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.