Abstract C42: Mitochondrial glutaminase regulation and small molecule inhibition of glutamine metabolism

Abstract

Abstract The mitochondrial enzyme glutaminase isoform C (GAC) has recently gained significant attention for its critical role in supporting glutamine dependent proliferation in a variety of cancer cells and has been a significant focus for therapeutic intervention (1,2). To this end, we have combined biophysical techniques with cellular metabolic assays to gain insight as to the mechanism of action of the novel GAC inhibitor, compound 968, shown previously to inhibit Dbl (for Diffuse B-cell Lymphoma) oncogene dependent transformation (2). We have developed a fluorescence resonance energy transfer (FRET) approach, along with X-ray crystallography and multi-angle light scattering (MALS), to study the oligomer dynamics of GAC in relation to its activation in vitro. To compliment in vitro biophysical studies of GAC activation and inhibition, we employed stable 13C-isotope tracing assays in a model Dbl-inducible cell system to monitor 13C incorporation in TCA cycle intermediates derived from 13C-glutamine. Using dynamic FRET assays, as well as novel GAC mutants that are trapped as constitutive dimer or monomer species, we show that the dimer-to-tetramer transition is indeed directly linked to activation of the enzyme. Subsequently we go on to show that the two previously described allosteric GAC inhibitors, 968 and BPTES, elicit their inhibitory effects through distinctly different mechanisms. Both, however, target specific oligomeric states of GAC: compound 968 preferentially binds to the monomeric form of GAC to disrupt critical oligomer contacts, in stark contrast to BPTES, which binds to and induces tetramer formation by interacting with a loop located near the glutamine binding site. The binding of compound 968 to the monomeric form of GAC was found to induce a conformational change that renders the enzyme inactive, however still able to participate in oligomer formation and dissociation. The consequence of this 968-bound GAC species in vivo was investigated by first showing that 968 treatment of a Dbl-inducible MEF cell line with 968 was capable of reversing Dbl-induced transformation as evidenced by the inhibition of focus formation and proliferation. In this same model cell system, we show that induction of the Dbl-oncogene leads to an increase in glutaminolysis and glutamine fueled anaplerosis by monitoring 13C-enrichment in TCA cycle intermediates derived from 13C-glutamine, and that this increased glutaminolysis is potently inhibited by 968 treatment. These results highlight advances in understanding the mechanism of glutaminase regulation as well as the potential future of small molecules targeting glutamine metabolism as therapeutics. (1) Wang, J.B. et al., (2010) Cancer Cell 18, 207-219 (2) Le, A. et al., (2012) Cell Metabolism 15, 110-121 This project was supported by the Chemical Biology Training Grant (Award Number T32GM008500) from the National Institute of General Medical Sciences and the NIH Citation Format: Clint Stalnecker, Jon Erickson, Sekar Ramachandran, Ralph DeBerardinis, Rick Cerione. Mitochondrial glutaminase regulation and small molecule inhibition of glutamine metabolism. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr C42.