Abstract 1881: Hypoxia Inducible Factor (HIF) promotes reductive carboxylation by regulating citrate levels and renders VHL-deficient and hypoxic cells sensitive to glutamine deprivation.
Authors: Gameiro Paulo , Metelo Ana , Pérez-Carro Rocio , Arreolla Alexandra , Baker Rania , Wang Zongwei , Olumi Aria , Rathmell Kimryn , López-Larrubia Pilar , Stephanopoulos Gregory , Iliopoulos Othon 
Published: August 2014

Abstract
Hypoxia is a hallmark of tumor microenvironment. We showed before that exposure of cells to hypoxia reprograms cell metabolism and promotes an IDH1-mediated reductive carboxylation (RC) of glutamine-derived alpha-ketoglutarate (a-KG) for lipogenesis. In addition, Von Hippel-Lindau (VHL)-deficient renal cell carcinoma (RCC) cells use glutamine to generate citrate and lipids through RC of a-KG.
To gain insights into the molecular mechanisms underlying RC we took advantage of a panel of disease-associated VHL mutants. The ability of VHL mutants to restore glucose oxidation and inhibit RC correlates with their ability to bind and inactivate HIF. In addition, expression of a VHL-independent HIF mutant in VHL-restored RCC cells is sufficient to promote RC. These results indicate that HIF expression is necessary and sufficient for the induction of RC, at least in human VHL-deficient RCC cells. Next we showed that HIF promotes RC by a “mass action” effect on citrate/a-KG equilibrium. HIF expression drastically reduces intracellular citrate levels. Feeding VHL-deficient RCC cells with acetate or citrate, or knocking-down PDK-1 and ACLY enzymes restored intracellular citrate levels and suppressed RC. These data suggest that HIF-induced low intracellular citrate levels promote the reductive flux by mass action, to maintain lipogenesis.
Expression of HIF renders hypoxic and VHL-deficient cells addicted to glutamine in vitro. VHL-deficient cells displayed significantly less growth than their isogenic counterparts in media containing low glutamine concentration or in the presence of a glutaminase inhibitor. Systemic administration of a glutaminase inhibitor (BPTES) suppressed the growth of VHL-deficient human RCC cell lines as xenografts in nude mice.
Lastly, we investigated whether RC occurs in vivo. We metabolically labeled mice bearing VHL-deficient tumors by infusing them with 13C-1-Glutamine for up to 6 hours and we detected the early formation of labeled citrate in the tumors; thus we showed for the first time RC in vivo.
Our data provide mechanistic insights into the signaling events that mediate hypoxia-induced RC, strongly suggest that RC is an in vivo phenomenon in growing tumors and highlight potential novel therapeutic approaches for treatment of hypoxic and VHL-deficient tumors based on their metabolic signature.
Citation Format: Paulo Gameiro, Ana Metelo, Rocio Pérez-Carro, Alexandra Arreolla, Rania Baker, Zongwei Wang, Aria Olumi, Kimryn Rathmell, Pilar López-Larrubia, Gregory Stephanopoulos, Othon Iliopoulos. Hypoxia Inducible Factor (HIF) promotes reductive carboxylation by regulating citrate levels and renders VHL-deficient and hypoxic cells sensitive to glutamine deprivation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1881. doi:10.1158/1538-7445.AM2013-1881