Combination Of CB-839 Research Articles

GLS Inhibitor CB-839 Modulates Cellular Metabolism in AML and Potently Suppresses AML Cell Growth when Combined with 5-Azacitidine

Abstract Glutamine (Gln) is required for growth and proliferation of several tumor types including AML. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes conversion of Gln to glutamate (Glu), which provides carbons for the TCA cycle and regulates redox homeostasis through production of glutathione and NADH. CB-839 is a highly selective, reversible, allosteric inhibitor of GLS. In this study we studied metabolic and cellular consequences of GLS inhibition in AML cells cultured in normoxic or hypoxic conditions. First, we performed metabolomic analysis of HL-60 cells co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs). Consistent with the known mechanism of GLS inhibition, CB-839 caused a rapid and extensive decrease in intracellular Glu in both HL60 and MSC and a corresponding increase in intracellular Gln in both cell types. Unexpectedly, CB-839-treated cells exhibited a rapid increase in intracellular and extracellular concentrations of multiple amino acids (Phe, kynurenine, Trp, Leu, Ile, Met, Tyr, Val, Thr, Ala, Gln, Asn, and His), possibly reflecting inhibition of global protein synthesis. CB-839 suppressed cysteine consumption from the extracellular compartment and caused rapid increase in intracellular taurine in HL-60 cells, suggesting altered redox homeostasis (Fig. 1A). CB-839 inhibited cellular growth of HL-60 and MV4;11 AML cells cultured alone or co-cultured with MSC, under conditions mimicking the BM microenvironment (Fig. 1B). Stable isotope-resolved metabolomics (SIRM) analysis with 13C5, 15N2-Gln in HL-60 cells indicated that treatment with CB-839 severely hindered Gln anaplerosis to similar extent under normoxic or hypoxic conditions. Moreover, Gln is predominantly used to carry out oxidative metabolism. The enriched fraction of aspartate in treated cells dropped dramatically (to approximately 20% or less of the pool), suggesting that leukemia cells require Krebs cycle-derived oxaloacetate transamination for the generation of aspartate (Fig. 1C). Limiting Gln supply using CB-839 caused reduction in the concentration of alpha-ketoglutarate (α-KG) and the oncometabolite 2-hydroxyglutarate (2-HG), known to play a role in the pathogenesis of AML. We have previously shown that the leukemic BM microenvironment is highly hypoxic (Benito PLoS One 2011), andhypoxia has been reported to induce production of the L-enantiomer of 2-HG (L-2HG) (Intlekofer Cell Metabolism 2015). In AML cells, hypoxia selectively induced the production of L-2HG measured by LC-MS/MS in HL-60 (6.2 fold) and OCI-AML3 cells (2.9 fold) with wt-IDH. This increase in L-2HG was potently inhibited by CB-839, implicating Gln as a source for L-2HG production by AML cells under hypoxia. HL-60 and OCI-AML3 AML cells produced very limited amounts of the D-enantiomer of 2HG (D-2HG), and neither hypoxia nor CB-839 significantly affected D-2HG levels. We recently reported that CB-839 increased hydroxymethylation (hmc) levels using a HELP-GT assay (Velez ASH 2015), and the implications of those observations are the subject of ongoing studies. Prompted by the observation of increased hmc in response to CB-839 treatment, we next examined the efficacy of CB-839 in combination with the DNMT3A inhibitor 5-azacitidine (5-AZA). Treatment with 1µM CB-839 and escalating doses of 5-AZA caused additive or synergistic inhibition of cellular growth after 5 days of culture, both under normoxia and hypoxia, in AML cell lines (OCI-AML3, HL-60, MV4;11) and in primary AML cells (n=3) (Fig. 1D). To test the efficacy of both compounds in vivo, we injected NSG-S mice with genetically engineered MV4;11/Luc cells. Bioluminescent imaging (BLI) demonstrated significantly reduced leukemia burden in treated groups compared to controls, more prominently in the CB-839 plus 5-AZA co-treated mice. CB-839 and 5-AZA co-treatment resulted in significant extension of survival compared with 5-AZA single agent, p In summary, GLS inhibition causes AML growth arrest by multiple mechanisms, including inhibition of protein synthesis and disruption of redox homeostasis. Gln contributes to hypoxia-induced production of L-2HG and possibly epigenome regulation in AML, and concomitant blockade of GLS by CB-839 and DNMT3A with 5-AZA potently suppresses AML cell growth in vitro and in vivo. The clinical trial examining the efficacy of this combination is ongoing (Calithera, NCT02071927). Download : Download high-res image (328KB) Download : Download full-size image Disclosures Lorenzi: Erytech Pharma: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties: NIH-held patent related to L-asparaginase. DiNardo: Novartis: Research Funding; Abbvie: Research Funding; Agios: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding. Konopleva: Calithera: Research Funding; Cellectis: Research Funding.

Abstract 430: Combination of CB-839 and everolimus is effective in inhibiting growth of endocrine resistant breast cancer in vivo

Abstract About 70% of all breast cancers are estrogen receptor alpha positive (ER+). Anti-hormone therapy such as antiestrogens are often used to treat ER+ breast cancer but breast cancer cells can develop resistance to these drugs (endocrine resistance). Unfortunately, ~50% percent of all antiestrogen treated tumors eventually develop endocrine resistance, and therefore, there is an urgent need to find ways to treat this incurable disease. We have shown that endocrine resistant breast cancer cells show an increased dependence on the amino acid glutamine and this process is regulated by MYC activation via the unfolded protein response (UPR). Metabolites of glutamine such as glutamate and proline are significantly elevated in endocrine resistant cells. Cellular changes in glutamine are sensed by the mammalian target of rapamycin (mTOR) complex, mTORC1, which is known to be deregulated in endocrine resistant breast cancer. In this study, we used human antiestrogen (Fulvestrant/Faslodex or Tamoxifen) sensitive and resistant ER+ breast cancer cells and xenografts to test the efficacy of CB-839, an anti-glutaminase, and everolimus (Afinitor), a mTORC1 inhibitor. Combination of CB-839 and everolimus, but not each drug alone, inhibited growth of antiestrogen resistant tumors compared to vehicle alone at 4 weeks post-treatment. The combination treatment did not significantly inhibit growth of antiestrogen sensitive tumors compared with its respective vehicle alone treatment. Thus, a combination strategy that targets glutamine dependence and increased mTOR activation may be a novel strategy in treating endocrine resistant breast cancer. Citation Format: Yassi Fallah, Diane M. Demas, Susan Demo, Ayesha N. Shajahan-Haq. Combination of CB-839 and everolimus is effective in inhibiting growth of endocrine resistant breast cancer in vivo [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 430. doi:10.1158/1538-7445.AM2017-430

Abstract 2851: Cellular glutamine pool size change in response to glutaminase inhibition detected by kinetic analysis of [18F](2S,4R)4-fluoroglutamine PET

Abstract INTRODUCTION Oncogene-dependent reliance on glutamine is a cancer vulnerability that can be exploited for therapeutic gain. Inhibitors of glutaminase, the enzyme that catalyzes the conversion of glutamine to glutamate, have been developed and have shown antitumor effects in several tumor models. The specific and potent glutaminase inhibitor CB-839 demonstrated marked antitumor efficacy in a triple-negative breast cancer (TNBC) cell line with inherently high glutaminase activity (HCC-1806), but not in an estrogen receptor-positive (ER+) cell line with inherently low glutaminase activity. An early phase 1 clinical trial of CB-839 in combination with paclitaxel has shown encouraging results in TNBC patients. Cell-line specific efficacy of glutaminase inhibitors supports the need for clinical biomarkers that can predict and evaluate therapeutic efficacy. As a minimally metabolized glutamine analog with similar transport properties, [18F](2S,4R)4-Fluoroglutamine ([18F]4F-Gln) is an ideal radiotracer to infer glutamine pool size through estimates of tracer distribution volume (DV). In this study, we demonstrate differences in DV of [18F]4F-Gln in two xenografts with different levels of glutaminolysis (TNBC vs. ER+) using dynamic PET imaging, as well as show the effect of anti-glutaminase therapy on [18F]4F-Gln DV. METHODS TNBC (HCC-1806) and ER+ (MCF-7) xenografts were established in athymic nu/nu mice. Mice were scanned in a dedicated animal PET scanner at baseline and after CB-839 administration. Dynamic imaging was obtained for one hour upon injection of [18F]4F-Gln (300-350 μCi) via the tail vein. Kinetic analysis was performed with PMOD. RESULTS [18F]4F-Gln uptake was largely reversible with Logan plots demonstrating late linearity and k3 in a two-compartment (trapping) model was low (less than 0.01/min in most cases). Strong correlation was seen in DV estimates by Logan plot and a single-compartmental model (R2>0.9), but not with estimates from a two-compartment model. MCF-7 xenografts demonstrated greater than 60% larger DV at baseline than TNBC xenografts indicative of increased cellular glutamine concentrations in MCF-7 xenografts. An increase in DV was detected in TNBC xenografts post-glutaminase inhibition (>30% mean change), but not in MCF-7 xenografts. CONCLUSION Estimates of [18F]4F-Gln DV offer the ability to non-invasively infer tumor glutaminolysis. Low [18F]4F-Gln uptake in highly glutaminolytic tumors and an increase in [18F]4F-Gln uptake with glutaminase inhibition is concordant with changes in cellular glutamine concentration as measured by MR spectroscopy of tumor extracts. Our studies indicate promise for the use of [18F]4F-Gln as a predictive and pharmacodynamic marker for glutaminase-targeted therapy. Supported by: Komen SAC130060, DE-SE0012476, R21-CA198563 We thank Calithera Inc. for providing CB-839 and Dr. Susan Demo for discussions. Citation Format: Austin R. Pantel, Hsiaoju Lee, Shihong Li, Robert K. Doot, Robert H. Mach, David A. Mankoff, Rong Zhou. Cellular glutamine pool size change in response to glutaminase inhibition detected by kinetic analysis of [18F](2S,4R)4-fluoroglutamine PET [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 2851. doi:10.1158/1538-7445.AM2017-2851

Abstract 3551: Targeting glutamine addiction of PIK3CA mutant colorectal cancers

Abstract Glutamine addiction is a major metabolic reprogramming event that occurs in cancer cells. Many tumors exhibit oncogene-dependent addiction to glutamine. PIK3CA, which encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase α, is the most frequently mutated oncogene in human cancers. However, whether PIK3CA mutations reprogram cancer metabolism is an important unaddressed question. Here we show that oncogenic PIK3CA mutations render colorectal cancers (CRCs) more dependent on glutamine to growth. As a metabolite, glutamine is first converted to glutamate by glutaminase (GLS) and then to α-ketoglutarate (α-KG) by either a transaminase or a glutamate dehydrogenase. Calithera Biosciences recently developed a potent GLS inhibitor called CB-839, which is currently in phase I clinical trials in cancer patients. Using isogenic colorectal cancer cell lines with either WT or mutant PIK3CA allele knockout, we demonstrated that CRCs with PIK3CA mutations are more sensitive to growth inhibition by CB-839. Remarkably, combination of CB-839 with 5-FU induces regression xenograft tumors in PIK3CA mutant CRC models, suggesting that this combinational therapy may be effective approach to treat CRC patients whose tumors harbor PIK3CA mutations. Mechanistically, mutant p110α up-regulates gene expression of glutamate pyruvate transaminase 2 (GPT2) through an AKT-independent PDK1-RSK2-ATF4 signaling axis, thereby facilitating conversion of glutamate to α-KG. Together, our data establish oncogenic PIK3CA mutations as a cause of glutamine addiction in CRCs and that targeting glutamine metabolism may be effective approach to treat CRCs with PIK3CA mutations. Citation Format: Yiqing Zhao. Targeting glutamine addiction of PIK3CA mutant colorectal 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 3551. doi:10.1158/1538-7445.AM2017-3551

Targeted Inhibition of EGFR and Glutaminase Induces Metabolic Crisis in EGFR Mutant Lung Cancer

Cancer cells exhibit increased use of nutrients, including glucose and glutamine, to support the bioenergetic and biosynthetic demands of proliferation. We tested the small-molecule inhibitor ofglutaminase CB-839 in combination with erlotinib on epidermal growth factor receptor (EGFR) mutant non-small cell lung cancer (NSCLC) as a therapeutic strategy to simultaneously impair cancer glucose and glutamine utilization and thereby suppress tumor growth. Here, we show that CB-839 cooperates with erlotinib to drive energetic stress and activate the AMP-activated protein kinase (AMPK) pathway in EGFR (del19) lungtumors. Tumor cells undergo metabolic crisis and cell death, resulting in rapid tumor regression invivo in mouse NSCLC xenografts. Consistently, positron emission tomography (PET) imaging with 18F-fluoro-2-deoxyglucose (18F-FDG) and 11C-glutamine (11C-Gln) of xenografts indicated reduced glucose and glutamine uptake in tumors following treatment with CB-839+ erlotinib. Therefore,PET imaging with 18F-FDG and 11C-Gln tracers can be used to non-invasively measure metabolic response to CB-839 and erlotinib combination therapy.

Glutaminase Inhibitor CB-839 Enhances Proteasome Inhibitor Sensitivity in Multiple Myeloma Cells

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. DisclosuresNo relevant conflicts of interest to declare.

Gls Inhibitor CB-839 Modulates Cellular Metabolism in AML and Potently Suppresses AML Cell Growth When Combined with 5-Azacitidine

Glutamine (Gln) is required for growth and proliferation of several tumor types including AML. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes conversion of Gln to glutamate (Glu), which provides carbons for the TCA cycle and regulates redox homeostasis through production of glutathione and NADH. CB-839 is a highly selective, reversible, allosteric inhibitor of GLS. In this study we studied metabolic and cellular consequences of GLS inhibition in AML cells cultured in normoxic or hypoxic conditions.First, we performed metabolomic analysis of HL-60 cells co-cultured with bone marrow (BM)-derived mesenchymal stem cells (MSCs). Consistent with the known mechanism of GLS inhibition, CB-839 caused a rapid and extensive decrease in intracellular Glu in both HL60 and MSC and a corresponding increase in intracellular Gln in both cell types. Unexpectedly, CB-839-treated cells exhibited a rapid increase in intracellular and extracellular concentrations of multiple amino acids (Phe, kynurenine, Trp, Leu, Ile, Met, Tyr, Val, Thr, Ala, Gln, Asn, and His), possibly reflecting inhibition of global protein synthesis. CB-839 suppressed cysteine consumption from the extracellular compartment and caused rapid increase in intracellular taurine in HL-60 cells, suggesting altered redox homeostasis (Fig. 1A). CB-839 inhibited cellular growth of HL-60 and MV4;11 AML cells cultured alone or co-cultured with MSC, under conditions mimicking the BM microenvironment (Fig. 1B).Stable isotope-resolved metabolomics (SIRM) analysis with 13C5, 15N2-Gln in HL-60 cells indicated that treatment with CB-839 severely hindered Gln anaplerosis to similar extent under normoxic or hypoxic conditions. Moreover, Gln is predominantly used to carry out oxidative metabolism. The enriched fraction of aspartate in treated cells dropped dramatically (to approximately 20% or less of the pool), suggesting that leukemia cells require Krebs cycle-derived oxaloacetate transamination for the generation of aspartate (Fig. 1C). Limiting Gln supply using CB-839 caused reduction in the concentration of alpha-ketoglutarate (α-KG) and the oncometabolite 2-hydroxyglutarate (2-HG), known to play a role in the pathogenesis of AML.We have previously shown that the leukemic BM microenvironment is highly hypoxic (Benito PLoS One 2011), andhypoxia has been reported to induce production of the L-enantiomer of 2-HG (L-2HG) (Intlekofer Cell Metabolism 2015). In AML cells, hypoxia selectively induced the production of L-2HG measured by LC-MS/MS in HL-60 (6.2 fold) and OCI-AML3 cells (2.9 fold) with wt-IDH. This increase in L-2HG was potently inhibited by CB-839, implicating Gln as a source for L-2HG production by AML cells under hypoxia. HL-60 and OCI-AML3 AML cells produced very limited amounts of the D-enantiomer of 2HG (D-2HG), and neither hypoxia nor CB-839 significantly affected D-2HG levels. We recently reported that CB-839 increased hydroxymethylation (hmc) levels using a HELP-GT assay (Velez ASH 2015), and the implications of those observations are the subject of ongoing studies.Prompted by the observation of increased hmc in response to CB-839 treatment, we next examined the efficacy of CB-839 in combination with the DNMT3A inhibitor 5-azacitidine (5-AZA). Treatment with 1µM CB-839 and escalating doses of 5-AZA caused additive or synergistic inhibition of cellular growth after 5 days of culture, both under normoxia and hypoxia, in AML cell lines (OCI-AML3, HL-60, MV4;11) and in primary AML cells (n=3) (Fig. 1D).To test the efficacy of both compounds in vivo, we injected NSG-S mice with genetically engineered MV4;11/Luc cells. Bioluminescent imaging (BLI) demonstrated significantly reduced leukemia burden in treated groups compared to controls, more prominently in the CB-839 plus 5-AZA co-treated mice. CB-839 and 5-AZA co-treatment resulted in significant extension of survival compared with 5-AZA single agent, p<0.001 (Fig. 1E).In summary, GLS inhibition causes AML growth arrest by multiple mechanisms, including inhibition of protein synthesis and disruption of redox homeostasis. Gln contributes to hypoxia-induced production of L-2HG and possibly epigenome regulation in AML, and concomitant blockade of GLS by CB-839 and DNMT3A with 5-AZA potently suppresses AML cell growth in vitro and in vivo. The clinical trial examining the efficacy of this combination is ongoing (Calithera, NCT02071927). [Display omitted] DisclosuresLorenzi:Erytech Pharma: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Patents & Royalties: NIH-held patent related to L-asparaginase. DiNardo:Novartis: Research Funding; Abbvie: Research Funding; Agios: Research Funding; Celgene: Research Funding; Daiichi Sankyo: Research Funding. Konopleva:Calithera: Research Funding; Cellectis: Research Funding.

Abstract 1004: GLS inhibitor CB-839 modulates cellular metabolism in AML and potently suppresses AML cell growth when combined with 5-azacitidine

Abstract Glutamine (Gln) is required for growth and proliferation of several tumor types including AML. Glutaminase (GLS) is a mitochondrial enzyme that catalyzes conversion of Gln to glutamate (Glu), which provides carbons for the TCA cycle and regulates redox homeostasis through production of glutathione and NADH. CB-839 is a highly selective, reversible, allosteric inhibitor of GLS. In this study we studied metabolic and cellular consequences of GLS inhibition in AML cells cultured in normoxic or hypoxic conditions. First, we performed metabolic analysis of HL-60 cells co-cultured with bone marrow-derived mesenchymal stem cells (MSCs). Consistent with the known mechanism of GLS inhibition, CB-839 caused a rapid and extensive decrease in intracellular Glu in both HL60 and MSC and a corresponding increase in intracellular Gln in both cell types. Unexpectedly, CB-839-treated cells exhibited a rapid increase in intracellular and extracellular concentrations of multiple amino acids, possibly reflecting inhibition of global protein synthesis. CB-839 suppressed Cys consumption from the extracellular compartment and caused rapid increase in intracellular taurine in HL-60 cells, suggesting altered redox homeostasis. CB-839 inhibited cellular growth of HL-60 and MV4;11 AML cells cultured alone or co-cultured with MSC, demonstrating activity under conditions mimicking BM microenvironment. We have previously shown that the leukemic bone marrow microenvironment is highly hypoxic (Benito PLoS One 2011), and hypoxia has been reported to induce production of the L-enantiomer of 2-HG (L-2HG) (Intlekofer Cell Metabolism 2015). In AML cells, hypoxia selectively induced the production of L-2HG (measured by LC-MS/MS) in HL-60 (6.2+/- fold) and OCI-AML3 cells (2.9+/- fold) with wt-IDH. That increase in L-2HG was potently inhibited by CB-839. AML cells produced very little D-2HG, and neither hypoxia nor CB-839 significantly affected D-2HG levels. We recently reported that CB-839 increased hydroxymethylation (hmc) levels using a HELP-GT assay (Velez ASH 2015), and the implications of those observations are the subject of ongoing studies. Prompted by the observation of increased hmc in response to CB-839 treatment, we next examined the efficacy of CB-839 in combination with the DNMT3A inhibitor 5-azacitidine (5-AZA). Treatment with 1μM CB-839 and escalating doses of 5-AZA caused additive or synergistic inhibition of cellular growth after 5 days of culture, both under normoxia and hypoxia, in AML cell lines (OCI-AML3, HL-60, MV4;11) and primary AML cells (n = 6). In summary, GLS inhibition causes AML growth arrest by multiple mechanisms, including inhibition of protein synthesis and disruption of redox homeostasis. Gln contributes to hypoxia-induced production of L-2HG and possibly epigenome regulation in AML, and concomitant blockade of GLS by CB-839 and DNMT3A with 5-AZA potently suppresses AML cell growth. Citation Format: Tianyu Cai, Philip L. Lorenzi, Dinesh Rakheja, Michael Pontikos, Lina Han, Qi Zhang, Helen Ma, Thomas D. Horvath, Amit K. Verma, Marina Konopleva. GLS inhibitor CB-839 modulates cellular metabolism in AML and potently suppresses AML cell growth when combined with 5-azacitidine. [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 1004.

Phase 1 Study of CB-839, a First-in-Class, Glutaminase Inhibitor in Patients with Multiple Myeloma and Lymphoma

Phase 1 Study of CB-839, a First-in-Class, Glutaminase Inhibitor in Patients with Multiple Myeloma and Lymphoma

Abstract C49: Phase 1 study of CB-839, a first-in-class, orally administered small molecule inhibitor of glutaminase in patients with refractory solid tumors

Abstract Background: CB-839 is a first-in-class highly selective inhibitor of glutaminase (GLS), a key enzyme in the utilization of glutamine by many cancer cells. Inhibition of GLS blocks the ability of tumor cells to use glutamine as a source of energy and anabolic building blocks. CB-839 has broad activity, inducing cell death or growth arrest in triple negative breast cancer (TNBC), KRAS-mutant non-small cell lung cancer (NSCLC), clear cell renal cell cancer (RCC), and mesothelioma cell lines. GLS inhibition with CB-839 has also been shown to synergistically combine with several standard of care (SOC) chemotherapies and signal transduction inhibitors. Methods: CX-839-001 is an ongoing Phase 1 study of escalating doses of CB-839 given continuously in 21 day cycles in advanced and/or treatment-refractory solid tumor patients (pts) to evaluate safety and tolerability, and to identify the recommended Phase 2 dose (R2PD). It was initially given three times daily (TID) without food, but based on PK and safety data, the drug is now being given twice daily (BID) with meals. Pharmacokinetics (PK) is monitored on Days 1 and 15; pharmacodynamics is measured in platelets and in tumor biopsies. Pts are currently being enrolled at the RP2D, 600 mg BID, with the tumor types noted above as well as pts receiving CB-839 in combination with SOC agents, including paclitaxel in TNBC, docetaxel in KRAS-mutant NSCLC, erlotinib in EGFR-mutant NSCLC, and everolimus in RCC. Results: Safety data from 85 pts enrolled in the trial revealed a low rate of Gr3/4 AEs, with 9.4% (8/85) of pts experiencing a Gr3/4 AE related to CB-839. One DLT occurred on study, a drug-related Gr3 creatinine elevation in a patient with diabetic nephropathy at the 250 mg TID dose level. Reversible, asymptomatic Gr3 elevations in transaminases occurred mostly on the TID schedule in 6/32 pts (18.8%), with only one Gr3 event occurring among 53 pts (1.9%) receiving the BID regimen. Clear exposure-dependent inhibition of GLS in platelets was seen 4 hr after the first dose of CB-839, with &amp;gt;90% inhibition at PK trough for most pts at the RP2D. A total of 93 pts have been enrolled on the monotherapy cohorts of the trial as of 24 July 2015, including 32 pts on TID dosing (100-800 mg) and 61 pts on BID dosing (600-1000 mg). Radiographic stable disease (SD) was observed in 6 (19%) of 31 efficacy-evaluable pts receiving CB-839 on the TID schedule and 17 (40%) of 43 efficacy-evaluable pts on the BID schedule. Pts with SD have been on study for a median of &amp;gt;5 cycles (range 3-18); 15 (65%) of these pts remain on treatment. Seven of 11 (64%) evaluable RCC pts on the BID dose schedule showed SD, 5 of whom remain on study. A TNBC patient has maintained SD for more than one year and has had a 23% reduction in tumor burden. GLS expression was evaluated by IHC and found to be moderate to high in most patient tumor samples. Additional correlative studies will also be presented. Conclusions: CB-839 has been well tolerated when administered BID with food at and above doses that produced robust inhibition of GLS in platelets and in tumors. Evidence of prolonged SD in pts receiving single agent CB-839, together with strong preclinical data in combination with SOC agents including paclitaxel, everolimus and erlotinib, provides a clear rationale for continued evaluation of CB-839 in several tumor types, as a single agent and in combination with SOC therapies. Citation Format: Funda Meric-Bernstam, Angela DeMichele, Melinda L. Telli, Pamela Munster, Keith W. Orford, George D. Demitri, Gary K. Schwartz, Othon Iliopoulos, James W. Mier, Taofeek K. Owonikoko, Mark K. Bennett, Manish R. Patel, Jeffery R. Infante, James J. Harding. Phase 1 study of CB-839, a first-in-class, orally administered small molecule inhibitor of glutaminase in patients with refractory solid tumors. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C49.

Abstract C115: Targeting glutamine metabolism in colorectal cancers with PIK3CA mutations

Abstract Glutamine addiction is a major metabolic reprogramming event that occurs in cancer cells. Many tumors exhibit oncogene-dependent addiction to glutamine. PIK3CA, which encodes the p110α catalytic subunit of phosphatidylinositol 3-kinase α, is the most frequently mutated oncogene in human cancers. However, whether PIK3CA mutations reprogram cancer metabolism is an important unaddressed question. Here we show that oncogenic PIK3CA mutations render colorectal cancers (CRCs) more dependent on glutamine to growth. As a metabolite, glutamine is first converted glutamate by glutaminase (GLS) and then to α-ketoglutarate (α-KG) by either a transaminase or a glutamate dehydrogenase. Calithera Biosciences recently developed a potent GLS inhibitor called CB-839, which is currently in phase I clinical trials in cancer patients. Using isogenic colorectal cancer cell lines with either WT or mutant PIK3CA allele knockout, we demonstrated that CRCs with PIK3CA mutations are more sensitive to growth inhibition by CB-839. Remarkably, combination of CB-839 with 5-FU induces regression xenograft tumors from a CRC with a PIK3CA mutation, suggesting that this combinational therapy may be effective approach to treat CRC patients whose tumors harbor PIK3CA mutations. Mechanistically, mutant p110α up-regulates gene expression of glutamate pyruvate transaminase 2 (GPT2) in CRC cells, thereby facilitate conversion of glutamate to α-KG. Using [13C5-]glutamine isotope-tracing technology, we showed that PIK3CA mutant CRCs produce more α-KG from glutamine to α-KG to replenish the tricarboxylic acid (TCA) cycle to generate ATP. Moreover, aminooxyacetate, which inhibits enzymatic activity of transaminases including GPT2, suppresses xenograft tumor growth of CRCs with PIK3CA mutations, but not CRCs with WT PIK3CA. Mutant p110α up-regulates the transcription of GPT2 through an AKT-independent PDK1-RSK2-ATF4 signaling axis. We showed that ATF4 is a transcription factor that activates GPT2 gene expression. We further demonstrated that mutant p110α activates RSK2 kinase through PDK1. Activated RSK2 then phosphorylates ATF4 at the serine residue 245, which in turn recruits deubiquitinase USP8 and protects ATF4 from ubiquitin-mediated degradation. Together, our data establish oncogenic PIK3CA mutations as a cause of glutamine addiction in CRCs and that targeting glutamine metabolism may be effective approach to treat CRCs with PIK3CA mutations. Citation Format: Zhenghe John Wang, Yujun Hao. Targeting glutamine metabolism in colorectal cancers with PIK3CA mutations. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr C115.

Glutaminase Inhibitor CB-839 Synergizes with Pomalidomide in Preclinical Multiple Myeloma Models

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. DisclosuresParlati: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.

Anti-Myeloma Activity of a Novel Glutaminase Inhibitor CB-839

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. DisclosuresMunshi: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.