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 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.