Abstract 2179: Increased BCAT1 expression creates a pseudo-hypoxic environment that promotes cell proliferation in a subtype of glioblastoma

Abstract

Abstract Background: BCAT1 (Branched Chain Amino Transferase 1) has been shown to be upregulated in human IDH (isocitrate dehydrogenase) wild-type glioblastoma (GBM) and BCAT1 knockdown leads to reduced cell proliferation in vitro and in vivo1. Here we investigated the role of BCAT1 in human patient-derived glioblastoma cells and the corresponding orthotopically implanted xenografts to understand how upregulation of the enzyme leads to a more aggressive phenotype. Methods: The expression of BCAT1 was investigated in a panel of patient derived IDH wild-type glioblastoma cells and BCAT1 expression modulation was achieved using lentiviral expression of a doxycycline-inducible shRNA, to knockdown BCAT1 and expression of a BCAT1 cDNA to overexpress BCAT1. Effects of BCAT1 knockdown and overexpression were investigated in vitro in cell culture and in vivo, following intracranial implantation of mice with the relevant cells. Results: Variable levels of BCAT1 expression were identified in different patient-derived cell lines. RNA sequencing analysis of BCAT1 knockdown in BCAT1 expressing glioblastoma cells revealed that knockdown led to significant downregulation of HIF 1α (Hypoxia Inducible Factor 1α) and its targets, which was confirmed by Western Blot analysis of Carbonic Anhydrase IX (CAIX) and Hexokinase II (HKII) in cell and tumor lysates. The opposite was observed following BCAT1 overexpression. BCAT1 knockdown also led to higher DNA demethylation levels. Treatment with membrane permeant dimethyl-alpha ketoglutarate mimicked the effect of BCAT1 knockdown. These data suggest that decreased cell proliferation following BCAT1 knockdown in BCAT1 expressing cells is mediated by accumulation of alpha-ketoglutarate, a substrate of BCAT1. Cells that had no BCAT1 expression showed a similar growth profile and overexpression had no effect on proliferation, suggesting that these cells are not dependent on BCAT1 expression. Conclusion: We conclude that in a subtype of GBM characterized by upregulated BCAT1, BCAT1 expression results in lowered levels of alpha-ketoglutarate, leading to reduced prolyl hydroxylase activity and stabilization of HIF 1α and therefore a pseudo-hypoxic state, resulting in a transcriptome that favors a high proliferation rate. This makes BCAT1 a potential therapeutic target, for tumors that show BCAT1 upregulation.1. Tönjes M, Barbus S, Park YJ, et al. BCAT1 promotes cell proliferation through amino acid catabolism in gliomas carrying wild-type IDH1. Nat Med. 2013;19(7):901-908. doi:10.1038/nm.3217 Citation Format: Maria Fala, Susana Ros, Ashley Sawle, Kevin M. Brindle. Increased BCAT1 expression creates a pseudo-hypoxic environment that promotes cell proliferation in a subtype of glioblastoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2179.