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Mitochondria play a multifaceted role in tumorigenesis through regulation of energy production, biomass, redox state, and engagement of cell death pathways. The mitochondrial Q (coenzyme Q10)-pool facilitates electron transport from Complexes I and II to III and is essential for regulating these activities. Therefore, altering mitochondrial Q-pool homeostasis represents a potential therapeutic strategy in cancer. BPM 31510 is a Coenzyme Q10 containing lipid nanodispersion currently in clinical trials for pancreatic cancer. Here, we used BPM 31510 to assess how modulation of mitochondrial Q-pool homeostasis impacts mitochondrial electron transport chain function to activate regulated cell death. Pancreatic cancer cell lines (MIA PaCa-2 and Panc-1) represent models with sensitivity to BPM 31510 both in vitro and in vivo. Treatment with BPM 31510 (EC50 dose, 24 h) resulted in significant mitochondrial enrichment of CoQ10 compared to other subcellular compartments, and quantitatively, CoQ10 levels were 100 times higher in mitochondria isolated from BPM 31510 treated cells over untreated controls (MIA PaCa-2 untreated, 0.31 nmol/mg; treated, 39.4 nmol/mg; Panc1 untreated, 0.21 nmol/mg; treated, 29.3 nmol/mg). Alterations in mitochondrial respiration characterized by dose-dependent decreases in succinate- or glycerol-3-phosphate-fueled respiration were observed in cells treated with BPM 31510, while pyruvate or TMPD/ascorbate-fueled respiration was only modestly affected, suggesting that BPM31510 specifically impairs respiratory responses dependent on Complexes II/III. Moreover, in the presence of multiple mitochondrial substrates, total respiratory capacity was decreased and reliance on pyruvate (Complex I)-fueled respiration was increased with BPM 31510 treatment, indicative of bioenergetic remodeling. Concomitantly to BPM 31510-dependent changes in mitochondrial respiratory responses, BPM 31510 exposure increased oxidation of the reactive oxygen species (ROS) probes, CellROX Green and DCF-DA, increased oxidized glutathione, and decreased levels of the cellular reducing equivalent NADPH. Importantly, BPM 31510-induced death could be partially rescued by agents which alleviate electron transport chain impairment linking respiratory function to the anti-cancer mechanism of action of BPM 31510. Together, these data indicate that BPM31510 directly impairs the mitochondrial Q-pool and respiratory function resulting in oxidative stress and consequential cell death and thus provide mechanistic understanding of the anti-cancer activity of BPM31510. Citation Format: Pallavi Awate, Tulin Dadali, Ryan Ng, Saie Mogre, Anne R. Diers, Hannah Rockwell, Justice McDaniel, Emily Chen, Fei Gao, Michael Kiebish, Stephane Gesta, Vivek Vishnudas, Niven R. Narain, Rangaprasad Sarangarajan. Coenzyme Q10 (BPM31510-IV in clinical trials) increases mitochondrial Q-pool and modulates electron transport chain function to elicit cell death in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3530.

BPM31510 is a metabolic modulating agent composed of a parenteral nanodispersion of ubidecarenone which is currently in clinical studies for glioblastoma. Glioblastoma is a highly metabolic and aggressive malignancy with limited treatment options and dismal median survival. Temozolomide (TMZ) as a first line treatment option, however, 90% of recurrent gliomas acquire TMZ chemoresistance. Recently, acquisition to TMZ resistance has been correlated to alterations in mitochondrial metabolism. Thus, in the present study we sought to investigate whether BPM31510 could elicit anti-cancer activity in TMZ naïve and TMZ-chemoresistant glioma models. In vitro, in a 2D model BPM31510 treatment demonstrated anti-cancer activity in a panel of glioma cell lines (rat C6 and human U251-MG and U87-MG), and this effect was translatable in spheroidal 3D cultures. Importantly, in an aggressive rat C6 orthotopic glioma model, treatment with BPM31510 (50mg/kg/day, b.i.d) starting between 4 and 8 days post-implantation resulted in a 32% cure rate compared to 0% in controls (P < 0.001, Fisher's exact test), demonstrating an improved survival (P < 0.01, log rank survival), despite producing a minimal change in median survival (13 vs. 12 days). A marked increase in caspase3 staining was observed in tumors from BPM31510 treated animals compared to controls assessed at a similar time point post-tumor implantation, suggesting a strong apoptotic effect of this agent in vivo. Next, BPM31510 was examined in a cellular model of acquired TMZ resistance (TMZ-R) generated by exposing parental (chemosensitive naïve) U251-MG and U87-MG cells to increasing concentrations of TMZ for 9-12 months. Similar to parental cells, BPM31510 displayed anti-cancer activity in both TMZ-R cell models, as decreased cell viability and an increase in the percentage of apoptotic cells was observed upon BPM31510 treatment. Consistent with prior studies, compared to parental cells, TMZ-R cells demonstrated metabolic rewiring characterized by increases in mitochondrial function parameters and decreased extracellular acidification rate, indicative of glycolytic flux. Regardless of chemosensitivity, BPM31510 decreased mitochondrial substrate oxidation (e.g., succinate, glycerol-3-phosphate) at doses which induce cell death. Concomitantly, increases in the reactive oxygen species production were observed with BPM 31510 treatment in both parental and TMZ-R cell lines. Together, these data define a link between regulation of mitochondrial function and the anti-cancer activity of BPM31510 in both TMZ chemo-sensitive and resistant glioblastoma models, demonstrating a distinct approach in targeting mitochondrial metabolism for the treatment of this clinically intractable disease. Citation Format: Tulin Dadali, Shreya Kulkarni, Ryan Ng, Pallavi Awate, Saie Mogre, Anne R. Diers, Taichang Jang, Milton Merchant, Jiaxin Sun, Stephane Gesta, Khampaseuth Thapa, Seema Nagpal, Lawrence Recht, Niven R. Narain, Rangaprasad Sarangarajan. BPM 31510, a clinical stage metabolic modulator, demonstrates therapeutic efficacy in glioblastoma models of temozolomide chemo-sensitive and resistance by targeting mitochondrial function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 873.

BPM31510, a parenteral nanodispersion of ubidecarenone that is in clinical testing for solid tumors, effectuates an anti-cancer effect in highly metabolic cancers by eliciting an anti-Warburg effect. Intraperitoneal (i.p.) administration of BPM31510 reverses paraplegia in a rat CNS leukemia model demonstrating the bioavailability of the drug to the central nervous system. The study describes data demonstrating the therapeutic utility of BPM31510 in glioblastoma multiforme (GBM). BPM31510 decreased cell proliferation rates in patient derived GBM cell lines compared to temozolamide (TMZ), pretreatment with BPM31510 followed by TMZ challenge was associated with significant synergy in reducing cell proliferation compared to monotherapy. In a C6 glioma allograft rat model treatment with BPM31510 alone (n=30/group) (i.p., 50mg/kg, b.i.d) increased overall survival compared to untreated control group. BPM31510 in combination with procarbazine (oral, 60mg/kg; d 8, 21) and vincristine (IV, 1.4mg/kg; d 8, 29) significantly improved overall survival compared to BPM31510 alone or chemotherapy. Furthermore, long term survival was achieved in four of twenty rats with C6 gliomas receiving BPM31510 doses between 10 and 50 mg/kg compared with none treated with either vehicle or irradiation (P < 0.02, Fisher exact test). Interestingly, the long term survival persisted for over two months after all treatment was discontinued; MR imaging confirmed the presence of static masses in these long term survivors. Collectively, the data demonstrate that BPM31510 has a robust effect in a preclinical model of GBM. Intravenous BPM31510 is currently in Phase 1/2 clinical trial for TMZ+BEV refractory GBM patients. Citation Format: Stephane Gesta, Semma Nagpal, Tulin Dadali, Milton Merchant, Taichang Jan, Anne R. Diers, Michael Kiebish, Joaquin Jiminez, Vivek K. Vishnudas, Niven R. Narain, Rangaprasad Sarangarajan, Lawrence Recht. BPM31510, a clinical stage metabolic modulator demonstrates therapeutic efficacy in an in vivo C6 rat glioma model and synergizes with temozolomide [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4067. doi:10.1158/1538-7445.AM2017-4067

BPM 31510, a clinical stage nanodispersion of ubidecarenone, demonstrates anti-tumor effects by eliciting an anti-Warburg metabolic switch in cancer. Previous studies in an immune compromised PaCa2 xenograft model has unequivocally demonstrated significant efficacy of BPM 31510 on tumor volume and survival. The fundamental property of BPM 31510 to influence mitochondrial bioenergetics and the recognized interplay between T cell metabolism and maturation prompted investigation into the effects of BPM 31510 on T lymphocyte functions in eliciting anti-cancer effects. In this study BPM 31510 selectively influenced activation and maturation of T cells in murine peripheral blood mononuclear cells (PBMCs). Moreover, in addition to determining changes in the CD3+ population, changes in surface expression of PD-1 and CTLA4 along with the IFN-γ secretion were examined. Murine cancer cell lines exposed to BPM 31510 were associated with variable sensitivity with highly metabolic tumor types being most sensitive. Next, the anti-cancer activity of BPM 31510 in an in vivo immunocompetent syngeneic Pan02 rodent model was investigated. Murine Pan02 pancreatic cancer cells were implanted subcutaneously into C57BL/6 mice. Tumors with mean volume of 80 mm3 were treated twice a day with vehicle control or BPM 31510 at 25, 50, 100 mg/kg, administered intraperitoneal. Tumor volumes were measured every 4 days. At day 21 post treatment, tumors were harvested and analyzed for the level of infiltrating immune cells by immunofluorescent staining with CD8+ for T cells and F4/80 for tumor macrophages. These results demonstrate a dose-dependent reduction in tumor volume following 21 days of BPM 31510 treatment. In summary, BPM 31510 exerts potent anti-tumor effects through its dual function of modulating tumor cell metabolism and potentially influencing immune check-point to improve overall survival outcomes. Citation Format: Shiva Kazerounian, Aishwarya Sarma, Nidhi Gaur, Maria D. Nastke, Tulin Dadali, Anne R. Diers, Stephane Gesta, Vivek K. Vishnudas, Rangaprasad Sarangarajan, Niven R. Narain. BPM 31510, a clinical stage candidate demonstrates potent anti-tumor effect in an immune-competent syngeneic pancreatic cancer model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5576. doi:10.1158/1538-7445.AM2017-5576

Dysregulated mitochondria play a multifaceted role in tumorigenesis through regulation of energy production, biomass, redox state, and engagement of cell death pathways. Perturbations in mitochondrial fluxes (i.e inhibition of electron chain complexes activity, impaired electron flow) have substantial effects on cell viability, suggesting that targeting mitochondrial function could be effective for therapeutic response in cancer. BPM31510, containing oxidized coenzyme Q10 elicits an anti-Warburg effect is currently in phase II clinical trials for solid tumors. Previously, we have demonstrated the anti-cancer properties of BPM31510 in breast and pancreatic xenograft models. Here, we examined the mechanism of action of BPM31510 in vitro. Using a multi-cancer cell panel, BPM31510 was shown to be consistently and selectively cytotoxic to cancer cells, compared to normal and non-tumorigenic controls, and sensitivity did not correlate to cell doubling time or mutational status. Treatment with BPM 31510 (EC50) in breast and pancreatic cancer cells resulted in a time- and dose-dependent decrease in mitochondrial membrane potential which preceded an increase in early and late apoptosis cells, suggesting BPM31510 initiates a mitochondrial mediated cell death pathway. Using a fluorescently labeled CoQ10 we were able to trace the subcellular location of the CoQ10, which predominantly accumulates in mitochondria and lipid droplets in a time dependent manner. Additionally, the mitochondrial enrichment of CoQ10 is accompanied by morphological changes that varied amongst the different cancer cell types. As CoQ10 is a redox molecule required for electron transfer activity between complexes, we hypothesized that disruption of Q-pool homeostasis would alter complex activity. To investigate this, Complex driven respiration was measured in cells treated with BPM31510 and compared to untreated. Alterations in mitochondrial respiration characterized by a dose-dependent decrease in succinate (Complex II) and glycerol-3-phosphate (Complex III)-fueled respiration were observed in cells treated with BPM31510, while no changes were seen in pyruvate driven respiration (Complex I), suggesting that BPM31510 specifically impairs respiration responses that are more dependent on Q-pool functionality. As impairment of the electron transport chain increases intracellular oxidative stress, we next investigated if BPM 31510 treatment increases ROS levels. After 24h treatment, BPM31510 significantly increased ROS levels in treated cancer cells compared to untreated. Furthermore, BPM31510 induced death could be in part prevented by co-treatment with antioxidants. Together, these data demonstrates BPM31510 has anti-cancer activity in multiple cancer cell types and define a unique and novel functional link between mitochondrial Q-pool disruption and the mechanism of action of BPM31510. Citation Format: Tulin Dadali, Katerina Krumova, Anne R. Diers, Pallavi Awate, Ryan Ng, Arleide Lee, Stephane Gesta, Vivek K. Vishnudas, Rangaprasad Sarangarajan, Niven R. Narain. BPM31510 modulates mitochondrial complex activity to influence oxidative stress in effectuating cell death in multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1497. doi:10.1158/1538-7445.AM2017-1497

By means of a phenomenon termed “the Warburg effect,” tumor cells shift their energy production by mitochondrial oxidative phosphorylation to aerobic glycolysis, resulting in the upregulation of glucose consumption and increased cellular oxidative and nitrosative stress. To compensate for such toxic levels of ROS/RNS, cancer cells rely heavily on their antioxidant defense mechanisms, which are largely controlled by the NAD(P)/NAD(P)H redox partners. We found that the modulation of NAD metabolism in vivo, specifically via the deletion of the NAD glycohydrolase CD38, resulted in increased production of intrinsic ROS and increased DNA damage following exposure to chemotherapeutics. Furthermore, in vitro experiments showed that CD38 knockdown in CD38-expressing tumor cells prevented the generation of stable transfectants, highlighting a role for CD38 in tumor cell survival. In light of these findings, we hypothesized that pharmacological inhibition of CD38 may be an effective therapy for the treatment of hematological cancers, in particular those which uniformly overexpress CD38, such as MM and chronic lymphocytic leukemia. Indeed, treatment of human MM cell lines LP-1 and KMS-12-PE with CD38 antagonists sensitized the cells to standard ROS-inducing chemotherapeutics. We conducted a high-throughput screening (HTS) campaign of over two hundred thousand unique and non-proprietary lead-like compounds using an optimized and miniaturized HTS based on a luminescent NAD quantitation platform. Five hundred active hits were analyzed for toxicity using a cell-based HTS assay purposely designed with CD38-negative HEK293 cells to avoid elimination of desirable compounds toxic to CD38-positive cells. Hits with non-specific properties, such as PAINS (Pan Assay Interference Compounds), were removed by computational filtering, and the remaining compounds were tested for inhibition of human CD38 activity in cells. The last phase of the compound progression pathway involved testing for non-selective inhibition of other NAD-consuming enzymes, namely Poly(ADP-ribose) polymerase-1, and Sirtuin-1, which led to the identification of two distinct chemical series that exhibit >10-fold selectivity for human CD38. Hit-to-lead chemistry is currently underway to synthesize key analogs by rational drug design. In summary, our data suggests that CD38 is an antioxidant protein selectively used to maintain a cellular redox balance, and proposes that targeting the enzymatic activity of CD38 may be a novel therapeutic strategy for chemosensitizing hematological cancers. Our HTS campaign efforts are paving the way for the discovery and development of potent and selective small-molecule inhibitors of CD38. Citation Format: Davide Botta, Tulin Dadali, Betty J. Mousseau, Fen Zhou, Michael D. Schultz, Esther Zumaquero, Anna Manouvakhova, Melinda I. Sosa, Sara N. McKellip, LaKeisha Woods, Nichole A. Tower, Larry J. Ross, Lynn Rasmussen, E. Lucille White, Indira Padmalayam, Wei Zhang, Maaike Everts, Corinne E. Augelli-Szafran, James R. Bostwick, Mark J. Suto, Frances E. Lund. High-throughput screening efforts for the identification of selective and potent inhibitors of CD38 for the treatment of hematological cancers. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-055.

De-regulated lipid homeostasis is a key feature of the altered metabolic phenotype observed in cancer, particularly in the adipose-rich microenvironment of breast tumors. Mitochondria serve as a central hub for lipid metabolism through mitochondrial β-oxidation pathways and biosynthesis through production of citric acid cycle-derived citrate and the activity of ATP citrate lyase, the rate-limiting enzyme in lipid biosynthesis. We have previously demonstrated BPM 31510 ability to effectuate Warburg switch from glycolysis to mitochondrial oxidative phosphorylation resulting in activation of apoptosis in multiple cancers including triple negative breast cancer. The present investigated the role of BPM 31510 in influencing lipid metabolism in cancer cells to arbitrate its anti-cancer effect. Cell viability was assessed in MDA-MB231 and SkBr-3 breast cancer cells exposed to BPM 31510 alone or in combination with fatty metabolism inhibitors including C75, etomoxir, and trimetazidine, inhibitors of fatty acid synthase (FASN), carnitine palmitoyltransferase 1 (CPT-1), and β-oxidation, respectively, as well as a more pleiotropic fatty acid metabolism modulator, metformin. Breast cancer cells were more sensitive to BPM 31510 when treated in combination with C75, etomoxir, and trimetazidine as indicated by a left-shift in the BPM 31510 dose-response curve in both MDA-MB231 and SkBr-3 cells. In contrast, combined treatment with metformin did not alter cytotoxic responses to BPM 31510, indicating specificity for responses in fatty acid metabolism pathway modulation. Interestingly, BPM31510 treatment was associated with a dose- and time-dependent increase in mRNA expression of fatty acid metabolism gene products (FASN, CPT1, ACSL1) and accumulation of the triglyceride backbone, glycerol, in MDA-MB-231 cells. Structural lipidomic analysis used to assess the metabolic fate of BPM 31510 liposomal formulation components demonstrated that these were readily incorporated into prevalent diacyl-glyerol (DAG) and triacyl-glycerol (TAG) species along with de novo fatty acid species such as palmitate. Together, these results demonstrate that BPM 31510 alters endogenous and exogenous lipid homeostasis in breast cancer cells and rationale for potential combination with fatty acid metabolism inhibitors for anti-cancer therapy. The results expands on BPM 31510 anti-cancer mechanism, suggesting a central role in arbitrating the convergence of glycolysis, glucose and fatty acid oxidation pathways within the cancer cell metabolism network. Citation Format: Bianca Jambhekar, Tulin Dadali, Anne R. Diers, Fei Gao, Hannah Rockwell, Emily Chen, Stephane Gesta, Vivek K. Vishnudas, Michael A. Kiebish, Niven R. Narain, Rangaprasad Sarangarajan. The anti-Warburg agent BPM 31510 arbitrates fatty acid metabolism in eliciting an anticancer response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1014.

Although only 15-20% of total breast cancer diagnoses are of the triple-negative breast cancer (TNBC) subtype, they account for a significant portion of the mortality rate due to their more aggressive phenotype and a high risk of reoccurrence. Metabolic rewiring supports breast cancer progression and metastasis, particularly in ER-negative and triple-negative (TNBC) breast tumors. Thus, we examined the effects of BPM 31510, a metabolic-modulating agent in clinical trials for solid tumors, in in vitro and in vivo ER-negative and TNBC models. BPM 31510 EC50/EC>90 values were determined for a panel of the breast cancer cell lines and compared to non-tumorigenic MCF12A cells in vitro, and the MDA-MB231 and SkBr-3, TNBC and ER-negative models respectively, were found to be the most sensitive to BPM 31510. Treatment with BPM 31510 (EC50 and EC>90 doses) resulted in a time- and dose-dependent decrease the viable cell population (PI- and Annexin V-negative) and a concomitant increase in cells in early and late apoptosis (PI-negative and PI-positive Annexin V-positive cells, respectively), suggesting that BPM 31510 activates regulated cell death pathways. Consistent with the in vitro data, MDA-MB231 tumor-bearing mice had smaller tumors after 30 days of treatment with BPM 31510 and increased cleaved caspase 3 staining in resected tumors. In vitro, BPM 31510-dependent breast cancer cell death was preceded by mitochondrial membrane potential depolarization (TMRE flow cytometry) and alterations in mitochondrial respiration characterized by a consistent, dose-dependent decrease in succinate (Complex II)-fueled respiration with more varied responses to BPM 31510 in cells provided the Complex I substrates (pyruvate or palmitoyl carnitine). To investigate the role of Complex II in BPM 31510-mediated cell death, pharmacological inhibitors of the dicarboxylate site (malonate) and Qp site (atpenin A5) of Complex II were used in combination with BPM 31510 to assess the resultant effects on cell death in MDA-MB231 cells. Co-treatment with malonate significantly attenuated BPM 31510-mediated cell death while atpenin A5 did not affect BPM 31510-induced cell death, indicating succinate oxidation at the dicarboxylate site of Complex II is required, in part, for induction of cell death by BPM 31510. Together, these data demonstrate BPM 31510 has a potent anti-cancer activity in preclinical breast cancer models and define a functional link between Complex II activity and the mechanism of action for BPM 31510. Citation Format: Tulin Dadali, Anne R. Diers, Arleide Lee, Ezer Benaim, Joaquin J. Jimenez, Stephane Gesta, Vivek K. Vishnudas, Rangaprasad Sarangarajan, Niven R. Narain. BPM 31510-induced alteration in Complex II activity is functionally linked to cell death activation pathway in a preclinical model of triple-negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 208.

Pancreatic adenocarcinoma (PanCa) is associated with poor prognosis and overall survival. Current first-line therapeutics are cytotoxic agents targeting DNA based mechanistic end-points for efficacy, their use limited by dose associated toxicity. There is a critical need for therapeutics with novel mechanisms amenable to combination with current standard-of-care chemotherapy to improve outcomes. BPM 31510 is a drug that targets cellular metabolism networks, effectuating an anti-Warburg effect in cancer. The documented high metabolic phenotype observed in PanCa provided rationale for investigation of BPM 31510 alone and in combination with gemcitabine in in vitro and in vivo PanCa models. Based on BPM 31510 EC50/EC>90 values for MIA-PaCa-2 and Panc-1 PanCa cell lines in vitro, the PanCa cells were significantly more sensitive to BPM 31510 compared to fibroblasts. BPM 31510 treatment in a time- and dose-dependent manner decreased the PI- and Annexin V-negative (viable) population and a concomitant increase in the percentage of Annexin V-positive cells, with PI indicative of early and late apoptosis. The anti-cancer activity of BPM 31510 was assessed in vivo using MIA-PaCa-2 tumor-bearing immune-compromised mice. Treatment with increasing doses of BPM 31510 (0.5-50 mg/kg IP, 3X/week) significantly improved survival outcomes, with the highest dose extending median survival by more than 36 days compared to saline control. Moreover, combined treatment with BPM 31510 and gemcitabine (150 mg/kg IV, 1X/week, given on cycles, 3 weeks on 1 week) resulted in further extension of median survival over either treatment alone. The mechanistic underpinnings for the enhanced efficacy of combination treatment were explored in vitro. In MIA-PaCa-2 cells, co-treatment with BPM 31510 and gemcitabine increased indices of regulated cell death higher than observed for either treatment alone. In contrast, although treatment with either BPM 31510 or gemcitabine alone increased caspase-3 activity, co-treatment did not enhance caspase-3 activation, suggesting that BPM 31510 augments gemcitabine cytotoxicity through independent mechanisms. In fact, BPM31510, and not gemcitabine, increased the mitochondrial uncoupling efficient ratio and Stateapparent in MIA-PaCa-2 cells. Nonetheless, co-treatment with BPM 31510 and gemcitabine synergistically decreased the mitochondrial membrane potential (ΔΨm) in cells prior to cell death. Taken together, these data indicate that BPM 31510-driven bioenergetic alterations are separate from the effects of gemcitabine; however, their effects converge at the mitochondrion to dissipate ΔΨm and activate regulated cell death. The data suggests that combination of BPM 31510 with gemcitabine in pancreatic cancer will effectuate an efficacy response via independent mechanisms with improvement in therapeutic outcome. Citation Format: Tulin Dadali, Anne R. Diers, Rakib Ouro-Djobo, Justin Bourdelais, Ezer Benaim, Bianca Jambhekar, Tony E. Walshe, Vivek K. Vishnudas, Joaquin J. Jimenez, Rangaprasad Sarangarajan, Niven R. Narain. BPM 31510 synergizes gemcitabine efficacy in pancreatic adenocarcinoma via mechanism independent of its anti-Warburg influence on metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 212.

The cellular redox state, which is controlled by the NAD/NADH and NADP/NADPH redox partners, is critical for numerous cellular activities including energy metabolism, signaling and transcription. Hematopoietic tumors have higher levels of intrinsic reactive oxygen species (ROS) due to increased NAD biosynthesis and metabolism required to further stimulate tumorigenesis. To compensate for such toxic levels of ROS, cancer cells rely heavily on their antioxidant defense mechanisms to prevent ROS-induced death/senescence and to promote neoplastic cell behavior. It has been postulated that pairing drugs that block the tumor antioxidant pathways with ROS-inducing chemotherapeutics may kill tumors more effectively. We hypothesized that the modulation of NAD metabolism dramatically alters the cellular redox state, and that drugs affecting NAD metabolism may be effective anti-cancer therapeutics. Using an in vivo model of Streptozotocin (STZ)-induced oxidative stress, we show that the NAD glycohydrolase CD38, which is expressed by many B-cell malignancies, acts as an antioxidant by lowering intrinsic ROS levels and by protecting cells from extrinsic ROS, DNA alkylating chemotherapeutics and ROS-induced senescence. In addition, we show that the overexpression of human CD38 increases the viability of murine Ba/F3 pro-B cells cultured in the absence of the survival factor IL-3. To examine the role of CD38 in tumor cell growth and survival, we reduced CD38 expression in human multiple myeloma KMS-12-PE cells by RNA interference (RNAi) targeting three separate regions of the CD38 coding sequence. The knockdown of CD38 expression resulted in a significant decrease in the number of stable transformants generated, suggesting that CD38 plays a key role in tumor cell survival. Lastly, we show that CD38 antagonists synergize with the chemotherapeutic agent Melphalan to block tumor cell survival in vitro. In summary, this work suggests that CD38 is an antioxidant protein selectively used to maintain a cellular redox balance, and proposes that targeting the enzymatic activity of CD38 may be a novel therapeutic strategy for chemosensitizing B-cell malignancies. We are currently conducting a high-throughput screening (HTS) campaign of over 200 thousand small-molecule compounds to identify inhibitors of CD38 that can be used in combination with standard ROS-inducing chemotherapies to treat CD38+ B-cell malignancies, such as multiple myeloma and chronic lymphocytic leukemia. Citation Format: Davide Botta, Tulin Dadali, Betty Mousseau, Anna Manouvakhova, Melinda I. Sosa, Sara N. MKellip, LaKeisha Woods, Nichole A. Tower, Larry J. Ross, Lynn Rasmussen, E Lucile White, James R. Bostwick, Frances E. Lund. Modulating the cellular redox state by targeting the NAD glycohydrolase CD38: A novel therapeutic approach for chemosensitizing B-cell malignancies. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1242. doi:10.1158/1538-7445.AM2015-1242