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PIK3CA encodes the p110α catalytic subunit of PI3K and is frequently mutated in human cancers, including ∼30% of colorectal cancer. Oncogenic mutations in PIK3CA render colorectal cancers more dependent on glutamine. Here we report that the glutaminase inhibitor CB-839 preferentially inhibits xenograft growth of PIK3CA-mutant, but not wild-type (WT), colorectal cancers. Moreover, the combination of CB-839 and 5-fluorouracil (5-FU) induces PIK3CA-mutant tumor regression in xenograft models. CB-839 treatment increased reactive oxygen species and caused nuclear translocation of Nrf2, which in turn upregulated mRNA expression of uridine phosphorylase 1 (UPP1). UPP1 facilitated the conversion of 5-FU to its active compound, thereby enhancing the inhibition of thymidylate synthase. Consistently, knockout of UPP1 abrogated the tumor inhibitory effect of combined CB-839 and 5-FU administration. A phase I clinical trial showed that the combination of CB-839 and capecitabine, a prodrug of 5-FU, was well tolerated at biologically-active doses. Although not designed to test efficacy, an exploratory analysis of the phase I data showed a trend that PIK3CA-mutant patients with colorectal cancer might derive greater benefit from this treatment strategy as compared with PIK3CA WT patients with colorectal cancer. These results effectively demonstrate that targeting glutamine metabolism may be an effective approach for treating patients with PIK3CA-mutant colorectal cancers and warrants further clinical evaluation. Significance: Preclinical and clinical trial data suggest that the combination of CB-839 with capecitabine could serve as an effective treatment for PIK3CA-mutant colorectal cancers.

Immune-checkpoint inhibitors and adoptive tumor-infiltrating lymphocyte (TIL) therapies have profoundly improved the survival of patients with melanoma. However, a majority of patients do not respond to these agents, and many responders experience disease relapse. Although numerous innovative treatments are being explored to offset the limitations of these agents, novel therapeutic combinations with immunotherapies have the potential to improve patient responses. In this study, we evaluated the antimelanoma activity of immunotherapy combinations with Telaglenastat (CB-839), a potent glutaminase inhibitor (GLSi) that has favorable systemic tolerance. In in vitro TIL:tumor coculture studies, CB-839 treatment improved the cytotoxic activity of autologous TILs on patient-derived melanoma cells. CB-839 treatment decreased the conversion of glutamine to alpha-ketoglutarate (αKGA) more potently in tumor cells versus TILs in these cocultures. These results suggest that CB-839 may improve immune function in a tumor microenvironment by differentially altering tumor and immune cell metabolism. In vivo CB-839 treatment activated melanoma antigen–specific T cells and improved their tumor killing activity in an immune-competent mouse model of adoptive T-cell therapy. Additionally, the combination of CB-839 with anti-PD1 or anti-CTLA4 antibodies increased tumor infiltration by effector T cells and improved the antitumor activity of these checkpoint inhibitors in a high mutation burden mouse melanoma model. Responsiveness to these treatments was also accompanied by an increase of interferon gamma (IFNγ)–associated gene expression in the tumors. Together, these results provide a strong rationale for combining CB-839 with immune therapies to improve efficacy of these treatments against melanoma.

PURPOSE. The purpose of this study was to translate our in vitro therapy approach to an in vivo model. Increased glutamine uptake is known to drive cancer cell proliferation, making tumor cells glutamine-dependent. Studying lymph-node aspirates containing malignant lung tumor cells showed a strong correlation between glutamine consumption and glutathione (GSH) excretion. Subsequent experiments with A549 and H460 lung tumor cell lines provided additional evidence for glutamine's role in driving synthesis and excretion of GSH. Using stable-isotope-labeled glutamine as a tracer metabolite, we demonstrated that the glutamate group in GSH is directly derived from glutamine, linking glutamine utilization intimately to GSH syntheses. MATERIALS AND METHODS. To understand the possible mechanistic link between glutamine consumption and GSH excretion, we studied GSH metabolism in more detail. Inhibition of glutaminase (GLS) with BPTES, a GLS-specific inhibitor, effectively abolished GSH synthesis and excretion. Since our previous work, several novel GLS inhibitors became available and we report herein effects of CB-839 in A427, H460 and A549 lung tumor cells and human lung tumor xenografts in mice. RESULTS. Inhibition of GLS markedly reduced cell viability, producing ED(50) values for inhibition of colony formation of 9, 27 and 217 nM in A427, A549 and H460, respectively. Inhibition of GLS is accompanied by ~30% increased response to radiation, suggesting an important role of glutamine-derived GSH in protecting tumor cells against radiation-induce injury. In subsequent mouse xenografts, short-term CB-839 treatments reduced serum GSH by >50% and increased response to radiotherapy of H460-derived tumor xenografts by 30%. CONCLUSION. The results support the proposed mechanistic link between GLS activity and glutathione synthesis and suggest that GLS inhibitors are effective radiosensitizers.

// Ravyn M. Thompson 1 , Dominik Dytfeld 2 , Leticia Reyes 1 , Reeder M. Robinson 1 , Brittany Smith 1 , Yefim Manevich 1 , Andrzej Jakubowiak 3 , Mieczyslaw Komarnicki 2 , Anna Przybylowicz-Chalecka 2 , Tomasz Szczepaniak 2 , Amit K. Mitra 5 , Brian G. Van Ness 5 , Magdalena Luczak 4 , Nathan G. Dolloff 1, * 1 Department of Cell and Molecular Pharmacology & Experimental Therapeutics, Medical University of South Carolina, Charleston, SC, USA 2 Karol Marcinkowski University of Medical Sciences, Poznan, Poland 3 University of Chicago, Chicago, IL, USA 4 Institute of Bioorganic Chemistry, Polish Academy of Sciences, Poznan, Poland 5 University of Minnesota, Minneapolis, MN, USA * These authors contributed equally to this work Correspondence to: Nathan G. Dolloff, email: dolloffn@musc.edu Keywords: multiple myeloma, proteasome inhibitor, glutaminase, carfilzomib Received: December 08, 2016 Accepted: March 01, 2017 Published: March 16, 2017 ABSTRACT Curative responses in the treatment of multiple myeloma (MM) are limited by the emergence of therapeutic resistance. To address this problem, we set out to identify druggable mechanisms that convey resistance to proteasome inhibitors (PIs; e.g., bortezomib), which are cornerstone agents in the treatment of MM. In isogenic pairs of PI sensitive and resistant cells, we observed stark differences in cellular bioenergetics between the divergent phenotypes. PI resistant cells exhibited increased mitochondrial respiration driven by glutamine as the principle fuel source. To target glutamine-induced respiration in PI resistant cells, we utilized the glutaminase-1 inhibitor, CB-839. CB-839 inhibited mitochondrial respiration and was more cytotoxic in PI resistant cells as a single agent. Furthermore, we found that CB-839 synergistically enhanced the activity of multiple PIs with the most dramatic synergy being observed with carfilzomib (Crflz), which was confirmed in a panel of genetically diverse PI sensitive and resistant MM cells. Mechanistically, CB-839 enhanced Crflz-induced ER stress and apoptosis, characterized by a robust induction of ATF4 and CHOP and the activation of caspases. Our findings suggest that the acquisition of PI resistance involves adaptations in cellular bioenergetics, supporting the combination of CB-839 with Crflz for the treatment of refractory MM.

Nearly all metastatic melanoma patients who respond to targeted therapies will relapse with the disease within a year. Although more durable responses are seen with immune therapies, about half of the melanoma patients do not respond to them, and a significant number of responders eventually relapse. Most relapsed melanomas also exhibit post-treatment resistance to these treatments. Hence, there is a clear and present need to develop therapeutics that counteract resistance associated with relapse. We and others earlier showed that melanomas with elevated mitochondrial activity possess improved cellular rigor and are intrinsically resistant to the antitumor effects of BRAF and MEK inhibitors. In many other instances, melanomas that initially respond to these inhibitors acquire resistance by elevating mitochondrial activity. Mitochondrial activity is elevated in part by increased cellular uptake of glutamine, and its conversion to alpha-ketoglutarate in the TCA cycle, with glutaminase enzyme playing a rate-limiting role. In this study, we show that BRAFV600E-mutant melanomas with intrinsic or acquired resistance to MAPK pathway inhibitors have lower glucose uptake and increased glutamine uptake compared to those that are sensitive. Treatment of these resistant melanomas with single-agent glutaminase inhibitor, CB-839, moderately inhibited their growth. However, a more robust inhibition of their growth was achieved when CB-839 was combined with BRAF and MEK inhibitors. In addition, CB-839 increased the in vivo activity of tumor-infiltrating lymphocytes (TILs) in a mouse vaccine model and also enhanced the proapoptotic effect of human autologous patient-derived TILs on their cognate melanoma cells. Seahorse bioenergetics stress tests showed that CB-839 inhibited mitochondrial OxPhos in tumor cells to a greater extent than in activated TILs. Additional molecular studies are currently in progress. A recent clinical trial in melanoma patients showed that combination treatment with CB-839 and the immune checkpoint blocker, nivolumab, caused an objective response in three melanoma patients who had earlier progressed on treatment with immune checkpoint blockade. Our preclinical results complement this clinical finding and suggest that CB-839 combination could potentiate the efficacy of targeted and immune therapies in refractory melanomas. Citation Format: Sruthy Varghese, Snigdha Pramanik, Rishika Prasad, Hannah Hodges, Leila Williams, Weiyi Peng, Hussein Tawbi, Vashisht Yennu Nanda. The glutaminase inhibitor CB-839 potentiates antimelanoma activity of standard-of-care targeted therapies and immunotherapies [abstract]. In: Proceedings of the AACR Special Conference on Melanoma: From Biology to Target; 2019 Jan 15-18; Houston, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(19 Suppl):Abstract nr PR06.

Purpose: Proliferation rates of basal breast cancers are dependent on glutamine metabolism as previously demonstrated by glutamine withdrawal or glutaminase inhibition by CB-839 in cell line models. Early and late adaptive signaling and metabolic responses to glutamine stress may enable cells to proliferate or evade cell death. We hypothesized that inhibition of these pathways would be synergistic in combination with glutaminase inhibition by CB-839. Methods: Protein signaling responses in HCC1806 cells (basal A breast cancer) were quantified using reverse phase protein arrays (RPPA) at early (4 h) and late (24 h) time points in response to low glucose or low glutamine concentrations as well as treatment with CB-839, a selective glutaminase inhibitor. Signaling pathways evaluated included mTORC1/2, PI3K/AKT, MAPK, and AMPK. ADP and ATP were quantified by ion exchange chromatography. Drug combinations predicted by RPPA were evaluated at therapeutically relevant concentrations using proliferation or apoptosis assays after 72 h of continuous treatment. Results: In agreement with previous reports of glutamine withdrawal, low glutamine and glutaminase inhibition resulted in inhibition of proliferation and early inhibition of mTORC1 signaling including pmTOR, pP70S6K, p4EBP1, and pS6. In contrast, for cells incubated in low glucose, proliferation and mTOR signaling were maintained at early time points despite robust pAMPK/pACC signaling and increased ADP/ATP ratios (2-orders of magnitude higher than baseline); mTOR signaling was only inhibited after 24 h when ATP concentrations were reduced. Bioenergetic stress as detected by pAMPK and pACC was not observed with glutaminase inhibition – ATP concentrations were increased by CB-839 treatment. Importantly, 2-deoxyglucose, a compound used to reduce ATP levels and activate AMPK signaling, was antagonistic in combination with CB-839. In contrast, hydroxychloroquine, an inhibitor of autophagy activated by mTOR inhibition, was synergistic with CB-839. Consistent with mTORC1-mediated negative feedback loops, CB-839 treatment also increased AKT and ERK signaling. Combinations of CB-839 with AKT or MEK inhibitors (AZD5363 and GSK1120212B) were at synergistic and additive, respectively, for cell proliferation. The MEK inhibitor in combination with CB-839 resulted in supra-additive effects using apoptosis as an endpoint. Conclusion: Glutaminase inhibition by CB-839 acutely inhibited mTORC1 signaling independently of AMPK signaling in HCC1806 cells. MEK and AKT signaling increased, likely in response to negative feedback loops with mTORC1. Combinatorial approaches with CB-839 targeting these compensation pathways resulted in increased responses supporting further preclinical and clinical studies. Citation Format: Jennifer B Dennison, Zhenlin Ju, Mills B Gordon. Signaling consequences and rational therapeutic combinations with glutaminase inhibitor, CB-839, in basal breast cancer [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P1-08-01.

Glutamine serves as an important source of energy and building blocks for many tumor cells. The first step in glutamine utilization is its conversion to glutamate by the mitochondrial enzyme glutaminase. CB-839 is a potent, selective, and orally bioavailable inhibitor of both splice variants of glutaminase (KGA and GAC). CB-839 had antiproliferative activity in a triple-negative breast cancer (TNBC) cell line, HCC-1806, that was associated with a marked decrease in glutamine consumption, glutamate production, oxygen consumption, and the steady-state levels of glutathione and several tricarboxylic acid cycle intermediates. In contrast, no antiproliferative activity was observed in an estrogen receptor–positive cell line, T47D, and only modest effects on glutamine consumption and downstream metabolites were observed. Across a panel of breast cancer cell lines, GAC protein expression and glutaminase activity were elevated in the majority of TNBC cell lines relative to receptor positive cells. Furthermore, the TNBC subtype displayed the greatest sensitivity to CB-839 treatment and this sensitivity was correlated with (i) dependence on extracellular glutamine for growth, (ii) intracellular glutamate and glutamine levels, and (iii) GAC (but not KGA) expression, a potential biomarker for sensitivity. CB-839 displayed significant antitumor activity in two xenograft models: as a single agent in a patient-derived TNBC model and in a basal like HER2+ cell line model, JIMT-1, both as a single agent and in combination with paclitaxel. Together, these data provide a strong rationale for the clinical investigation of CB-839 as a targeted therapeutic in patients with TNBC and other glutamine-dependent tumors. Mol Cancer Ther; 13(4); 890–901. ©2014 AACR.

Background: c-MYC activation is an early event of myeloma pathogenesis. It upregulates the expression of the glutaminase 1 (GLS1) enzyme which converts glutamine to glutamate in the mitochondria. Glutamate is required for the biosynthesis of various molecules in the tricarboxylic acid (TCA) cycle (i.e., glutamine anaplerosis). CB-839 HCl is a first-in-class, orally available, selective, noncompetitive inhibitor of GLS1. This inactivation of GLS1 results in an increase of glutamine and a decrease of glutamate and several TCA cycle intermediates within cancer cells, leading to a decrease in their proliferation and/or an increase in cell death. In the phase 1 study, CX-839-002, the safety and tolerability of CB-839 HCl was evaluated in patients with hematological tumors (multiple myeloma (MM) and non-Hodgkin's lymphoma), either as monotherapy or in combination with pomalidomide and dexamethasone or with dexamethasone alone. It was determined to be well tolerated and the maximal tolerated dose (MTD) was not reached. Proteasome inhibitors (PI) are the cornerstone agents in the treatment of myeloma. They disrupt normal protein homeostasis causing an induction of cellular proteotoxic stress, thus, making it an effective strategy against myeloma plasma cells, which naturally mass-produce large quantities of immunoglobulin proteins. PI-resistant MM cells are associated with changes in cellular bioenergetics that favor the increased use of mitochondrial respiration for energy production. Given the increased reliance of PI-resistant MM cells on mitochondrial respiration, and the critical role of glutamine for cellular respiration, inhibition of glutamine metabolism is a rational molecular strategy for the treatment of PI-resistant MM. Furthermore, pre-clinical studies demonstrate the in vitro and ex vivo synergism of CB-839 HCl with carfilzomib (CFZ) in terms of its cytotoxicity and anti-proliferation capacity in various primary human myeloma cell lines and primary patient myeloma cells respectively. As a result, this novel combination of glutaminase inhibition with proteasome inhibition appears promising as a therapeutic combination in MM and warrants further clinical investigation. Methods: This study is a phase 1, multicenter clinical trial of CB-839 HCl in combination with carfilzomib and dexamethasone for patients with relapsed and/or refractory myeloma. Part A of this trial is a 3+3 dose escalation design and Part B is a dose expansion cohort at the RP2D determined in Part A. Up to a maximum of 42 patients will be enrolled at participating ETCTN sites. CFZ will be administered in its usual weekly dosing schedule of days 1, 8 and 15 of a 28 day schedule along with dexamethasone on days 1, 8, 15 and 22. CB-839 will be started at a dose level of 400 mg twice daily and will be investigated to a maximum dose of 800 mg twice daily. Prior to day 1 of Cycle 1, we will administer a 7 day lead in of CB-839 monotherapy before combining it with CFZ. Key inclusion criteria are having relapsed/refractory myeloma with at least 2 prior lines of therapy and prior exposure to PIs, immunomodulators and Anti-CD38 monoclonal antibodies, having measurable disease, adequate hematologic reserve, kidney function and liver function. Key exclusion criteria are being refractory or intolerant to CFZ, adverse cardiac history, central nervous system disease and AL amyloidosis. The primary objective of this trial is to determine the MTD or recommended phase II dosing (RP2D) of CB-839 HCl in combination with carfilzomib and dexamethasone. The secondary objective is to evaluate the safety and tolerability as well as the overall response rate (ORR) associated of CB-839 HCl in combination with carfilzomib and dexamethasone. Correlative objectives will evaluate plasma pharmacokinetic profiles of CB-839 HCl and carfilzomib when used in combination. They will also evaluate potential predictive and prognostic biomarkers as well as resistance mechanisms using genomic DNA, RNA, flow cytometry, immunohistochemistry and metabolomics-based assessment platforms. Recruitment is ongoing and this trial is registered on clinicaltrials.gov: NCT03798678. Disclosures Baz: Bristol-Myers Squibb: Research Funding; Sanofi: Research Funding; Karyopharm: Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding; AbbVie: Research Funding; Merck: Research Funding. Neparidze:Janssen Scientific Affairs, LLC: Research Funding; Eidos Therapeutics: Other: Member of Independent Diagnostic Committee; MMRF/Synteract: Membership on an entity's Board of Directors or advisory committees. Kumar:Janssen: Consultancy, Research Funding; Celgene: Consultancy, Research Funding; Takeda: Research Funding.

Glutamine (Gln) serves as an important source of energy and building blocks for many tumor cells. The first step in Gln utilization is its conversion to glutamate (Glu) by the mitochondrial enzyme glutaminase. CB-839 is a potent, selective, and orally bioavailable inhibitor of both splice variants of glutaminase (KGA and GAC). CB-839 inhibits recombinant GAC with an IC50 of 25) that included a mixture of TNBC and ER/Her2-positive subtypes. The TNBC subtype displayed the greatest sensitivity to CB-839 treatment (IC50s ranging from 5-100 nM) and this sensitivity was correlated with: i) dependence on extracellular Gln for growth (r = 0.83, p Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P2-09-03.

Many tumor cells utilize the amino acid glutamine to meet the elevated bioenergetic and biosynthetic demands of rapid cell growth. The enzyme glutaminase converts glutamine to glutamate, which is used to fuel the TCA cycle, synthesize amino acids and nucleotides, and balance cellular oxidative stress. We developed CB-839, a potent and orally bioavailable small molecule inhibitor of glutaminase, that blocks production of glutamate and generation of downstream metabolites glutathione, malate and aspartate. Mice treated with CB-839 had decreased levels of nucleotides in their tumors, likely due to glutamine-derived aspartate being required for nucleotide biosynthesis. Consistent with our finding that CB-839 decreases nucleotide pools, CB-839 treatment delayed cancer cells from either entry into S-phase or progression through S-phase. Based on this observation, the ability of CB-839 to synergize with therapies that block cell cycle progression was tested. CB-839 synergized with the CDK4/6 inhibitor palbociclib in colorectal carcinoma (CRC), triple negative breast cancer (TNBC) and ER+ breast cancer cell lines resulting in anti-proliferative activity. The combination of CB-839 with palbociclib also led to decreased cell cycle progression through S-phase and caused an accumulation of cells in G1 as measured by an EdU DNA incorporation assay. In vivo, the combination of CB-839 with palbociclib resulted in enhanced anti-tumor activity in both an ER+ breast cancer and CRC xenograft tumor model. We next investigated whether CB-839 treatment would enhance the anti-tumor effects of DNA repair inhibitors, given the ability of CB-839 treatment to decrease nucleotide pools. CB-839 treatment in combination with the PARP inhibitors niraparib and talazoparib led to synergistic anti-proliferative activity in TNBC, CRC, non-small cell lung carcinoma, ovarian and prostate cancer cells. In vivo, the combination of CB-839 with PARP inhibitors showed enhanced anti-tumor activity compared to single agent treatment in a CRC tumor xenograft model. CB-839 is currently undergoing evaluation for efficacy in the treatment of cancer in several phase I/II clinical trials. These encouraging pre-clinical results support the testing of CB-839 with CDK4/6 or PARP inhibitors in cancer patients. Citation Format: Ethan D. Emberley, Mark Bennett, Jason Chen, Matthew Gross, Tony Huang, Amani Makkouk, Gisele Marguier, Alison Pan, Sandra M. Spurlock, Susanne Steggerda, Francesco Parlati. The glutaminase inhibitor CB-839 synergizes with CDK4/6 and PARP inhibitors in pre-clinical tumor models [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 3509.

2562Background: Colorectal cancers (CRCs) harboring a PIK3CA mutation demonstrate glutamine dependency in both in vitro and in vivo models, including those known to be fluoropyrimidine (FP) resista...

Multiple myeloma (MM) remains largely incurable due to the emergence of therapeutic resistance. We therefore set out in this study to identify druggable molecular mechanisms that convey resistance to proteasome inhibitors (PIs; e.g., bortezomib/VELCADE, carfilzomib/KYPROLIS), which are cornerstone agents in the treatment of MM. In comparing isogenic pairs of PI sensitive and resistant cells, we observed stark differences in cellular bioenergetics between the divergent phenotypes. While glycolysis rates between cell lines were similar, PI resistant cells exhibited increased mitochondrial respiration characterized by higher basal oxygen consumption rates (OCR) and overall respiratory capacity. Additionally, PI resistant cells were found to have lower activation of the AMP kinase (AMPK) energy stress pathway and increased levels of NAD(P)H, which serve as electron carriers in the mitochondrial process of oxidative phosphorylation. We determined that glutamine was the principle source of fuel driving mitochondrial respiration as removal of glutamine completely inhibited OCR as well as cell proliferation, whereas glucose and pyruvate were dispensable. Given the propensity for mitochondrial respiration in PI resistant cells and the dependence on glutamine for this process, we hypothesized that targeting glutamine utilization by PI resistant cells would restore their sensitivity to the cytotoxic effects of PIs. To test this possibility, we inhibited glutamine metabolism using the small molecule GLS1 inhibitor CB-839. CB-839 repressed basal OCR and total respiratory capacity and reduced cell viability to varying degrees in a panel of PI sensitive and resistant MM cell lines. Most notably, we found that CB-839 synergistically enhanced the cytotoxic activity of multiple PIs, including bortezomib, carfilzomib, ixazomib, and oprozomib, in a genetically diverse panel of 15 PI sensitive and resistant MM cell lines. The effects of CB-839 were the most apparent in combination with carfilzomib (Crflz), where it enhanced Crflz-induced death by >4-fold. CB-839 enhanced Crflz-induced apoptosis as measured by the activation of caspase 3, 7, 8 and the cleavage of the caspase-3 substrate PARP. Mechanistically, the combination of CB-839 and Crflz induces a strong and synergistic ER stress response, characterized by the induction of ATF4 and CHOP. Our findings suggest that the acquisition of PI resistance may involve adaptations in cellular bioenergetics that may be exploited therapeutically by targeting glutamine metabolism. Furthermore, our results support the combination of clinical stage compound CB-839 with PIs, particularly Crflz, for the treatment of refractory MM. Disclosures No relevant conflicts of interest to declare.

Introduction Glutaminase is a mitochondrial enzyme that converts glutamine to glutamate to support several metabolic processes including amino acid and nucleotide synthesis, maintenance of cellular redox homeostasis, and the replacement of TCA cycle intermediates. Selective glutaminase inhibitors BPTES and CB-839 have anti-proliferative activity in several pre-clinical cancer models including breast, pancreatic, lung, renal, brain, leukemia, and lymphoma. Across a panel of twenty-nine multiple myeloma cell lines, we found that glutaminase inhibition with CB-839 caused tumor cell death or growth inhibition in only a subset of cell lines. To identify biomarkers that predict sensitivity to CB-839 in multiple myeloma cells, we profiled cellular metabolites, mRNA transcripts, and signaling pathways in eight multiple myeloma cell (four CB-839-sensitive and four CB-839-resistant). Results Proteomic analysis showed that CB-839 treatment suppressed the activity of the amino-acid sensing kinase mTORC1 in CB-839-sensitive cells, leading to down regulation of protein synthesis and expression of metabolic genes. Analysis of steady-state levels of intra-cellular metabolites revealed that CB-839-sensitive cells had more profound decreases in nucleotide levels and less pronounced increases in essential amino acids upon CB-839 treatment compared to CB-839-resistant cells. This suggests that the metabolic response to glutaminase inhibition is fundamentally different in sensitive versus resistant multiple myeloma cell lines. Consistent with the in vitro data, in a xenograft model with the CB-839-sensitive cell line RPMI8226, CB-839 treatment produced a 71% reduction in tumor growth that was associated with reduced levels of intratumoral nucleotides and no changes in the levels of essential amino acids. We next explored protein biomarkers that predict resistance to CB-839 and found that pyruvate carboxylase (PC) expression strongly correlated with resistance. siRNA-mediated knockdown of PC reduced TCA cycle activity and sensitized cells to CB-839 treatment, suggesting that PC can rescue cells from glutaminase inhibition by supporting anapleurotic utilization of glucose. This hypothesis was further substantiated by the observation that treatment of CB-839-resistant cells with the AKT inhibitor MK2206 led to a decrease in glucose utilization, and when combined with CB-839, produced a significant decrease in TCA cycle activity and a profound synergistic anti-proliferative response. Conclusion Multiple myeloma cells show varying anti-proliferative responses to glutaminase inhibition by CB-839. CB-839 treatment inhibits mTORC1 pathway signaling and causes decreases in nucleotides in sensitive multiple myeloma cells. Multiple myeloma cells that are resistant to glutaminase inhibition have high expression of PC, which may allow these cells to utilize glucose instead of glutamine to resupply TCA cycle intermediates. Knockdown of PC or treatment with an AKT inhibitor causes cells to utilize less glucose and sensitizes resistant cells to glutaminase inhibition with CB-839. CB-839 is currently being evaluated in Phase 1 clinical trials for the treatment of various solid and hematological cancers including multiple myeloma. We are exploring the utility of PC and mTORC1 pathway signaling biomarkers to identify multiple myeloma patients that may respond to CB-839 treatment. Disclosures MacKinnon: Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership. Gross:Calithera Biosciences: Employment, Equity Ownership. Janes:Calithera Biosciences: Employment, Equity Ownership. Li:Calithera Biosciences: Employment, Equity Ownership. Rodriquez:Calithera Biosciences: Employment, Equity Ownership. Wang:Calithera Biosciences: Employment, Equity Ownership. Zhang:Calithera Biosciences: Employment, Equity Ownership. Parlati:Calithera Biosciences: Employment, Equity Ownership.

Glutamine is an abundant nutrient used by cancer cells to support energetic and biosynthetic pathways. Given that many cancer cells exhibit glutamine dependence, there has been interest in developing inhibitors targeting glutaminase, which catalyzes conversion of glutamine to glutamate. CB-839 is a glutaminase inhibitor with preclinical efficacy in suppressing cancer cell growth in culture and in vivo. It is now in Phase I clinical trials for solid and hematological malignancies, including non-small cell lung cancer (NSCLC). Here we set out to identify biomarkers predicting sensitivity of NSCLC cells to CB-839, using a highly annotated panel of 81 cell lines. These lines had heterogeneous oncogenotypes and displayed about a 5-fold range in their ability to tolerate glutamine deprivation. Assessing CB-839 sensitivity in ∼40 lines revealed a 5-fold difference in growth suppression among cell lines. There was a strong correlation between CB-839 sensitivity and both glutamine dependence and glutamate secretion. In a subset of 4 representative cell lines, CB-839 suppressed [U-13C]glutamine metabolism in both sensitive and insensitive cells suggesting other mechanisms contribute to CB-839 sensitivity. To identify potential biomarkers predicting CB-839 sensitivity, the glutamine dependence and drug sensitivity phenotypes were correlated with several orthogonal high-content data sets including mutational analysis of commonly-mutated oncogenes and tumor suppressors; sensitivity to chemotherapeutic agents; gene expression arrays; and reverse phase protein arrays (RPPA). Although we did not identify a strong correlation between mutational status and CB-839 sensitivity, expression of phospho-Rb (S807/811) and Smad3 did correlate with drug sensitivity. In addition, sensitivity to glutamine withdrawal correlated with expression of c-Myc and MEK2 protein. While further work is necessary to examine the importance of these proteins in glutamine metabolism, we hypothesize that one or more of these may represent novel biomarkers predicting sensitivity to glutaminase inhibition. Citation Format: Lindsey K. Boroughs, Pei-Hsuan Chen, Ling Cai, Mirna Rodriguez, Winter Zhang, Kenneth E. Huffman, Lauren A. Byers, Luc Girard, Adi F. Gazdar, John V. Heymach, Michael A. White, John D. Minna, Ethan Emberley, Alison Pan, Frank Parlati, Ralph J. DeBerardinis. Evaluation of the glutaminase inhibitor CB-839 in non-small cell lung cancer. [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 4710. doi:10.1158/1538-7445.AM2015-4710

Many hematological tumor cells are dependent on glutamine for growth and survival. Glutamine is the most abundant amino acid in plasma and can be utilized by tumor cells for production of energy and generation of building blocks for the synthesis of macromolecules. Small molecule CB-839 inhibits glutaminase (GLS) activity thereby blocking cellular glutamine utilization resulting in an anti-tumor effect in several hematological tumor types including multiple myeloma (MM), acute lymphocytic leukemia, and several types of non-Hodgkin’s lymphoma [Parlati et al. Blood 2013 122:4226]. Phase 1 clinical trials have been initiated to test the safety, pharmacokinetics, pharmacodynamics, and clinical activity of single agent CB-839 in several hematological malignancies. In anticipation of potential combinations of CB-839 with standard of care agents in future MM clinical trials, we tested the effects of CB-839 in combination with the IMiD, pomalidomide (POM). POM caused complete growth inhibition in MM.1S cells with an EC50 of 16 nM as opposed to partial growth inhibition in RPMI8226 cells, with an EC50 of 130 nM. CB-839 caused complete growth inhibition in MM.1S cells with an EC50 value of 26 nM and produced a cytotoxic effect in RPMI8226 cells with an EC50 of 160 nM. When combined, CB-839 enhanced the anti-proliferative activity of POM in both POM-sensitive MM.1S and POM-resistant RPMI8226 cells resulting in a synergistic anti-tumor effect as demonstrated by combination index values between 0.18-0.62 (mean= 0.36) for the MM.1S and 0.25-0.72 (mean= 0.38) for the RPMI8226 cells. To investigate the mechanism that underlies the observed synergy, RPMI8226 cells were treated for 24 hours and changes in proteins and metabolites were measured by reverse-phase-protein array and LC/MS, respectively. When treated with CB-839 alone, RPMI8226 cells respond by decreasing mTOR pathway signaling proteins (e.g. phospho-mTOR, phospho-p70S6K, phospho-PRAS40, phospho-S6), decreasing the amount of oncogenic proteins (c-Myc and c-Kit), and increasing programmed cell death pathway proteins (e.g. cleaved caspase 7, cleaved PARP), consistent with the cytotoxic activity observed for CB-839. Several of these changes were further enhanced in the presence of POM (e.g. phospho-p70S6K, phospho-S6, phospho-PRAS40, c-kit, c-Myc), however only the enhanced decrease in c-Myc reached statistical significance. Metabolite analysis showed changes with CB-839 consistent with GLS inhibition (e.g. decreases in glutamate, aspartate, succinate and malate and increases in glutamine). On the other hand, single agent POM caused very modest changes in the metabolite profile. When the two agents were combined, metabolite levels were consistent with those observed with single agent CB-839, with the notable exception of carbamoyl-aspartate where lower levels were measured in the combination group in comparison to cells treated with either agent alone. Carbamoyl-aspartate is an intermediate in the pyrimidine biosynthesis pathway and is synthesized by the multi-catalytic enzyme CAD (carbamoyl-phosphate synthetase 2, aspartate transcarbamylase, dihydroorotase), an enzyme that is regulated by mTOR [Ben-Sahra et al. (2013) Science339: 1323-8]. These observations suggest that CB-839 dampens mTOR signaling and POM may further attenuate this response, possibly contributing to the synergistic anti-tumor effect. These data motivated testing the anti-tumor effect of the combination of CB-839 and POM in mice bearing RPMI8226 xenografts. Oral dosing with single agent CB-839 and POM resulted in tumor growth inhibition (TGI) of 64% and 46%, respectively, whereas the combination of the two agents resulted in a TGI of 97%. Efficacious doses of CB-839 and POM alone or in combination were well tolerated with no effect on animal body weight. These promising results indicate that GLS inhibition with CB-839 in combination with POM may provide therapeutic benefit in MM and provide motivation for future clinical studies. Disclosures Parlati: Calithera Biosciences: Employment, Equity Ownership. Gross:Calithera Biosciences: Employment, Equity Ownership. Janes:Calithera Biosciences: Employment, Equity Ownership. Lewis:Calithera Biosciences: Employment, Equity Ownership. MacKinnon:Calithera Biosciences: Employment, Equity Ownership. Rodriguez:Calithera Biosciences: Employment, Equity Ownership. Shwonek:Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership.

Inhibition of glutaminase (GLS), the principal enzyme in the glutamine utilization pathway that coverts glutamine (Gln) to glutamate (Glu), is an attractive therapeutic approach in many cancers. Gln plays a unique role in the metabolism of proliferating cancer cells, providing building blocks to sustain cell proliferation and regulating redox homeostasis and signal transduction pathways. Recent findings indicate that leukemic cells depend on Gln as a major carbon source for growth and survival [Willems et al., Blood, 2013]. We previously reported that a subset of acute myeloid leukemia (AML) cell lines are sensitive to Gln deprivation as well as inhibition of glutaminase by the small molecule BPTES [Matre et al. ASH 2013 #606]. Here we report the efficacy of CB-839, a novel, potent, orally bioavailable GLS inhibitor currently under clinical investigation, in the Gln-dependent subset of AML. First, expression of GLS gene splice variants glutaminase C (GAC) and kidney glutaminase (KGA) and of the GLS2 gene was determined through analysis of RNA sequencing data from 173 newly diagnosed AML patients in the TCGA dataset. Of the GLS gene splice variants, the expression levels of GAC were much higher than those of KGA; GLS2 was expressed at low levels. Levels of both GAC and KGA mRNA were significantly higher (two-sample Wilcoxon test) in AML patients with complex cytogenetics and monosomal karyotype (n=31) than in those with diploid AML (n=88, p=0.019 and p=0.01); GAC levels were higher in core-binding factor AML (n=14) than in diploid AML (p=0.018). These findings indicate high expression of the GLSGAC splice variant in specific AML subsets. Analysis of a panel of AML cell lines showed that, in a subset of leukemia cells, CB-839 treatment decreased viable cell number and induced apoptosis. In sensitive cell lines (Molm14, OCI-AML3, MV4;11), CB-839 decreased viable cell number with IC50s between 10nM and 100nM and induced significant apoptosis. HL60, MOLM13, KG1α, and OCI-AML2 cells were less sensitive (IC50 100-1000nM) and responded with minor induction of cell death. CB-839 decreased viability by >40% in blasts from 9 of 20 (45%) primary AML samples. GC- or LC-MS metabolic profiling of OCI-AML3 and THP1 cell lines as well as primary patient samples revealed that GLS inhibition by BPTES or CB-839 was accompanied by concomitant decrease in concentration of downstream GLS metabolites such as glutamate, α-ketoglutarate (a-KG), aspartate, fumarate, and malate. Investigation of the effects of CB-839 on mitochondrial OXPHOS by the Seahorse Bioscience XF96 Analyzer showed that CB-839 exposure for 16 h caused a dose-dependent decrease in maximal respiratory capacity in OCI-AML3 cells, indicating reduced availability of the substrates for OXPHOS. Similar results were obtained upon treatment with BPTES and in AML cells stably transduced with GLS shRNA. Gln, through Glu, is a precursor for cellular α-KG, which can undergo further metabolism through the Krebs cycle or be further metabolized to 2-hydroxyglutarate (2-HG) by mutant isocitrate dehydrogenase (IDH). In THP1 cell lines stably transduced with doxycycline-inducible mutant IDH1-R132H or IDH2-R140Q construct, CB-839 exposure for 4 days reduced intracellular 2-HG oncometabolite levels by >50%. This was associated with induction of differentiation marker CD11b and morphological signs of differentiation in CB-839–treated IDH2-R140Q cells [30%±2% increase in CD11b mean fluorescent intensity (p In summary, these results indicate that GLS is a relevant therapeutic target in AML, warranting future inclusion of GLS inhibitors in the armamentarium of multi-agent therapeutic approaches. In particular, reduction of production of the oncometabolite 2-HG in conjunction with therapeutic blockade of Gln metabolism may serve as a tailored therapeutic strategy in IDH-mutated AML cells. Disclosures Konopleva: Calithera Biosciences: Research Funding.

Background and Rationale: Cancer cells possess different metabolic requirements than normal cells, such as increased fatty acid synthesis and increased rates of glutamine metabolism, which facilitate their growth and survival. Glutamine is one of the key metabolites required by different cancer cell types for their survival. It is a precursor for α-keto glutarate (αKG) of tricarboxylic acid cycle. Intracellular glutamine is metabolized by a biochemical process glutaminolysis: glutamine is converted to glutamate by glutaminase (GLS), followed by glutamate conversion to αKG by glutamate dehydrogenase (GDH). Three mammalian GLS have been identified: the Liver-type (LGA or GLS2), the Kidney-type (KGA) and Glutaminase C (GAC). Elevated GAC mRNA levels have been detected in gliomas, colorectal carcinomas, adenomas and breast tumor cell lines. GLS activity is linked to tumor growth, since its inhibition by small molecule inhibitors or siRNA knockdown suppresses tumor growth in solid tumors. Elevated glutaminolysis confers drug resistance. These findings suggest that targeting GLS activity inhibits oncogenic transformation and overcomes drug resistance. Here we evaluated anti-MM activity of CB-839, a novel potent orally bioavailable inhibitor of both KGA and GAC splice variants of glutaminase. Materials and methods: MM cell lines, patient MM cells, and peripheral blood mononuclear cells (PBMCs) from normal healthy donors were utilized to assess the anti-MM activity of CB-839. All studies involving human samples were performed under approved protocols at Dana-Farber Cancer Institute. Cell viability was assessed by WST-1 (Chemicon International) assay, as per manufacturer’s instructions. MM cells from patients were isolated by CD138-positive selection using CD138 microbeads and the auto MACS magnetic cell sorter. Total cell lysates were subjected to immunoblot analysis. HUVEC cell angiogenesis assay and transwell cell migration assays were performed, as previously described (Chauhan et al., Blood, 2010, 115(4): 834-45). Glutamine and glutamate determination kit was purchased from Sigma, USA. Drug source: CB-839 was obtained from Calithera Biosciences, USA. Results: KGA and GAC are highly expressed in MM cell lines than in normal PBMCs. We first confirmed the functional specificity of glutamines (GLS) inhibitor CB-839 using MM cells. Specifically, a significant increase in glutamine and a concomitant decrease in glutamate levels were observed in CB-839-treated RPMI-8226 MM cells (p < 0.05; n=3). Moreover, treatment of MM cells with CB-839 significantly decreased both basal and active α-keto glutarate levels (p < 0.05; n=3). Treatment of MM cell lines (MM.1S, MM.1R, RPMI-8226, Dox-40, U266, ARP-1, INA-6, ANBL6.WT, ANBL6.R, and LR5) and primary patient cells for 72h significantly decreased their viability (IC50 range 1.25 μM-10 μM) (p < 0.05; n=3) without markedly affecting PBMCs from normal healthy donors, suggesting specific anti-MM activity and a favorable therapeutic index for CB-839. Conversely, pretreatment of MM cells with α-keto glutarate, a downstream product of glutamine breakdown abrogates anti-MM activity of CB-839. Moreover, CB-839 inhibits proliferation of MM cells even in the presence of BM stromal accessory cells. Mechanistic studies show that CB-839 triggered MM cell death occurs in a caspase-dependent manner, associated with induction of autophagy in MM cells via inhibition of PI3K/Akt/mTOR signaling pathway without concurrent activation of mTORC1. CB-839 induced autophagy was evident by increased levels of autophagy markers Beclin-1, Atg-7 and LC3B. Combining CB-839 with a biochemical inhibitor of autophagy 3-methyl adenine or chloroquine triggered antagonism, suggesting that both autophagy and caspase-mediated apoptotic pathway contribute to anti-MM activity of CB-839. Furthermore, CB-839 blocked migration of MM cells and angiogenesis. Finally, combination of CB-839 with pomalidomide, bortezomib or SAHA induces synergistic anti-MM activity. Conclusion: Our preclinical studies showing efficacy of CB-839 in MM disease models provide the framework for a clinical trial of CB-839, either alone or in combination, to improve outcome in relapsed and refractory MM patients. Disclosures Munshi: Ocopep: Consultancy, Equity Ownership, Patents & Royalties; Sanofi-Aventis: Consultancy; Janssen: Consultancy; Onyx: Consultancy; Celgene: Consultancy. Richardson:Oncopeptides AB: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Millennium: Membership on an entity's Board of Directors or advisory committees. Chauhan:Triphase Accelerator: Consultancy. Anderson:Celgene: Consultancy; Millenium: Consultancy; Onyx: Consultancy; Gilead: Consultancy; Sanofi Aventis: Consultancy; BMS: Consultancy; Oncopep/Acetylon: Equity Ownership.

CB-839 is a potent, orally bioavailable small-molecule inhibitor of the tumor metabolism target glutaminase (GLS) that is currently in Phase 1 clinical trials for the treatment of solid and hematological malignancies. GLS is a mitochondrial enzyme that converts Gln to glutamate (Glu) to support several metabolic processes including amino acid synthesis, maintenance of cellular redox homeostasis, and the replacement of TCA cycle intermediates. CB-839 has in vitro antiproliferative activity across a sub-set of cell lines from diverse hematological tumor types including multiple myeloma (MM), acute lymphocytic leukemia, and non–Hodgkin’s lymphoma [Parlati et al. Blood 2013 122:4226]. To identify biomarkers that would predict sensitivity to CB-839 in MM, we profiled cellular metabolites, mRNA transcripts, and oncogenic signaling pathways in eight MM cell lines representing four CB-839-sensitive cell lines (RPMI8226, MM.1S, KMS-11, and IM-9) and four CB-839-resistant cell lines (AMO-1, L-363, KMS-28PE, and OPM-2). CB-839 treatment for 4 hours significantly decreased the levels of amino acids (Glu, aspartate, proline) as well as TCA cycle intermediates (fumarate, malate, succinate) across all cell lines. However, prior to treatment, CB-839-sensitive cells had significantly lower baseline levels of pyruvate-, fumarate-, and succinyl-CoA-derived amino acids compared to CB-839-resistant cells. In addition, both the adenylate and guanylate energy charges, a measure of cellular metabolic activity [Atkinson and Walton, J. Biol. Chem. 1967 242: 3239-41], were significantly lower in CB-839 sensitive cells. These observations suggest that cells with low levels of amino acids and/or low cellular energy charge are more susceptible to the pharmacological effects of CB-839. Reverse phase protein array and immunoblot analysis were used to evaluate the impact of CB-839 on signaling pathways across the panel of cell lines. Consistent with the observed decreases in amino acid pools, CB-839 treatment for 4 hours led to an acute inhibition of the amino-acid sensing kinase mTORC1 across all cell lines, as evidenced by decreased phosphorylation of p70S6K and S6. CB-839 treatment also reduced the levels of c-Myc across all cell lines, consistent with a block in five-prime cap-dependent translation as a likely consequence of mTORC1 inhibition. However, untreated sensitive cells had lower baseline levels of both 4E-BP1 and phospho-4E-BP1 in comparison to resistant cells, and at 24 hours following treatment, the mTORC1-dependent activation of CAD, a key enzyme in pyrimidine biosynthesis (Ben-Sahra et al., Science 2013 15: 1323-8), was reduced in CB-839-sensitive but not in resistant cells. These results suggest that CB-839 inhibits mTORC1 signaling in MM cells and that sensitive cells may lack compensatory mechanisms to overcome this inhibition. Consistent with differential regulation of mTORC1 in CB-839-sensitive versus resistant cells, RNAseq analysis showed greater down-regulation of several mTORC1-regulated transcripts encoding glycolytic enzymes (such as ALDOA and TPI1, Düvel et al., 2010 Mol. Cell 39: 171-83) in sensitive cells versus resistant cells following CB-839 treatment. RNAseq analysis also revealed elevated levels of pyruvate carboxylase (PC) in CB-839 resistant cells as compared to sensitive cells. Immunoblot analysis of twenty four MM cell lines supported this observation; PC protein levels were inversely correlated with response to CB-839 (r = 0.64, p = 0.001) and PC protein and mRNA transcript levels were highly correlated (r = 0.94, p = 0.0004). PC is an anaplerotic enzyme that converts pyruvate to oxaloacetate. Therefore, sufficiently high levels of glycolytic enzymes and PC could lessen the dependence of a MM cell line on Gln metabolism for replenishment of TCA intermediates. The studies described herein elucidate differences in metabolic and signaling pathways among MM cell lines. These differences in metabolites, mTORC1 signaling, glycolytic enzymes and PC may account for differential sensitivity to CB-839. We are exploring the potential utility of PC expression as a biomarker in our ongoing clinical studies of CB-839. Disclosures Mackinnon: Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership. Rodriguez:Calithera Biosciences: Employment, Equity Ownership. Parlati:Calithera Biosciences: Employment, Equity Ownership.

Many tumor cells are dependent on glutamine (Gln) for growth and survival. Gln is the most abundant amino acid in plasma and tumor cells can utilize Gln for energy production and generation of building blocks for the synthesis of macromolecules. A key step in Gln utilization is its conversion to glutamate (Glu) by the mitochondrial enzyme glutaminase (GLS). Suppressing GLS activity via either siRNA knockdown or small molecule inhibitor treatment has anti-proliferative activity in several tumor types. GLS mRNA levels are elevated in several hematological malignances, particularly those derived from the B-cell lineage including multiple myeloma (MM), acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL) and several types of non-Hodgkin’s lymphoma (NHL). Interestingly, these B-cell derived tumors express low levels of glutamine synthetase (GLUL), an enzyme that reverses the action of GLS by converting Glu to Gln. A high GLS/low GLUL mRNA expression pattern may be indicative of tumor-dependence on extracellular Gln for growth. This expression pattern is observed in triple negative breast cancer (TNBC), a tumor type that we and others have shown to be highly dependent on exogenous Gln for cell proliferation and sensitive to GLS inhibitor treatment. CB-839 is a novel, potent, orally bioavailable GLS inhibitor that inhibits recombinant human GLS with an IC50 of 15 nM. The anti-proliferative activity of CB-839 on a panel of breast cell lines is correlated with a high ratio of GLS to GLUL expression, a pattern associated with the TNBC subtype. Given the similarity of the GLS/GLUL expression pattern observed in TNBC with some hematologic malignancies, we evaluated the anti-tumor activity of CB-839 in cell culture and xenograft models of hematological malignancies predicted to be dependent on Gln for growth. Across a panel of 18 cell lines derived from MM, ALL and diffuse large B-cell lymphoma (DLBCL), Gln deprivation resulted in a minimum of 70% reduction in growth for 16 cell lines and induced cell death in 5 cell lines. Across the same panel of cell lines, the majority were sensitive to CB-839 treatment with 16 cell lines exhibiting an antiproliferative IC50 of Disclosures: Parlati: Calithera BIosciences: Employment. Bromley-Dulfano:Calithera Biosciences: Employment. Demo:Calithera Biosciences: Employment. Janes:Calithera Biosciences: Employment. Gross:Calithera Biosciences: Employment. Lewis:Calithera Biosciences: Employment. MacKinnon:Calithera Biosciences: Employment. Rodriguez:Calithera Biosciences: Employment. Yang:Calithera Biosciences: Employment. Zhao:Calithera Biosciences: Employment. Bennett:Calithera Biosciences: Employment.