 6 HMCLs (BCN, KMS12PE, MM1S and KMM1) to S63845, leading to a 1.7- to 4.6-fold reduction in S63845 LD50 (Table 3A). Interestingly, Efsevin overcame the resistance of BCN cell line to S63845 BH3 mimetic. Bliss synergy scores in KMM1 (39.7 ± 2.9) and KMS12PE (34.3 ± 2.3) confirmed a strong synergy between Efsevin and S63845 (Figure 4B).

[0109] Similarly, we also found a significant synergy between Efsevin and Venetoclax in 4 t(l l;14) HMCLs (KARPAS620, MDN, XG5 and KMS12PE), resulting in a 1.8- to 6.8-fold decrease in Venetoclax LD50 (Figure 4C, Table 3B), with a Bliss score of 39.7 ± 2.9 in KMS12PE (Figure 4D). Notably, Efsevin enabled the achievement of Venetoclax LD50 values well below 100 nM, which is the highest ex vivo concentration associated with a favorable in vivo clinical response to Venetoclax (Matulis SM. Leukemia. 2019 May;33(5): 1291-6)

[0110] Interestingly, the combination of Efsevin and BH3 -mimetics was effective irrespective of TP53 status. Sensitization to MCL1 or BCL2 BH3-mimetics was observed in both TP53+ / + and TP53- / mut HMCLs (Table 3A and 3B). Consistently, Efsevin reduced S63845 LD50 values from 80nM to 40nM in the isogenic NCI-H929 TP53- / -, although this remained higher than the S63845 LD50 observed in the NCI-H929 TP53+ / + cells (Figure 4E).

[0111] Since MM is mainly dependent on MCL1 for survival (12,13), we assessed the effect of Efsevin (15 pM) in combination with S63845 (25 nM) in myeloma cells from 27 consecutive patients, 14 at diagnosis and 13 at relapse from the MYRACLE cohort. Median cell death induced by Efsevin, S63845 and their combination was 6.9%, 31.7%, and 54.7%, respectively (Figure 5A). Because it was not possible to perform synergy assays in patient samples, we analyzed the differences between the expected and the observed values (Figure 5B) The combination of S63845 and Efsevin was indeed effective in 96% of the samples, being additive (-10%< observed minus expected death <10%) in 16 samples (59%) and synergistic (observed minus expected death >10%) in 10 samples (37%).

[0112] Efsevin sensitizes Myeloma cell lines to the BH3 mimetic targeting BCLXL (Al 155463)

[0113] ANBL6 and MDN human myeloma cell lines were incubated 96 hours with Efsevin and a BH3 mimetic targeting BCLXL (Al 155463) was added for the last 24 hours. Efsevin significantly decreased the Al 155463 LD50 in both cell lines (Figure 6).

[0114] Conclusion:

[0115] We have demonstrated that VDAC2 is a non-BCL2 family protein that plays a role in apoptosis regulation controlling BAK effector rather BAX effector in MM cells. Accordingly, VDAC2 protein expression correlated with the protein levels of BAK but not with BAX in MM. Interestingly, VDAC2 KO cells lost their global and specific mitochondrial priming, probably due to the drastic BAK decrease observed in these cells. We demonstrated for the first time that Efsevin, a VDAC2 compound, identified through library screening for chemicals capable to reverse the arrhythmia phenotype in the tremblor zebrafish model, was able to modulate the apoptotic response to BH3 mimetics S63845 (MCL1 specific) or Venetoclax (BCL2 specific) in MM cell lines. This effect was directly correlated to the level of VDAC2 protein expression and independent of the p53 status of MM cells. The sensitization effect of Efsevin on S63845 apoptosis induction was also confirmed ex-vivo in myeloma cells from patients’ samples as well as a Mantle cell lymphoma cell line. Efsevin enhanced global and specific mitochondrial priming, indicating that VDAC2 regulates apoptosis upstream mitochondrial outer membrane permeabilization. REFERENCES:

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Claims

CLAIMS:

1. A combination of a VDAC2 modulator and a BH3-mimetic compound for use in the treatment of a cancer in a subject in need thereof.

2. A i) VDAC2 modulator and ii) a BH3-mimetic compound, 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 Multiple myeloma or a Mantle cell lymphoma.

4. A combination for use according to the claim 1 or a combined preparation for use according to the claim 2 wherein the VDAC2 modulator is efsevin and the BH3 -mimetics compound is an inhibitor of BCL2 like Venetoclax or and inhibitor of MCL1 like S63845.

5. A method for treating a cancer comprising administering to a subject in need thereof a therapeutically effective amount of a VDAC2 modulator and a BH3 -mimetic compound.

6. A therapeutic composition comprising a VDAC2 modulator and a BH3-mimetic compound for use in the treatment of cancer in a subject in need thereof.

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