Project Summary Metabolic reprogramming in cancer is an attractive source of therapeutic targets because it fuels tumor growth and metastasis through enzymes that are in principle amenable to inhibition with small molecules. Metabolic reprogramming is intrinsic to renal cell carcinoma (RCC). In fact, few tumors are as profoundly linked to metabolic derangement as RCC and in particular, clear cell RCC (ccRCC). This is shown by: the clear cell phenotype, which arises from lipid/glycogen accumulation; direct metabolic reprogramming by the ccRCC signature event, VHL inactivation; and the observation that germline mutations in metabolic enzymes cause RCC, but few other tumor types. The two main barriers to targeting metabolic reprograming are the lack of knowledge about RCC metabolism in patients and the absence of validated translational platforms. To address these challenges, we executed 5 major activities in Years 1 – 5. First, we pioneered intraoperative infusions of 13C-labeled nutrients in patients to directly report on RCC metabolism in humans, which revealed, among others, suppressed glucose oxidation. Second, we showed, mechanistically, that suppressed glucose oxidation is due to deficient oxidative phosphorylation. Third, we determined that additive-free, orthotopically implanted, patient tumors (tumorgrafts, TG) are valid models to study human RCC metabolism. Fourth, we established the In Vivo Metabolism Lab, an innovative translational platform to detect metabolic reprogramming in human tumors, nominate therapeutic strategies, test them in TG models and primary human tumor tissue, and advance the most promising leads. Fifth, we demonstrated that both primary ccRCC tumors and metastases use glutamine to maintain redox balance and produce essential biosynthetic intermediates. Building upon discoveries by us and others implicating glutamine in cancer, the CB-839 glutaminase inhibitor was developed. However, results in ccRCC trials have been disappointing. One possible explanation is that glutaminase is only one of several enzymes that catabolize glutamine. Our new data not only explain CB-839 lack of efficacy, but also identify new opportunities for intervention. Indeed, while CB-839 inhibits carbon metabolism by targeting glutaminase, glutamine is also a source of nitrogen in RCC, which is processed via amidotransferases, which are not inhibited by CB-839. In preliminary data, we show that pan-glutamine inhibition with JHU-083 not only effectively inhibits amidotransferase reactions, but also significantly blocks ccRCC TG growth. To advance effective glutamine targeting to the clinic, in Years 6 – 10, we will pursue the following Aims. Aim 1. Probing the role of amidotransferases in mediating resistance to CB-839 glutaminase inhibitor. Aim 2. Targeting IDH enzymes to maximize glutamine blockade. Aim 3. To maximize the impact of glutamine targeting by leveraging the tumor microenvironment using next-generation models.