Abstract
CB-839 is a potent, orally bioavailable small-molecule inhibitor of the tumor metabolism target glutaminase (GLS) that is currently in Phase 1 clinical trials for the treatment of solid and hematological malignancies. GLS is a mitochondrial enzyme that converts Gln to glutamate (Glu) to support several metabolic processes including amino acid synthesis, maintenance of cellular redox homeostasis, and the replacement of TCA cycle intermediates. CB-839 has in vitro antiproliferative activity across a sub-set of cell lines from diverse hematological tumor types including multiple myeloma (MM), acute lymphocytic leukemia, and non–Hodgkin’s lymphoma [Parlati et al. Blood 2013 122:4226]. To identify biomarkers that would predict sensitivity to CB-839 in MM, we profiled cellular metabolites, mRNA transcripts, and oncogenic signaling pathways in eight MM cell lines representing four CB-839-sensitive cell lines (RPMI8226, MM.1S, KMS-11, and IM-9) and four CB-839-resistant cell lines (AMO-1, L-363, KMS-28PE, and OPM-2).
CB-839 treatment for 4 hours significantly decreased the levels of amino acids (Glu, aspartate, proline) as well as TCA cycle intermediates (fumarate, malate, succinate) across all cell lines. However, prior to treatment, CB-839-sensitive cells had significantly lower baseline levels of pyruvate-, fumarate-, and succinyl-CoA-derived amino acids compared to CB-839-resistant cells. In addition, both the adenylate and guanylate energy charges, a measure of cellular metabolic activity [Atkinson and Walton, J. Biol. Chem. 1967 242: 3239-41], were significantly lower in CB-839 sensitive cells. These observations suggest that cells with low levels of amino acids and/or low cellular energy charge are more susceptible to the pharmacological effects of CB-839.
Reverse phase protein array and immunoblot analysis were used to evaluate the impact of CB-839 on signaling pathways across the panel of cell lines. Consistent with the observed decreases in amino acid pools, CB-839 treatment for 4 hours led to an acute inhibition of the amino-acid sensing kinase mTORC1 across all cell lines, as evidenced by decreased phosphorylation of p70S6K and S6. CB-839 treatment also reduced the levels of c-Myc across all cell lines, consistent with a block in five-prime cap-dependent translation as a likely consequence of mTORC1 inhibition. However, untreated sensitive cells had lower baseline levels of both 4E-BP1 and phospho-4E-BP1 in comparison to resistant cells, and at 24 hours following treatment, the mTORC1-dependent activation of CAD, a key enzyme in pyrimidine biosynthesis (Ben-Sahra et al., Science 2013 15: 1323-8), was reduced in CB-839-sensitive but not in resistant cells. These results suggest that CB-839 inhibits mTORC1 signaling in MM cells and that sensitive cells may lack compensatory mechanisms to overcome this inhibition.
Consistent with differential regulation of mTORC1 in CB-839-sensitive versus resistant cells, RNAseq analysis showed greater down-regulation of several mTORC1-regulated transcripts encoding glycolytic enzymes (such as ALDOA and TPI1, Düvel et al., 2010 Mol. Cell 39: 171-83) in sensitive cells versus resistant cells following CB-839 treatment. RNAseq analysis also revealed elevated levels of pyruvate carboxylase (PC) in CB-839 resistant cells as compared to sensitive cells. Immunoblot analysis of twenty four MM cell lines supported this observation; PC protein levels were inversely correlated with response to CB-839 (r = 0.64, p = 0.001) and PC protein and mRNA transcript levels were highly correlated (r = 0.94, p = 0.0004). PC is an anaplerotic enzyme that converts pyruvate to oxaloacetate. Therefore, sufficiently high levels of glycolytic enzymes and PC could lessen the dependence of a MM cell line on Gln metabolism for replenishment of TCA intermediates.
The studies described herein elucidate differences in metabolic and signaling pathways among MM cell lines. These differences in metabolites, mTORC1 signaling, glycolytic enzymes and PC may account for differential sensitivity to CB-839. We are exploring the potential utility of PC expression as a biomarker in our ongoing clinical studies of CB-839.
Disclosures
Mackinnon: Calithera Biosciences: Employment, Equity Ownership. Bennett:Calithera Biosciences: Employment, Equity Ownership. Rodriguez:Calithera Biosciences: Employment, Equity Ownership. Parlati:Calithera Biosciences: Employment, Equity Ownership.
The FBXW7-SHOC2-Raptor Axis Controls the Cross-Talks between the RAS-ERK and mTORC1 Signaling Pathways
