PROJECT SUMMARY Cancer cells exploit multiple metabolic strategies to generate biosynthetic precursors that fuel malignant proliferation. Such metabolic redundancy and plasticity hampers effectiveness of therapies that target cancer cell metabolism and underscores the importance of identifying tumor types most likely to respond to metabolic inhibitors. The goal of this proposal is to test the hypothesis that tumors with impaired metabolic pathways will have limited metabolic plasticity in generating critical biosynthetic intermediates, rendering them susceptible to metabolic inhibition. We focus on the tricarboxylic acid (TCA) cycle, which is a central metabolic hub that supports cell growth and yet is truncated or impaired in several forms of human renal cell cancer (RCC). A subset of RCC tumors arise from germline deficiencies in core TCA cycle enzymes succinate dehydrogenase (SDH) or fumarate hydratase (FH); more commonly, RCC tumors have hyperactive hypoxia-inducible factor (HIF) signaling that blunts TCA cycle metabolism. The goal of this proposal is to determine whether these RCC tumors with altered TCA cycle activity are dependent upon ATP citrate lyase (ACL) as an alternative source of critical metabolic intermediates. Aspartate, synthesized from TCA cycle intermediate oxaloacetate, supports nucleotide and protein synthesis and has emerged as a critical limitation for tumor growth. Nevertheless, how tumors with impaired TCA cycle flux sustain aspartate generation—and whether these compensatory pathways represent a targetable liability—remains largely unknown. My preliminary data demonstrate that SDH/FH-deficient or HIF-active RCC cells have reduced aspartate pools relative to their isogenic controls and that ACL inhibition selectively impairs survival of these cells with defective TCA cycle metabolism. In Aim 1, I will leverage a panel of isogenic RCC lines to test whether genetic and pharmacologic ACL inhibition specifically impairs growth of SDH-/FH-deficient RCC cells in vitro and in vivo. I will exploit genetic tools that supply intracellular aspartate to test the hypothesis that aspartate provision underlies the ACL requirement in SDH-/FH-deficient cells. In Aim 2, I will use RCC cells with hyperactive HIF driven by loss of the von Hippel Lindau tumor suppressor to determine whether cells with suppressed oxidative TCA cycle activity depend on ACL to produce aspartate and enable growth in vitro and in vivo. These studies will shed light not only on a potential metabolic Achilles heel in SDH-/FH-/VHL-null tumors but will also serve as proof of principle that cancer cells with TCA cycle dysfunction engage ACL as an alternative route of synthesizing anabolic precursors. The work and training plan outlined in this proposal will be completed in the laboratory of Dr. Lydia Finley with the co-advisement of Dr. Ross Levine at Memorial Sloan Kettering Cancer Center and will ideally prepare the applicant for further clinical training and a care...