Julie Janes

Antitumor Activity of the Glutaminase Inhibitor CB-839 in Triple-Negative Breast Cancer

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.

Inhibition of arginase by CB-1158 blocks myeloid cell-mediated immune suppression in the tumor microenvironment

BackgroundMyeloid cells are an abundant leukocyte in many types of tumors and contribute to immune evasion. Expression of the enzyme arginase 1 (Arg1) is a defining feature of immunosuppressive myeloid cells and leads to depletion of L-arginine, a nutrient required for T cell and natural killer (NK) cell proliferation. Here we use CB-1158, a potent and orally-bioavailable small-molecule inhibitor of arginase, to investigate the role of Arg1 in regulating anti-tumor immunity.MethodsCB-1158 was tested for the ability to block myeloid cell-mediated inhibition of T cell proliferation in vitro, and for tumor growth inhibition in syngeneic mouse models of cancer as a single agent and in combination with other therapies. Tumors from animals treated with CB-1158 were profiled for changes in immune cell subsets, expression of immune-related genes, and cytokines. Human tumor tissue microarrays were probed for Arg1 expression by immunohistochemistry and immunofluorescence. Cancer patient plasma samples were assessed for Arg1 protein and L-arginine by ELISA and mass spectrometry, respectively.ResultsCB-1158 blocked myeloid cell-mediated suppression of T cell proliferation in vitro and reduced tumor growth in multiple mouse models of cancer, as a single agent and in combination with checkpoint blockade, adoptive T cell therapy, adoptive NK cell therapy, and the chemotherapy agent gemcitabine. Profiling of the tumor microenvironment revealed that CB-1158 increased tumor-infiltrating CD8+ T cells and NK cells, inflammatory cytokines, and expression of interferon-inducible genes. Patient tumor samples from multiple histologies expressed an abundance of tumor-infiltrating Arg1+ myeloid cells. Plasma samples from cancer patients exhibited elevated Arg1 and reduced L-arginine compared to healthy volunteers.ConclusionsThese results demonstrate that Arg1 is a key mediator of immune suppression and that inhibiting Arg1 with CB-1158 shifts the immune landscape toward a pro-inflammatory environment, blunting myeloid cell-mediated immune evasion and reducing tumor growth. Furthermore, our results suggest that arginase blockade by CB-1158 may be an effective therapy in multiple types of cancer and combining CB-1158 with standard-of-care chemotherapy or other immunotherapies may yield improved clinical responses.

Abstract 552: Immuno-oncology agent CB-1158 is a potent and selective arginase inhibitor and causes an immune-mediated anti-tumor response

L-arginine is a critical metabolite for T-cell receptor signaling and subsequent T-cell proliferation, and depletion of arginine arrests T-cell growth. In the tumor microenvironment, infiltrating myeloid-derived suppressor cells (MDSCs), macrophages, and neutrophils produce arginase, which depletes local arginine concentrations and dampens T cell-mediated immune surveillance. Pharmacological inhibition of arginase is expected to restore arginine levels and allow T-cells to proliferate, thereby leading to an immune-mediated anti-tumor response. CB-1158 is a potent inhibitor of human arginase (IC50 = 98 nM). In culture, human granulocytes release arginase and deplete media arginine to levels that inhibit T-cell proliferation. In a co-culture system of human granulocytes and T-cells, CB-1158 potently blocks granulocyte-derived arginase activity, maintains extracellular arginine levels, and restores proliferation of T-cells. CB-1158 has high oral bioavailability in rodents and is very well tolerated. BID oral dosing of CB-1158 leads to dose-dependent pharmacodynamic increases in plasma and tumor arginine levels resulting in single agent anti-tumor efficacy in mouse syngeneic tumor models including Lewis Lung carcinoma (LLC) and Madison 109. The anti-tumor effects of CB-1158 are consistent with promoting a proinflammatory tumor microenvironment. Following CB-1158 treatment, multiple Th1 T-cell, NK-cell, and M1 macrophage-associated chemokines, cytokines, and activation markers are elevated in the LLC tumor microenvironment. The anti-tumor efficacy of CB-1158 requires an intact tumor microenvironment since CB-1158 has no effect on LLC cell growth in vitro. Furthermore, CB-1158 treatment of immunocompromised C57/SCID mice bearing LLC tumors has no anti-tumor effect, supporting an immune-mediated anti-tumor mechanism. Immunosuppression in the tumor microenvironment can occur via multiple mechanisms, including arginine depletion, and our data support the combination of checkpoint inhibitors and arginase inhibition by CB-1158. In mice bearing LLC tumors, CB-1158 in combination with checkpoint inhibitors reduced tumor growth, increased the number of tumor infiltrating CD8+ T-cells, and increased the level of Th1/NK/M1-associated chemokines, cytokines, and activation markers in the tumor microenvironment. In mice bearing 4T1 tumors, a tumor type that is highly refractory to checkpoint inhibition, the combination of CB-1158 with anti-PD-1 and anti-CTLA-4 reduces tumor growth and lung metastases. These results support the development of CB-1158, a first-in-class arginase inhibitor, as a novel immuno-oncology agent targeting the immunosuppressive effects of tumor-infiltrating myeloid cells. Citation Format: Melissa Works, Mark Bennett, Jason Chen, Ethan Emberley, Tony Huang, Julie Janes, Weiqun Li, Andy Mackinnon, Gisele Marguier, Silinda Neou, Alison Pan, Francesco Parlati, Mirna Rodriguez, Susanne Steggerda, Tracy Wang, Jing Zhang, Winter Zhang, Matthew Gross. Immuno-oncology agent CB-1158 is a potent and selective arginase inhibitor and causes an immune-mediated anti-tumor response. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 552.

Abstract 1416: CB-839, a novel potent and selective glutaminase inhibitor, has broad antiproliferative activity in cell lines derived from both solid tumors and hematological malignancies

Many tumor cells utilize altered metabolic pathways to meet the bioenergetic and biosynthetic demands of rapid and sustained growth. One of the key nutrients that fuels tumor growth is the amino acid glutamine. It has long been recognized that the growth and survival of many tumor cell lines in vitro is dependent on extracellular glutamine. A critical step in the utilization of glutamine is its conversion to glutamate by the mitochondrial enzyme glutaminase. Glutamate and glutamate-derived metabolites in turn support a number of crucial cellular pathways including the citric acid cycle, redox balance and amino acid synthesis. CB-839 is a novel and selective inhibitor of glutaminase that has antitumor activity in preclinical models of triple negative breast cancer (TNBC), a tumor type that is particularly dependent on glutamine. Across a panel of breast cancer cell lines, the activity of CB-839 correlates with high expression of glutaminase, specifically the GAC splice variant but not the KGA splice variant, and low expression of glutamine synthetase (GLUL), an enzyme that converts glutamate to glutamine. This expression pattern is found in primary TNBC tumors suggesting a reliance on exogenous glutamine and glutaminase activity in vivo. To determine if other tumor types have a similar expression pattern, we undertook a systematic evaluation of GAC, KGA and GLUL expression across a diverse set of primary tumors using a normalized microarray dataset allowing comparison across a range of tumor and normal tissue samples. Elevated GAC and decreased GLUL expression relative to other tumor types or corresponding normal tissue was identified in a number of tumor types including non-small cell lung cancer, multiple myeloma, and non-Hodgkin9s lymphoma. To explore whether this expression pattern predicts a reliance on glutamine and glutaminase, we tested the glutamine dependence and the activity of CB-839 on a panel of 72 cell lines representing 5 tumor types indicated by the primary tumor expression analysis. Across this cell line panel, the majority were dependent on glutamine showing either cell death, growth arrest or slowed growth after glutamine withdrawal. Similarly, CB-839 had antiproliferative activity in the majority of cell lines with IC50s in the range of 10-300 nM. Importantly, there was a strong correlation between glutamine dependence and response to 1 µM CB-839 as measured by relative cell growth or death (correlation coefficient 0.72, p Citation Format: Francesco Parlati, Susan D. Demo, Matthew I. Gross, Julie R. Janes, Evan R. Lewis, Andy L. MacKinnon, Mirna L.M. Rodriguez, Peter J. Shwonek, Taotao Wang, Jinfu Yang, Dong Zhang, Frances Zhao, Mark K. Bennett. CB-839, a novel potent and selective glutaminase inhibitor, has broad antiproliferative activity in cell lines derived from both solid tumors and hematological malignancies. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1416. doi:10.1158/1538-7445.AM2014-1416

Abstract 2329: Glutaminase inhibition with CB-839 enhances anti-tumor activity of PD-1 and PD-L1 antibodies by overcoming a metabolic checkpoint blocking T cell activation

Recent studies have highlighted the importance of the tumor metabolic environment for controlling immune activation. T-cells activated through the TCR/CD28 receptor switch to a highly glycolytic metabolism and increase their requirement for glucose and glutamine. Consequently, limited availability of glucose or glutamine can block T-cell activation and proliferation. Likewise, immune checkpoints proteins, PD-1 and CTLA-4, suppress T cell metabolism by inhibiting glycolysis, glutamine uptake and glutaminolysis (Patsoukis et al Nat Comm. 2015). Chang et al (Cell. 2015) recently demonstrated that glucose consumption by tumors restricts glucose availability and blocks activation of T cells, and that treatment with CTLA-4, PD-1, or PD-L1 antibodies can re-activate T cell glycolysis. CB-839 is a glutaminase inhibitor currently in Phase 1 trials in patients with solid and hematological malignancies. CB-839 blocks glutamine consumption in tumors and causes a significant elevation of tumor glutamine levels. Therefore, we hypothesized that CB-839 might enhance the activity of immune checkpoint inhibitors via metabolic modulation of the tumor microenvironment. We first confirmed that T- cell proliferation is dependent on glutamine but is only minimally inhibited by CB-839. In the absence of glutamine, splenic mouse T cells stimulated with anti-CD3/CD28 had reduced glucose consumption and did not proliferate. In contrast, CB-839 treatment did not mimic the effects of glutamine withdrawal on T-cells. CB-839 had no effect on glucose consumption by activated T-cells and only a minimal effect on proliferation. We also confirmed in the OVA vaccinia model that CB-839 had minimal effects on CD4 and CD8 T-cell proliferation in vivo, while the non-specific glutamine inhibitor DON caused a dramatic reduction in the number of CD4 and CD8 T-cells. To determine if CB-839 could enhance the anti-tumor efficacy of immune checkpoint inhibitors, we treated mice bearing syngeneic CT26 colon carcinoma tumors with anti PD-1 or anti PD-L1 alone or in combination with CB-839. The addition of CB-839 to either anti PD-1 or anti PD-L1 treatment enhanced anti-tumor activity, augmenting tumor regression and promoting survival. Depletion of CD8+ T-cells from CT26 tumors reversed the anti-tumor effects of PD-L1 and CB-839, demonstrating that the combination targets CD8+ T-cells in the immune microenvironment. These data are the first demonstration that modulation of glutamine metabolism in tumors can enhance the activity of checkpoint inhibitors and provide a rationale for combining CB-839 with immune checkpoint inhibitors in the clinic. Overall, these data highlight a new therapeutic approach to treating cancer by targeting tumor metabolism as a means of enhancing the efficacy of immunotherapy. Citation Format: Matt Gross, Jason Chen, Judd Englert, Julie Janes, Robert Leone, Andy MacKinnon, Francesco Parlati, Mirna Rodriquez, Peter Shwonek, Jonathan Powell. Glutaminase inhibition with CB-839 enhances anti-tumor activity of PD-1 and PD-L1 antibodies by overcoming a metabolic checkpoint blocking T cell activation. [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 2329.

Abstract A195: CB-1158 inhibits the immuno-oncology target arginase and causes an immune mediated anti-tumor response

The role of myeloid derived suppressor cells (MDSCs) has become increasingly recognized as an important mechanism of tumor immune evasion. However, to date there are no effective means to antagonize the immunosuppressive activity of these cells in patients. MDSCs suppress cytotoxic T-cells and Natural Killer cells through the secretion of the enzyme arginase which depletes local arginine concentrations. The depletion of arginine in the tumor microenvironment renders cytotoxic T-cells unable to proliferate and therefore unable to effectively mount an anti-tumor attack. Similarly, M2 macrophages and polymorphonuclear cells (PMNs) express high levels of arginase and may contribute to the local suppression of immune responses. Restoration of arginine levels in the tumor microenvironment via arginase inhibition would be expected to allow T-cell activation and proliferation to occur and result in T-cell mediated anti-tumor responses. We have developed novel, potent, and specific inhibitors of arginase. Our clinical candidate CB-1158 has an IC 50 of less than 100 nM in a recombinant human arginase assay. As expected, inhibition of arginase in cell culture does not have a direct anti-proliferative effect on any cell type tested. However, in Peripheral Blood Mononuclear Cells (PBMCs) from a patient with renal cell carcinoma containing both suppressive MDSCs and cytotoxic T-cells, the addition of an arginase inhibitor to the MDSC/T-cell co-culture resulted in a dose-dependent increase in T-cell proliferation relative to vehicle controls. CB-1158 has high oral bioavailability in mice and rats. In mice bearing Lewis Lung Carcinoma (LLC) syngeneic tumors, treatment with CB-1158 results in a 3-4 fold increase in tumor arginine levels with a clear pharmacokinetic/pharmacodynamic relationship. The pharmacodynamic effect of arginase inhibition in tumors was sustained throughout a 24-hour period using a twice-daily oral dosing schedule and has been observed in multiple syngeneic models. Moreover, systemic plasma arginine levels are significantly increased in mice following dosing with CB-1158. Importantly, oral dosing with CB-1158 results in single agent anti-tumor efficacy in the LLC model in C57.Bl/6 mice. In contrast, treatment of immunocompromised C57/scid mice bearing LLC tumors with CB-1158 had no effect on tumor growth. This finding is consistent with the observed anti-tumor efficacy in immune competent mice being mediated through an immune mechanism. Evaluation of tumors treated with arginase inhibitors revealed an increase in CD3+ T-cell infiltrates further supporting an immune-based mechanism of action. CB-1158 has been very well tolerated in rodents with no impact on body weights or serum chemistry enzymes following multi-week dosing schedules. CB-1158 is a first-in-class arginase inhibitor that targets the immunosuppressive effects of myeloid cells in the tumor microenvironment and is currently in development as a novel immuno-oncology strategy. Based on this novel mechanism of action there is also a potential for enhanced therapeutic benefit by combining CB-1158 with other immune checkpoint inhibitors. Citation Format: Matthew Gross, Jason Chen, Ethan Emberley, Julie Janes, Weiqun Li, Andy Mackinnon, Alison Pan, Francesco Parlati, Mirna Rodriguez, Susanne Steggerda, Tracy Wang, Melissa Works, Jing Zhang, Winter Zhang, Mark Bennett. CB-1158 inhibits the immuno-oncology target arginase and causes an immune mediated anti-tumor response. [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 A195.

Abstract P1-08-07: A novel pharmacodynamic assay to measure glutaminase inhibition following oral administration of CB-839 in triple negative breast cancer biopsies

Triple negative breast cancer (TNBC) cell lines are highly dependent on glutamine (Gln) for growth and survival. A critical step in Gln utilization is its conversation to glutamate (Glu) by the mitochondrial enzyme glutaminase (GLS). CB-839 is a potent inhibitor of GLS that has anti-proliferative activity in TNBC cell lines and antitumor activity in TNBC xenograft models (Gross et al., Mol. Cancer Ther. 13:890). Across a panel of breast cancer cell lines derived from both receptor positive and TNBC tumors, sensitivity to CB-839 was associated with (i) elevated GLS expression, (ii) elevated GLS activity, and (iii) the TNBC subtype. Importantly, many of the determinants of CB-839 sensitivity in cell lines are also present in primary tumor samples, including high mRNA and protein expression of GLS and a high Glu to Gln ratio in TNBC tumors as compared to receptor positive tumors. These observations motivate the Phase 1 clinical study of CB-839 in TNBC patients. To aid in the selection of a recommended Phase 2 dose, we sought to develop a pharmacodynamic (PD) assay to directly measure the GLS activity in breast tumor lysates in order to determine the extent of GLS inhibition in tumor biopsies from CB-839 treated patients. To develop a robust PD assay, we first identified conditions that maintain the GLS:CB-839 inhibitory complex during preparation of lysates from CB-839 treated samples. High concentrations of KCl (150 mM) and low concentrations of K-phosphate (15 mM) in the lysis buffer, as well as maintaining the lysate at a low temperature stabilized the inhibited complex. Following gel filtration of the lysate to remove unbound CB-839 and exchange the buffer, GLS activity was immediately measured with a coupled enzyme assay. The GLS activity measured at this step reflects the residual activity present in a sample that was exposed to CB-839. To quantify the amount of total GLS present in the sample, we incubated the same lysate for 3 hours at room temperature under conditions of low KCl and high phosphate to allow the the GLS:CB-839 complex to fully dissociate prior to measuring activity. This assay format allows quantitation of the % GLS inhibition from a single tumor lysate sample and eliminates the requirement for multiple biopsies as well as any assay variability due to tumor heterogeneity. We utilized this tumor PD assay to determine the plasma drug levels required for maximal tumor GLS inhibition in a preclinical TNBC model. Mice bearing HCC1806 TNBC tumors were first treated with a range of CB-839 doses. Four hours after oral administration, a 10 mg/kg dose of CB-839 resulted in >90% inhibition of tumor GLS. CB-839 plasma concentrations of 100 nM corresponded to 50% inhibition of tumor GLS, while maximal inhibition occurred at plasma concentrations ≥300 nM. In xenograft studies, maximal anti-tumor efficacy was achieved with BID dosing at 200 mg/kg, a dose and schedule that resulted in trough plasma levels of CB-839 of ≥300 nM and sustained GLS inhibition in tumors. As part of an ongoing Phase 1 trial, this assay will be utilized to monitor tumor PD responses in TNBC patients undergoing treatment. Citation Format: Andy MacKinnon, Mark Bennett, Ethan Emberley, Mathew Gross, Julie Janes, Evan Lewis, Alison Pan, Mirna Rodriguez, Peter Shwonek, Taotao Wang, Jinfu Yang, Frances Zhao, Francesco Parlati. A novel pharmacodynamic assay to measure glutaminase inhibition following oral administration of CB-839 in triple negative breast cancer biopsies [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 P1-08-07.

Abstract 4711: CB-839, a selective glutaminase inhibitor, synergizes with signal transduction pathway inhibitors to enhance anti-tumor activity

Many tumor cells utilize the amino acid glutamine to meet the elevated bioenergetic and biosynthetic demands of rapid cell growth. Glutamine is utilized by the mitochondrial enzyme glutaminase, which converts glutamine to glutamate. Glutamate is then used to fuel downstream metabolic pathways such as the TCA cycle, redox balance and amino acid synthesis. CB-839 is a novel and selective inhibitor of glutaminase that impacts multiple metabolic pathways downstream of glutamate and has anti-tumor activity in several preclinical models. In addition to its effect on metabolism, CB-839 treatment decreased mTORC1 signaling in triple negative breast cancer [Dennison et al. (2014) Keystone Tumor Metabolism Conference Abstract #1027] and multiple myeloma cells [MacKinnon et al. (2014) ASH Annual Meeting Abstract # 3429]. These studies suggest that nutrient deprivation by CB-839 treatment can be sensed by the mTORC1 pathway. This observation motivated an evaluation of whether inhibitors of receptor tyrosine kinase signaling, the MAP kinase pathway or the PI3 kinase/mTOR pathway could combine favorably with CB-839 to inhibit proliferation. The pan receptor tyrosine kinase inhibitor pazopanib, or the mTORC1 inhibitor everolimus synergized with CB-839 to produce anti-proliferative effects in several renal cell carcinoma (RCC) cell lines (CI range: 0.09-0.85 for pazopanib/CB-839 and 0.48-0.54 for everolimus/CB-839). The EGFR inhibitor erlotinib also synergized with CB-839 to produce strong anti-proliferative effects in several non-small cell lung cancer (NSCLC) cell lines (combination index (CI) range: 0.39-0.75). CB-839 and the MEK inhibitor selumetinib showed strong synergy in the KRAS mutant NSCLC cell line H2122 (CI = 0.46), but weak synergy in the KRAS wild-type cell line H661 (CI = 0.9). These antiproliferative effects were associated with diminished mTORC1 signaling measured in NSCLC cells treated with CB-839, erlotinib, selumetinib or combinations of these agents. The in vitro synergistic CB-839 combinations were also evalutated in an in vivo H2122 NSCLC xenograft model where enhanced anti-tumor activity was observed when CB-839 was dosed together with either erlotinib or selumetinib. CB-839 is currently in Phase 1 clinical development for the treatment of solid and hematological tumor types. This study is the first demonstration that CB-839 can be used in combination with signaling pathway inhibitors to increase the anti-tumor activity of these agents. These observations provide a rationale for testing combinations of CB-839 with either erlotinib or selumetinib in NSCLC patients and pazopanib or everolimus in RCC patients. Citation Format: Mirna Rodriguez, Winter Zhang, Mark Bennett, Ethan Emberley, Mathew Gross, Julie Janes, Andrew MacKinnon, Alison Pan, Susanne Steggerda, Melissa Works, Francesco Parlati. CB-839, a selective glutaminase inhibitor, synergizes with signal transduction pathway inhibitors to enhance anti-tumor activity. [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 4711. doi:10.1158/1538-7445.AM2015-4711

Abstract 966: Novel pharmacodynamic assays to measure glutaminase inhibition following oral administration of CB-839

Glutaminase is a mitochondrial enzyme that plays a crucial role in tumor growth and survival. Glutaminase converts glutamine (Gln) to glutamate (Glu) fueling multiple downstream metabolic pathways required for cellular proliferation. CB-839 is a novel, selective and potent inhibitor of glutaminase that displays antitumor activity in preclinical models of several hematologic and solid tumor types. Treatment of tumor cell lines in vitro with CB-839 caused dose-dependent decreases in Glu and increases in Gln with a potency similar to the IC50 of CB-839 on recombinant glutaminase and the EC50 of CB-839 in cellular proliferation assays. Oral administration of CB-839 to mice bearing human xenograft tumors resulted in similar changes in Glu and Gln levels in the tumor. To determine if these pharmacodynamics effects of CB-839 directly correlated with inhibition of glutaminase, we developed an assay to measure glutaminase activity in tumors. Since CB-839 is a reversible inhibitor, we first developed conditions that maintain the enzyme-inhibitor complex during preparation of tumor lysates. High concentrations of KCl (150 mM) and low concentrations of K-phosphate (15 mM) in the lysis buffer, as well as maintaining the lysate at a low temperature promoted stability of the inhibited complex. Following gel filtration to remove unbound CB-839 and exchange the buffer, glutaminase activity was measured with a coupled enzyme assay. This assay was used to study the dose-dependence of glutaminase inhibition in tumors. Four hours after oral administration, a 10 mg/kg dose of CB-839 resulted in >90% inhibition of tumor glutaminase and was associated with near maximal changes in tumor Gln and Glu. Moreover, CB-839 plasma concentrations of 100 nM corresponded to 50% inhibition of tumor glutaminase, while maximal inhibition occurred at plasma concentrations ≥300 nM. In xenograft studies, maximal anti-tumor efficacy was achieved with BID dosing at 200 mg/kg. This dose and schedule allowed for sustained plasma levels of CB-839 of ≥300 nM, and corresponded to sustained glutaminase inhibition in tumors. In an effort to develop a surrogate marker for inhibition of tumor glutaminase, we adapted the assay to measure glutaminase inhibition in platelets. Ex vivo treatment of human whole blood with CB-839 resulted in dose-dependent suppression of platelet glutaminase activity with an IC50 of 25 nM and >85% inhibition at concentrations at or above 300 nM. Furthermore, glutaminase activity in platelets isolated from mice treated with CB-839 showed dose-dependent inhibition that correlated with inhibition of tumor glutaminase. Thus, platelet glutaminase activity may represent a surrogate for monitoring inhibition of glutaminase in human tumors. These assays will be applied to the clinical study of CB-839 to monitor pharmacodynamic responses and to ensure maximal inhibition of glutaminase in patients undergoing therapy. Citation Format: Andy L. MacKinnon, Mark K. Bennett, Matthew I. Gross, Julie R. Janes, Evan R. Lewis, Mirna L.M. Rodriguez, Peter J. Shwonek, Wang Taotao, Jinfu Yang, Frances Zhao, Francesco Parlati. Novel pharmacodynamic assays to measure glutaminase inhibition following oral administration of CB-839. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 966. doi:10.1158/1538-7445.AM2014-966

Abstract 5604: Preclinical antitumor activity of novel small molecule glutaminase inhibitors in triple-negative breast cancer.

Many tumor cells are dependent on glutamine (Gln) and Gln-derived metabolites to meet bioenergetic and biosynthetic demands. A key cellular reaction in the utilization of Gln is its deamidation by the enzyme glutaminase to yield glutamate (Glu). We have developed a series of potent and selective small molecule glutaminase inhibitors for evaluation as novel cancer therapeutics. These inhibitors exhibit 5-20 nM potency against the broadly-expressed form of glutaminase (GLS) with minimal activity against the liver form of the enzyme (GLS2). Expression analysis (see below) has identified triple-negative breast cancer (TNBC) as a potential clinical target population for GLS inhibitors. TNBC is a poor prognosis breast cancer subtype lacking estrogen receptor (ER), progesterone receptor (PR) and the growth factor receptor Her2. TNBC is insensitive to approved targeted therapies (ER antagonists and anti-Her2 agents) and is currently treated with conventional cytotoxic drugs. Therefore, TNBC represents a critical unmet medical need for which new targeted therapeutics are urgently needed. Analysis of a primary breast tumor mRNA expression dataset (The Cancer Genome Atlas; n=756) revealed that low ER, PR, and Her2 expression is associated with high GLS expression and low expression of both GLS2 and glutamine synthetase, an enzyme that opposes the action of glutaminase by synthesizing Gln from Glu. This expression pattern, together with published work on the Gln dependence of breast tumor cell lines [Kung et al. (2011) PLOS Genet 7:e1002229] suggests that TNBC may be particularly dependent on GLS. To test this hypothesis, we evaluated the anti-tumor activity of our GLS inhibitors on a panel of breast tumor-derived cell lines (n>25) that included a mixture of TNBC and ER-positive subtypes. The TNBC subtype displayed the greatest sensitivity to GLS inhibitor treatment (IC50s ranging from 5-100 nM) and this sensitivity correlated with a dependence on extracellular Gln for cell growth. In the TNBC cell line MDA-MB-231 the antiproliferative effect of the GLS inhibitor was associated with a dose-dependent accumulation of Gln and depletion of Glu. Additionally, GLS inhibition showed additive in vitro activity in combination with paclitaxel, a standard-of-care treatment in TNBC. In a mouse orthotopic tumor xenograft model with MDA-MB-231 cells implanted in the mammary fat pad, oral delivery of a GLS inhibitor caused an accumulation of tumor Gln, a reduction in tumor Glu, and enhanced the anti-tumor efficacy of the paclitaxel. Experiments aimed at expanding this observation with other TNBC xenograft models are in progress. Overall, these results demonstrate that a selective inhibitor of GLS, either as a single agent or in combination with standard-of-care chemotherapeutics, may be effective as a targeted therapeutic in TNBC. Citation Format: Susan Demo, Tania Chernov-Rogan, Matthew Gross, Julie Janes, Raja Kawas, Evan Lewis, Francesco Parlati, Hector Rodriguez, Mirna Rodriguez, Jinfu Yang, Frances Zhao, Adam Richardson, Mark K. Bennett. Preclinical antitumor activity of novel small molecule glutaminase inhibitors in triple-negative breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5604. doi:10.1158/1538-7445.AM2013-5604