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.