Abstract 245: Glutamine fuels the TCA cycle in VHL and FH mutant renal cell carcinoma
Authors: Kaushik Akash K. , Yong Cissy , Ragavan Mukundan , Stevens Christina , Woolford Layton , Rao Aparna , Faubert Brandon , Pachnis Panayotis , Zacharias Lauren , Vu Hieu , Merritt Matthew , Brugarolas James , DeBerardinis Ralph J. 
Published: September 2020

Abstract
Renal cell carcinoma (RCC) is one of the top 10 diagnosed cancers in the United States, and it accounts for about 64,000 deaths every year. In recent years, combinations of surgery and targeted therapies have significantly improved patient outcomes; however, a subset of patients with advanced RCC poorly respond to current treatments. Therefore, we need a better understanding of the underlying molecular mechanisms of RCC to delineate novel therapeutic liabilities. A common feature across RCC subtypes is profound metabolic dysregulation, including massive lipid deposition in VHL mutant clear cell RCC (ccRCC), fumarate and succinate accumulation in FH- and SDH-mutant RCC respectively, and enhanced glucose metabolism. We recently confirmed the Warburg effect in human ccRCC patients intraoperatively infused with [U-13C]glucose, raising possibilities of other fuel sources feeding into the TCA cycle in ccRCC. We investigated if glutamine fuels the TCA cycle by infusing [U-13C]glutamine in NOD-SCID mice bearing orthotopic patient-derived xenografts (PDXs) generated from VHL- and FH-mutant RCC. We identified significant incorporation of 13C derived from [U-13C]glutamine into the TCA cycle in both VHL and FH-mutant RCC compared to kidney tissues. While VHL-mutant ccRCC engaged in both oxidative and reductive metabolism of glutamine, VHL-mutant sarcomatoid RCC exclusively demonstrated enhanced reductive metabolism, indicating the importance of reductive carboxylation of glutamine-derived α-ketoglutarate (α-KG) in an aggressive stage of the disease. Importantly, both IDH1 (cytosolic) and IDH2 (mitochondrial) regulate reductive carboxylation of glutamine-derived α-KG in cells derived from VHL-mutant PDX. In contrast, FH-mutant RCC demonstrated significant oxidative metabolism of [U-13C]glutamine, resulting in the enrichment of four 13C labeled fumarate, malate and aspartate, indicating a novel metabolic rewiring in FH-mutant tumors. We hypothesized that increased fumarate level in FH-mutant RCC induces reversal of enzymatic reactions that would typically generate fumarate by the breakdown of argininosuccinate and adenylosuccinate in the urea cycle and purine nucleotide cycle, respectively. Concurrent reversal of reactions upstream of argininosuccinate and adenylosuccinate can generate aspartate, which can form malate via malate-aspartate shuttle. Supporting our hypothesis, we confirmed a significant enrichment of 13C labeled argininosuccinate and adenylosuccinate in FH-mutant RCC. Finally, we demonstrate that inhibition of mitochondrial enzyme glutaminase with CB-839 decreases the growth of VHL- and FH-mutant RCC and contributions of glutamine into the TCA cycle. Overall, our study demonstrates that glutamine is a major fuel source for the TCA cycle, and targeting glutaminase with CB-839 has therapeutic benefits in VHL- and FH-mutant RCC.
Citation Format: Akash K. Kaushik, Cissy Yong, Mukundan Ragavan, Christina Stevens, Layton Woolford, Aparna Rao, Brandon Faubert, Panayotis Pachnis, Lauren Zacharias, Hieu Vu, Matthew Merritt, James Brugarolas, Ralph J. DeBerardinis. Glutamine fuels the TCA cycle in VHL and FH mutant renal cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 245.