PROJECT SUMMARY North America has the highest incidence of renal cancer in the world with the most common subtype being clear cell renal cell carcinoma (ccRCC). Alarmingly, the incidence of ccRCC is on the rise in the U.S. and globally. The overall five-year survival rate for ccRCC is only 10-12% once it becomes metastatic, demonstrating the need for new therapies. Metabolic dysfunction is common in ccRCC based on histologic and molecular analysis. New therapies to treat ccRCC could target its dysregulated metabolic pathways. The etiology of ccRCC is complex but a major risk factor is obesity. High levels of branched-chain amino acids (BCAAs) are present in the serum of patients with ccRCC and obesity, suggesting a potential mechanistic link. BCAAs (leucine, isoleucine, and valine) are essential amino acids, whose concentrations are regulated by dietary intake and catabolism. BCAA metabolism promotes tumor growth in many different types of cancer, but the role of BCAAs in ccRCC is unknown. BCAA catabolism occurs primarily via the first two enzymes, BCAA transaminase (BCAT) and branched chain ketoacid dehydrogenase (BCKDH), respectively. BCKDH is an enzyme complex that catalyzes the rate-limiting reaction. The products of BCAA catabolism are then oxidized within the mitochondria to produce succinyl-CoA and acetyl-CoA, which can be used by the TCA cycle for anaplerosis and mitochondrial respiration. My preliminary data demonstrate that BCAA catabolic enzyme BCAT2 and BCKDH subunits are frequently reduced in human ccRCC tumors compared to normal adjacent kidney tissue (NAT). This decreased expression occurs as early as stage 1 and is associated with reduced overall survival. Additionally, BCAAs and their catabolic metabolites are decreased in ccRCC. These results suggest that BCAA catabolism is reduced early in ccRCC and contributes to ccRCC aggressiveness. ccRCC may reduce BCAA metabolism to decrease mitochondrial respiration and ROS because ccRCC is susceptible to ROS due to the large amount of intracellular lipids that can undergo peroxidation. ccRCC decreases mitochondrial respiration and ROS by downregulating genes involved in fatty acid oxidation and oxidative phosphorylation. I hypothesize that reduced BCAA metabolism promotes ccRCC cell growth and tumorigenesis, and does so by decreasing mitochondrial respiration and the production of ROS. Aim 1 will determine how reduced BCAA catabolism promotes ccRCC cell growth in vitro. I will use genetic and pharmacologic approaches to assess how gain or loss of BCAA metabolism affects proliferation of ccRCC and immortalized renal epithelial cell lines. Aim 2 will identify how reduced BCAA catabolism contributes to ccRCC tumorigenesis in vivo. I will use novel genetic mouse models to determine the role of BCAA catabolic flux on renal epithelial cell function and ccRCC tumorigenesis. Together, these approaches will identify the mechanisms by which BCAA catabolism regulates ccRCC tumorigenesis and ident...