Selective Inhibitor Of Glutaminase Research Articles

Glutaminase Upregulation As a Mechanism of Chemotherapy Resistance in High-Risk Leukemia

Many types of cancers are characterized by elevated glutamine consumption. Glutaminase is a therapeutic target to arrest cancer cell development. Ikaros (IKZF1) is a DNA-binding, zinc finger protein that functions as a transcriptional regulator and a tumor suppressor in leukemia. Ikaros activity is essential for normal hematopoiesis and immune development and is expressed at high levels in most hematopoietic cells. Deletion of IKZF1 leads to the downregulation and upregulation of many important genes and is a predictor of poor outcomes in leukemia. We hypothesize that upregulation of glutaminase in cancer cells with IKZF1 deletion may be a mechanism whereby high-risk leukemias convey chemotherapy resistance. Conversely, Ikaros may help keep healthy hematopoietic cells in-check by preventing over-expression of glutaminase. To test this hypothesis, we exposed two lines of B-ALL cells to the allosteric and selective glutaminase inhibitor BPTES. One cell line had IKZF1 intact (Nalm-6 scramble) and one had IKZF1 deleted via a CRISPR knockout (Nalm-6 IK KO). Treatments were done in triplicate with an exposure time of 72 hours. WST-1 viability assay was used to obtain an IC50 value for each cell line. Leukemia cells with IKZF1 knocked out had a two-fold higher IC50 than cells with IKZF1 intact. This indicates that IKZF1 deletion may convey chemotherapy resistance by upregulating glutaminase and implies that strategies targeting glutaminase may improve treatment outcomes in high-risk leukemia patients.

Inhibition of glutaminase 1 activity reverses airway hyperresponsiveness and decreases IL-1β+ M1s and IL-17 producing ILC3s in high-fat diet-fed obese mice.

In obesity, disturbed glutamine metabolism contributes to enhanced inflammation by inducing alterations in immune cells. As macrophages and innate lymphoid cells (ILCs) are known to be involved in the pathogenesis of obesity-related asthma, we tested our hypothesis that altered glutamine metabolism may link obesity to airway hyperresponsivenss (AHR), a cardinal feature of asthma, focusing on these innate immune cells. Four-week-old male C57BL/6 mice were fed a high-fat diet (HFD) for 13 wk in the presence or absence of BPTES [Bis-2-(5-phenylacetamido-1,3,4-thiadiazol-2-yl)ethyl sulfide, a selective inhibitor of glutaminase 1 which converts glutamine to glutamate] and their blood, lung, and adipose tissues were analyzed. We then conducted in vitro experiments using bone marrow-derived macrophages (BMDMs) and mouse alveolar macrophage cell line. Furthermore, we investigated plasma glutamine and glutamate levels in obese and nonobese asthmatics. BPTES treatment prevented HFD-induced AHR and significantly decreased IL-1β+ classically activated macrophages (M1s) and type 3 ILCs (ILC3s) which increased in the lungs of HFD-fed obese mice. In in vitro experiments, BPTES treatment or glutamine supplement significantly reduced the proportion of IL-1β+NLRP3+ M1s in lipopolysaccharide-stimulated BMDMs and mouse alveolar macrophage cell line. BPTES treatment also significantly reduced the IL-17 producing ILC3s differentiated from ILCs in naïve mouse lung. In addition, plasma glutamate/glutamine ratios were significantly higher in obese asthmatics compared to nonobese asthmatics. Inhibition of glutaminolysis reverses AHR in HFD-induced obese mice and decreases IL-1β + NLRP3+ M1s and IL-17 producing ILC3s, which suggests altered glutamine metabolism may have a role in the pathogenesis of obesity-related AHR.

LBA54 - ENTRATA: Randomized, double-blind, phase II study of telaglenastat (tela; CB-839) + everolimus (E) vs placebo (pbo) + E in patients (pts) with advanced/metastatic renal cell carcinoma (mRCC)

LBA54 - ENTRATA: Randomized, double-blind, phase II study of telaglenastat (tela; CB-839) + everolimus (E) vs placebo (pbo) + E in patients (pts) with advanced/metastatic renal cell carcinoma (mRCC)

Abstract 2323: Involvement of glutamine in tolerance of human pancreatic cancer cell to inhibition of fatty acid synthesis

Abstract Pancreatic cancer (PC) is the fifth leading cause of cancer-related mortality in Japan. Although the standard chemotherapy regimens, FOLFIRINOX (fluorouracil, leucovorin, irinotecan and oxaliplatin), gemcitabine and nab-paclitaxel, are widely used to treat patients with advanced pancreatic cancer, their antitumor effects (progression and survival rates) are less potent against this cancer type than other solid tumors. Previously, we found that inhibition of acetyl-CoA carboxylase (ACC), a major rate-controlling enzyme of fatty acid synthetic pathway, suppressed the proliferation of PC cells. However, in this study, we found the tolerance to the inhibition in a part of PC cells. Therefore, we investigated the mechanism of the tolerance to ACC inhibition in PC cells. The human PC cell lines, AsPC-1, BxPC-3 and PANC-1 were obtained from the American Type Culture Collection. Live and apoptotic cell numbers were determined using the MUSE Annexin V and Dead Cell Kit according to the manufacturer's instructions. PANC-1 cells showed the tolerance to inhibition of fatty acid synthesis by ACC inhibitor, 5-(tetradecyloxy)-2-furoic acid (TOFA), whereas TOFA induced apoptosis in AsPC-1 and BxPC-3 cells. In addition, PANC-1 did not show autophagy even under the condition of fatty acid starvation. Next, we investigated whether PANC-1 has the tolerance to nutrient starvation in nutrient-depletion medium, Earle's Balanced Salt Solution (EBSS) and serum-free medium respectively. PANC-1 showed the survival even in such mediums, whereas AsPC-1 and BxPC-3 almost dead. This result suggests that the tolerance of fatty acid starvation of PANC-1 are related to that of nutrient starvation. Interestingly, proliferation of PANC-1 cultured in glutamine-depletion medium dramatically was suppressed, even though low glucose medium did not affect. BPTES, a selective inhibitor of glutaminase, also suppressed the proliferation and induced apoptosis in PANC-1, whereas there are little effects of 2-dexy-glucose, inhibitor of glycolysis, on them. These results suggest that the TCA cycle plays an important role on the tolerance to inhibition of fatty acid synthesis and nutrient starvation of PANC-1, and can be novel therapeutic target of PC cells which have the tolerance to nutrient starvation like PANC-1. Citation Format: Koji Nishi, Mina Suzuki, Noriko Yamamoto, Yumiko Iwase, Nagahiko Yumita. Involvement of glutamine in tolerance of human pancreatic cancer cell to inhibition of fatty acid synthesis [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 2323.

Abstract PD3-13: Phase 1 study of CB-839, a first-in-class oral inhibitor of glutaminase, in combination with paclitaxel in patients with advanced triple negative breast cancer

Abstract Background: CB-839 is a first-in-class oral highly selective inhibitor of glutaminase (GLS), a mitochondrial enzyme that plays a key role in cancer cell metabolism. Triple negative breast cancer (TNBC) has high GLS expression and demonstrates high glutamine utilization and glutamine dependence in clinical and preclinical studies. CB-839 has monotherapy antitumor activity in vitro and in vivo in preclinical models of TNBC, and also demonstrates synergistic anti-cancer activity with the standard of care agent paclitaxel (Pac). In an ongoing Phase 1 study, CB-839 combined with Pac (Pac-CB) is being evaluated in patients (pts) with metastatic TNBC. We previously reported that Pac-CB was well-tolerated and active in heavily pre-treated TNBC pts, including those previously refractory to taxane treatment for metastatic disease. We report here updated results from this ongoing study. Methods: Pts with refractory metastatic TNBC (prior taxane therapy allowed) received escalating doses of CB-839 (400-800 mg BID) in combination with full dose Pac of 80 mg/m2 Days 1, 8, 15 every 28 days. After demonstration of safety and tolerability, an expansion cohort was opened at the CB-839 recommended phase 2 dose of 800 mg PO BID. Results: As of the May 2017 data cut, 45 pts have enrolled across the dose escalation and expansion cohorts (7 pts at 400 mg, 12 at 600 mg, and 26 at 800 mg). 15 pts (33%) have received ≥5 prior lines of systemic therapy for metastatic disease (median 3 prior lines), and 39 pts (87%) have received prior taxane therapy. The Pac-CB regimen has been well tolerated with the most frequent treatment-related Grade ≥3 AEs being neutropenia (24%), anemia (7.6%), fatigue (2.6%), WBC decreased (2.6%), and peripheral neuropathy (2.6%). At CB-839 doses ≥600 mg BID (n=27 RECIST-evaluable), the ORR has been 22% and disease control rate (DCR=CR+PR+SD) 59%; in the subgroup of pts refractory to previous taxane therapy (n=19) the ORR has been 21% (4 pts) and DCR 47% (9 pts). The highest ORR in this study has been seen in pts of African ancestry (AA, n=11 RECIST evaluable) with an ORR of 36% (4 pts) and DCR 55% (6 pts), with all 4 AA responders being refractory to prior taxane treatment for advanced/metastatic disease. Conclusions: TNBC has elevated glutamine metabolism that requires mitochondrial GLS. In TNBC pts with heavy pretreatment and previous taxane exposure, the combination of CB-839 with Pac has demonstrated clinical activity including a 21% ORR in pts refractory to taxane. Notably, in patients of African ancestry, a population reported to have especially high glutamine utilization in TNBC tumors, the encouraging ORR of 36% suggests a potential genetic association with treatment response. The Pac-CB regimen has been well tolerated in late line TNBC pts, including a Gr 3 neuropathy signal of only 2.6%. A Phase 2 study, CX-839-007, has been initiated to further evaluate the activity and safety of Pac-CB in pts with TNBC, including defined cohorts of pts with 1st line and 3rd line+ metastatic disease in pts of African and non-African ancestry. Responses in relation to genetic background, molecular subtype of TNBC and glutamine biology will be studied. Citation Format: Kalinsky K, Harding JJ, DeMichele A, Infante JR, Gogineni K, Owonikoko TK, Isakoff S, Iliopoulos O, Patel MR, Munster P, Telli ML, Jenkins Y, Fiji GP, Whiting SH, Meric-Bernstam F. Phase 1 study of CB-839, a first-in-class oral inhibitor of glutaminase, in combination with paclitaxel in patients with advanced triple negative breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr PD3-13.

Abstract P6-11-05: Phase 1 study of CB-839, a small molecule inhibitor of glutaminase (GLS), in combination with paclitaxel (Pac) in patients (its) with triple negative breast cancer (TNBC)

Abstract Background: CB-839 is a first-in-class highly selective inhibitor of GLS, a key enzyme in the utilization of glutamine by cancer cells. TNBC has high GLS expression and is very dependent upon GLS-mediated conversion of glutamine to glutamate for tumor cell growth. CB-839 has antitumor activity in vitro and in vivo in preclinical models of TNBC. Recent studies demonstrate that glutamine utilization can contribute to resistance to paclitaxel, a therapy frequently used to treat TNBC patients. Paclitaxel sensitivity is dependent on down-regulation of the glutamine transporter, SLC1A5, and over-expression of SLC1A5 causes paclitaxel resistance. Consistent with these observations, inhibition of glutamine metabolism with CB-839 has demonstrated strong antitumor activity in combination with paclitaxel. CX-839-001 is an ongoing Phase 1 trial of CB-839 as monotherapy and in combination with approved agents. We previously reported pharmacodynamic studies demonstrating robust inhibition of GLS in pt blood and tumors and excellent tolerability of CB-839 monotherapy in a variety of tumor types including TNBC. In light of the preclinical rationale and monotherapy tolerability a combination arm was opened testing CB-839 with paclitaxel (Pac-CB) in patients with advanced TNBC. We report here updated results on the Pac-CB dose escalation and expansion cohorts. Methods: Patients with refractory advanced/metastatic TNBC (prior taxane therapy allowed) received escalating doses of CB-839 (400-800 mg BID) in combination with a fixed weekly Pac dose of 80 mg/m2 Days 1, 8, 15 of a 28 day cycle. Upon demonstration of safety and tolerability, an expansion cohort of TNBC pts was opened. Results: To date, 15 pts have received Pac-CB at three dose levels of CB-839: 7 pts at 400 mg BID, 5 at 600 mg BID and 3 at 800 mg BID with the latter dose level not completed. 40% of enrolled patients have received >5 prior lines of systemic therapy for adv/met disease, and 10 pts have received prior taxane therapy including 5 in the adv/met setting. The Pac-CB combination has been well tolerated with one DLT during dose escalation (G4 neutropenia at 400 mg BID) and a low rate of dose reductions (2 for Pac and 1 for CB-839). Of 15 pts, the best overall response rate (BORR, see Table) has been PR in 20% (3 pts), SD in 47% (7 pts) and PD in 33% (5 pts) with 5 patients remaining on study. At doses ≥600 mg BID (n=8) the BORR is 38% (3 pts), and disease control rate (CR + PR + SD) is 88% (7 pts). All 3 pts with PRs have received prior Pac, including 2 pts with disease that was refractory to Pac in the advanced/metastatic setting. Conclusions: The Pac-CB combination has been well tolerated and has demonstrated clinical activity in heavily pre-treated pts with TNBC. At doses ≥600 mg BID, BORR has been 38% and DCR 88%. Notably, PRs have occurred in pts with prior Pac therapy, including 2 pts with Pac-refractory disease in the adv/met setting. Updated data on the escalation and expansion cohorts will be presented. Dose LevelTotal400 mg BID600 mg BID800 mg BIDRECIST Response Evaluable (N)15753PR3 (20%)02 (40%)1 (33%)SD7 (47%)3 (43%)2 (40%)2 (67%)DCR (CR+PR+SD)10 (67%)3 (43%)4 (80%)3 (100%)PD5 (33%)4 (57%)1 (20%)0 Citation Format: DeMichele AM, Harding JJ, Telli ML, Münster P, McKay RR, Iliopoulos O, Whiting S, Orford KW, Bennett MK, Mier JW, Owonikoko TK, Patel MR, Kalinsky K, Carvajal RD, Infante JR, Merit-Bernstam F. Phase 1 study of CB-839, a small molecule inhibitor of glutaminase (GLS), in combination with paclitaxel (Pac) in patients (its) with triple negative breast cancer (TNBC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-11-05.

376 - CB-839, a selective glutaminase inhibitor, has anti-tumor activity in renal cell carcinoma and synergizes with everolimus and receptor tyrosine kinase inhibitors

376 - CB-839, a selective glutaminase inhibitor, has anti-tumor activity in renal cell carcinoma and synergizes with everolimus and receptor tyrosine kinase inhibitors

Phase 1 study of CB-839, a small molecule inhibitor of glutaminase (GLS), alone and in combination with everolimus (E) in patients (pts) with renal cell cancer (RCC).

4568Background: CB-839 is a selective inhibitor of GLS, a key enzyme in the utilization of glutamine by many cancer cells. CB-839 has broad activity in preclinical models, including RCC where GLS i...

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

Abstract 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 P1-08-01: Signaling consequences and rational therapeutic combinations with glutaminase inhibitor, CB-839, in basal breast cancer

Abstract 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.

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

Abstract 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-Hodgkin's 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<0.0001), suggesting that the glutamine requirement for tumor cell growth or survival is mediated by glutaminase and that this phenotype is consistent across numerous tumor types. These data suggest that CB-839 may have therapeutic activity in a range of tumor types that are glutamine dependent and motivate the clinical evaluation of this compound targeting a novel tumor survival pathway. 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 5604: Preclinical antitumor activity of novel small molecule glutaminase inhibitors in triple-negative breast cancer.

Abstract 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