PROJECT SUMMARY. Renal cell carcinoma (RCC) is among the 10 most common malignancies in both men and women. Although outcomes are excellent for patients with tumors confined to the kidney, patients with advanced disease face unfavorable outcomes despite several approved therapies. This underscores the need for new treatment strategies. We have identified elevations of the oncometabolite L-2-hydroxyglutarate (L-2HG) in a significant proportion of RCCs. Oncometabolites are small molecules that aberrantly accumulate in cancer cells and can impact tumors in many ways. Our studies reveal that L-2HG is a potent driver of RCC growth leading us to investigate the mechanisms by which this small molecule exerts its oncogenic activity. Delineating these effects will inform novel strategies for oncometabolite-driven kidney cancer. Our preliminary studies have uncovered two novel findings. First, L-2HG is a powerful regulator of the epitranscriptome. In particular, our preliminary studies demonstrate that L-2HG promotes accumulation of the RNA mark, N6-methyladenosine (m6A). m6A is the most prevalent nucleotide modification in eukaryotic mRNA and can profoundly impact gene expression (e.g. mRNA stability, translation). Second, L-2HG remodels tumor metabolism. Our compelling preliminary data indicate that these two findings are intricately linked leading us to propose the innovative hypothesis that L-2HG induced RNA hypermethylation remodels metabolism to drive tumor growth but creates metabolic liabilities. In Aim 1 we will determine the molecular underpinning by which L-2HG promotes RNA methylation and the functional significance of this effect. In Aim 2, we will dissect the mechanisms by which L- 2HG remodels metabolism with specific focus on glucose utilization and its support of anabolic activities. In Aim 3, we will assess the impact of L-2HG induced metabolic remodeling on tumor growth. In addition, we will determine if this remodeling creates metabolic vulnerabilities. Leveraging our expertise in the field, we will incorporate novel models generated by our group and apply state of the art methodologies such as ribosome profiling to study the biology of L-2HG. The proposal work is significant as it will likely lead to novel approaches that target either the epitranscriptome and/or tumor metabolism. The proposal is timely given recent proof-of- principle studies demonstrating that these approaches are pharmacologically targetable. We have assembled a multidisciplinary team required for the successful completion of the proposed studies. Given the prevalence of high L-2HG RCC, we are poised to advance the field with findings that will impact a significant proportion of kidney cancer patients.