Abstract 4450: CB839, an orally bioavailable glutaminase inhibitor, shows potent antitumor activity in vitro against models of soft tissue sarcoma and chondrosarcoma

Abstract

Abstract CB839 (Glutaminase inhibitor) is a potent, orally bioavailable and selective inhibitor of the first enzyme in glutamine utilization in Krebs tricarboxylic acid (TCA) cycle. CB839 has been shown to have significant anti proliferative activity in triple negative breast cancer cell lines in vitro as well as in mouse xenograft models. We tested the in vitro anti proliferative activity of CB839 in chondrosarcoma cell lines carrying a mutation in isocitrate dehydrogenase (IDH), a key enzyme in TCA cycle. These cell lines included JJ012 (IDH1 mutant) and SW1353 (IDH2 mutant). CH2879 cell line carrying wild-type IDH was used as a control. In addition to IDH mutant chondrosarcoma cell lines, we tested in vitro efficacy of CB839 in a panel of soft tissue sarcoma cell lines including malignant peripheral nerve sheath tumor (MPNST), Ewing sarcoma (CHP100) and Liposarcoma (LS141). Results from our in vitro proliferation assay showed that compared to wild-type IDH cell line CH2879, mutant IDH cell lines JJ012 and SW1353 were more sensitive to inhibition of proliferation by CB839. Among the soft tissue sarcoma cell lines tested, MPNST and ST8814, which carry an NF1 mutation (neurofibromatosis 1) or NF1 loss respectively, showed greater sensitivity to inhibition. NF1 negatively regulates Ras activity and NF1 mutation or loss in these cells is known to up regulate Ras/MAP kinase pathway. The extent of sensitivity observed in soft-tissue and chondrosarcoma cell lines correlated with their extracellular glutamine dependency. This was confirmed by cell proliferation assay in glutamine containing versus glutamine depleted media. Target inhibition by CB839 was confirmed by gas chromatography mass spectrometry (GC/MS) analysis, which showed reduced levels of intracellular intermediary metabolites including Glutamate, α-ketoglutarate, succinate, fumarate and 2-hydroxy-glutarate. Previous reports have suggested that α-ketoglutarate is a rate limiting factor in the activation of mTORC1. To explore whether CB839 affects mTORC1 signaling, we carried out western blot analysis to determine the phosphorylation levels of S6 (pS6), a direct substrate of mTORC1 activity. We observed down regulation of pS6 by CB839 only in IDH1 mutant cell line, JJ012, but not in wild-type IDH cell line, CH2879. This data strongly supports cross-talk between glutamine metabolism and mTORC activity in IDH mutant cell line. Some reports have suggested that under conditions of glutamine deprivation, cells use pyruvate carboxylase for anapleurosis. Treatment of chondrosarcoma cell lines by CB839 showed dose dependent suppression of pyruvate carboxylase only in IDH mutant but not wild type cell line. Taken together, our data strongly supports further pre-clinical research in order to explore the role played by IDH and other mutations such as NF1 in determining sensitivity to Glutaminase inhibition by CB839. Citation Format: Tahir N. Sheikh, Parag P. Patwardhan, Serge Cremers, Gary K. Schwartz. CB839, an orally bioavailable glutaminase inhibitor, shows potent antitumor activity in vitro against models of soft tissue sarcoma and chondrosarcoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4450. doi:10.1158/1538-7445.AM2015-4450