Richa Bajpai

Targeting the Metabolic Plasticity of Multiple Myeloma with FDA Approved Ritonavir and Metformin

Purpose: We have previously demonstrated that ritonavir targeting of glycolysis is growth inhibitory and cytotoxic in a subset of multiple myeloma cells. In this study, our objective was to investigate the metabolic basis of resistance to ritonavir and to determine the utility of cotreatment with the mitochondrial complex I inhibitor metformin to target compensatory metabolism. Experimental Design: We determined combination indices for ritonavir and metformin, impact on myeloma cell lines, patient samples, and myeloma xenograft growth. Additional evaluation in breast, melanoma, and ovarian cancer cell lines was also performed. Signaling connected to suppression of the prosurvival BCL-2 family member MCL-1 was evaluated in multiple myeloma cell lines and tumor lysates. Reliance on oxidative metabolism was determined by evaluation of oxygen consumption, and dependence on glutamine was assessed by estimation of viability upon metabolite withdrawal in the context of specific metabolic perturbations. Results: Ritonavir-treated multiple myeloma cells exhibited increased reliance on glutamine metabolism. Ritonavir sensitized multiple myeloma cells to metformin, effectively eliciting cytotoxicity both in vitro and in an in vivo xenograft model of multiple myeloma and in breast, ovarian, and melanoma cancer cell lines. Ritonavir and metformin effectively suppressed AKT and mTORC1 phosphorylation and prosurvival BCL-2 family member MCL-1 expression in multiple myeloma cell lines in vitro and in vivo. Conclusions: FDA-approved ritonavir and metformin effectively target multiple myeloma cell metabolism to elicit cytotoxicity in multiple myeloma. Our studies warrant further investigation into repurposing ritonavir and metformin to target the metabolic plasticity of myeloma to more broadly target myeloma heterogeneity and prevent the reemergence of chemoresistant aggressive multiple myeloma. Clin Cancer Res; 21(5); 1161–71. ©2014 AACR.

In Silico Modeling-based Identification of Glucose Transporter 4 (GLUT4)-selective Inhibitors for Cancer Therapy.

Background: GLUT4 is a promising target for multiple myeloma therapy. Results: In silico modeling of GLUT4, followed by virtual screening and validation, led to the identification of GLUT4-selective inhibitors. Conclusion: Despite significant homology between GLUT1 and GLUT4, we have identified GLUT4-selective inhibitors exhibiting cytotoxicity in myeloma. Significance: Potent selective GLUT4 inhibitors are promising cancer therapeutics with both cytostatic and chemosensitizing properties warranting further development. Tumor cells rely on elevated glucose consumption and metabolism for survival and proliferation. Glucose transporters mediating glucose entry are key proximal rate-limiting checkpoints. Unlike GLUT1 that is highly expressed in cancer and more ubiquitously expressed in normal tissues, GLUT4 exhibits more limited normal expression profiles. We have previously determined that insulin-responsive GLUT4 is constitutively localized on the plasma membrane of myeloma cells. Consequently, suppression of GLUT4 or inhibition of glucose transport with the HIV protease inhibitor ritonavir elicited growth arrest and/or apoptosis in multiple myeloma. GLUT4 inhibition also caused sensitization to metformin in multiple myeloma and chronic lymphocytic leukemia and a number of solid tumors suggesting the broader therapeutic utility of targeting GLUT4. This study sought to identify selective inhibitors of GLUT4 to develop a more potent cancer chemotherapeutic with fewer potential off-target effects. Recently, the crystal structure of GLUT1 in an inward open conformation was reported. Although this is an important achievement, a full understanding of the structural biology of facilitative glucose transport remains elusive. To date, there is no three-dimensional structure for GLUT4. We have generated a homology model for GLUT4 that we utilized to screen for drug-like compounds from a library of 18 million compounds. Despite 68% homology between GLUT1 and GLUT4, our virtual screen identified two potent compounds that were shown to target GLUT4 preferentially over GLUT1 and block glucose transport. Our results strongly bolster the utility of developing GLUT4-selective inhibitors as anti-cancer therapeutics.

Targeting glutamine metabolism in multiple myeloma enhances BIM binding to BCL-2 eliciting synthetic lethality to venetoclax

Multiple myeloma (MM) is a plasma cell malignancy that is largely incurable due to development of resistance to therapy-elicited cell death. Nutrients are intricately connected to maintenance of cellular viability in part by inhibition of apoptosis. We were interested to determine if examination of metabolic regulation of BCL-2 proteins may provide insight on alternative routes to engage apoptosis. MM cells are reliant on glucose and glutamine and withdrawal of either nutrient is associated with varying levels of apoptosis. We and others have demonstrated that glucose maintains levels of key resistance-promoting BCL-2 family member, myeloid cell leukemic factor 1 (MCL-1). Cells continuing to survive in the absence of glucose or glutamine were found to maintain expression of MCL-1 but importantly induce pro-apoptotic BIM expression. One potential mechanism for continued survival despite induction of BIM could be due to binding and sequestration of BIM to alternate pro-survival BCL-2 members. Our investigation revealed that cells surviving glutamine withdrawal in particular, enhance expression and binding of BIM to BCL-2, consequently sensitizing these cells to the BH3 mimetic venetoclax. Glutamine deprivation-driven sensitization to venetoclax can be reversed by metabolic supplementation with TCA cycle intermediate α-ketoglutarate. Inhibition of glucose metabolism with the GLUT4 inhibitor ritonavir elicits variable cytotoxicity in MM that is marginally enhanced with venetoclax treatment, however, targeting glutamine metabolism with 6-diazo-5-oxo-l-norleucine uniformly sensitized MM cell lines and relapse/refractory patient samples to venetoclax. Our studies reveal a potent therapeutic strategy of metabolically driven synthetic lethality involving targeting glutamine metabolism for sensitization to venetoclax in MM.

Specificity protein 1: Its role in colorectal cancer progression and metastasis

Specificity protein 1 (Sp1) is a widely expressed transcription factor that plays an important role in the promotion of oncogenes required for tumor survival, progression and metastasis. Sp1 is highly expressed in several cancers including colorectal cancer (CRC) and is related to poor prognosis. Therefore, targeting Sp1 is a rational for CRC therapy. In this review, we will recapitulate the current understanding of Sp1 signaling, its molecular mechanisms, and its potential involvement in CRC growth, progression and metastasis. We will also discuss the current therapeutic drugs for CRC and their mechanism of action via Sp1.

Co-inhibition of BET and proteasome enhances ER stress and Bim-dependent apoptosis with augmented cancer therapeutic efficacy

Agents that inhibit bromodomain and extra-terminal domain (BET) protein have been actively tested in the clinic as potential anticancer drugs. Proteasome inhibitors such as carfilzomib (CFZ) are FDA-approved for the treatment of patients with advanced multiple myeloma and have been tested against other cancers. The current study focuses on the combination of a BET inhibitor (e.g., JQ1) and a proteasome inhibitor (e.g., CFZ) as a novel cancer therapeutic strategy and the underlying mechanisms. The tested combination (JQ1 with CFZ) synergistically decreased cell survival and enhanced apoptosis in vitro and inhibited tumor growth in vivo. The dramatic induction of apoptosis was accompanied by enhanced elevation of Bim and ER stress. Bim knockout significantly attenuated apoptosis induced by the combination, suggesting a critical role of Bim induction in mediating the enhanced induction of apoptosis by BET and proteasome co-inhibition. The combination significantly increased Bim mRNA levels with limited effect on Bim protein stability, suggesting a primary transcriptional regulation of enhanced Bim expression. Our findings warrant further investigation of this combinatorial strategy as an effective regimen against cancer in the clinic.

Abstract 972: Targeting glucose and glutamine regulated BCL2 family members for multiple myeloma therapy

Multiple myeloma (MM) is a plasma cell malignancy accounting for approximately 11000 deaths annually in the US. Despite use of immunomodulatory drugs and proteasome inhibitors, MM is largely incurable with a median survival of 5 years due to the development of intrinsic and acquired resistance to these drugs. Targeting metabolic dependencies in a cancer cell may provide an effective strategy to target the molecular heterogeneity and chemo resistance characteristic of MM. Resistance is in part mediated by ineffective regulation of Bcl-2 family members. Myeloid cell leukemia factor 1 (MCL-1) is a key resistance promoting anti-apoptotic protein expressed in MM cells. We and others have previously established that glucose regulates expression of MCL-1. MM cells are heavily reliant upon glucose and glutamine and withdrawal of either nutrient is associated with varying levels of apoptosis. While glucose withdrawal was found to suppress MCL-1 expression uniformly, this did not always correlate with the extent of apoptosis induced. One potential mechanism for continued survival despite suppression of MCL-1 could be due to a shift in binding of pro-apoptotic BCL2 proteins to alternate pro-survival BCL2 members. Our investigation through co-immunoprecipitation experiments revealed that glucose or glutamine withdrawal enhances a shift in binding of pro-apoptotic BIM from MCL-1 to BCL2/ BCLxL. This shift in the association of BIM to BCL2 and BCLxL increased sensitivity to BH3 mimetics ABT-199/737. Induction of NOXA, that has a greater affinity for MCL-1 than BIM, is known to promote the redistribution of BIM to BCL2 and BCLxL. Indeed, glucose and glutamine withdrawal were found to increase NOXA expression associated with activation of the upstream GCN2/PERK/eIF2α/ATF4 axis in a cell type specific manner. Knock down of NOXA in MM cells successfully increased cell survival and reduced sensitivity towards ABT-199 in glucose or glutamine deprived cells. We have extended these observations to a panel of 7 myeloma cell lines and primary myeloma patient samples. Our study broadly divides MM cells into two subtypes on the basis of glucose or glutamine dependency and provides effective ways to their survival by targeting their specific metabolic dependencies in combination with the BH3 mimetic ABT-199. We also demonstrate that glucose transport and metabolism in MM can be targeted with the FDA approved GLUT4 inhibitor ritonavir and glutamine utilization can be inhibited by glutamine antagonist DON (6-Diazo-5-oxo-L-norleucine). Both ritonavir and DON treatments sensitized MM cell lines and primary patient samples to ABT-199, bolstering the utility of repurposing FDA-approved ritonavir and ABT-199 (in clinical trial) for MM therapy. Citation Format: Richa Bajpai, Shannon M. Matulis, Changyong Wei, Ajay K. Nooka, Lawrence H. Boise, Mala Shanmugam. Targeting glucose and glutamine regulated BCL2 family members for multiple myeloma therapy. [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 972. doi:10.1158/1538-7445.AM2015-972