Metabolomic, Proteomic and Genomic Profiling Identifies Biomarakers of Sensitivity to Glutaminase Inhibitor CB-839 in Multiple Myeloma

Introduction

Glutaminase is a mitochondrial enzyme that converts glutamine to glutamate to support several metabolic processes including amino acid and nucleotide synthesis, maintenance of cellular redox homeostasis, and the replacement of TCA cycle intermediates.  Selective glutaminase inhibitors BPTES and CB-839 have anti-proliferative activity in several pre-clinical cancer models including breast, pancreatic, lung, renal, brain, leukemia, and lymphoma.  Across a panel of twenty-nine multiple myeloma cell lines, we found that glutaminase inhibition with CB-839 caused tumor cell death or growth inhibition in only a subset of cell lines. To identify biomarkers that predict sensitivity to CB-839 in multiple myeloma cells, we profiled cellular metabolites, mRNA transcripts, and signaling pathways in eight multiple myeloma cell (four CB-839-sensitive and four CB-839-resistant).

Results

Proteomic analysis showed that CB-839 treatment suppressed the activity of the amino-acid sensing kinase mTORC1 in CB-839-sensitive cells, leading to down regulation of protein synthesis and expression of metabolic genes. Analysis of steady-state levels of intra-cellular metabolites revealed that CB-839-sensitive cells had more profound decreases in nucleotide levels and less pronounced increases in essential amino acids upon CB-839 treatment compared to CB-839-resistant cells. This suggests that the metabolic response to glutaminase inhibition is fundamentally different in sensitive versus resistant multiple myeloma cell lines.  Consistent with the in vitro data, in a xenograft model with the CB-839-sensitive cell line RPMI8226, CB-839 treatment produced a 71% reduction in tumor growth that was associated with reduced levels of intratumoral nucleotides and no changes in the levels of essential amino acids. We next explored protein biomarkers that predict resistance to CB-839 and found that pyruvate carboxylase (PC) expression strongly correlated with resistance. siRNA-mediated knockdown of PC reduced TCA cycle activity and sensitized cells to CB-839 treatment, suggesting that PC can rescue cells from glutaminase inhibition by supporting anapleurotic utilization of glucose. This hypothesis was further substantiated by the observation that treatment of CB-839-resistant cells with the AKT inhibitor MK2206 led to a decrease in glucose utilization, and when combined with CB-839, produced a significant decrease in TCA cycle activity and a profound synergistic anti-proliferative response.

Conclusion

Multiple myeloma cells show varying anti-proliferative responses to glutaminase inhibition by CB-839. CB-839 treatment inhibits mTORC1 pathway signaling and causes decreases in nucleotides in sensitive multiple myeloma cells. Multiple myeloma cells that are resistant to glutaminase inhibition have high expression of PC, which may allow these cells to utilize glucose instead of glutamine to resupply TCA cycle intermediates. Knockdown of PC or treatment with an AKT inhibitor causes cells to utilize less glucose and sensitizes resistant cells to glutaminase inhibition with CB-839. CB-839 is currently being evaluated in Phase 1 clinical trials for the treatment of various solid and hematological cancers including multiple myeloma. We are exploring the utility of PC and mTORC1 pathway signaling biomarkers to identify multiple myeloma patients that may respond to CB-839 treatment.