Project Summary Ewing's sarcoma (ES) is the second most common pediatric bone malignancy affecting ~10,000 children, adolescents, and young adults worldwide each year. Approximately half of all patients with Ewing's sarcoma will develop either recurrent or metastatic disease with less than 20% of such patients surviving long-term. The standard of care for ES patients includes multi-agent chemotherapy to treat documented or potential metastatic disease, coupled with surgery and/or irradiation to treat the primary tumor. Although some incremental advances have been made in the last three decades through intensification of conventional chemotherapy agents, more significant improvements will likely depend on the identification of novel treatment strategies. Two hallmark clinical features of ES are: 1) the accumulation of intracellular glycogen deposits that are Periodic acid-Schiff positive (PAS+) during pathological analysis, and 2) the EWS-FLI1 fusion oncogene. Contribution of EWS-FLI1 to tumorigenesis and epigenetics is well defined, but little is known about the biology and pathology of ES glycogen accumulation nor has ES glycogen metabolism been explored as an anti-ES target. The aims presented in this proposal are designed to interrogate the role of glycogen metabolism in promoting tumorigenesis in ES and extend my career in cancer metabolism. My preliminary data demonstrate glycogen in ES models has aberrant architecture with long chains, increased branching pattern, and high phosphate content. Thus, this glycogen is more similar to pathogenic polyglucosan bodies (i.e. ES-PGBs) and disrupts normal cellular processes. Excitingly, I have demonstrated that targeting ES-PGBs using a glycogen synthase inhibitor reduces in vivo tumor growth. I will expand on these findings in Aim 1 by demonstrating ES- PGBs alter cellular energy homeostasis utilizing high-throughput metabolomics profiling and innovative biochemical assays in vitro and in vivo. These results will advance our understanding of glycogen metabolism in ES and have broad implications for designing new therapeutic options for ES patients. In Aim 2, I plan to extend my predoctoral training in cellular metabolism of a rare cancer to my postdoctoral training in the emerging field of cancer metabolism's role in epigenetic regulation. I have received excellent training in the field of glycogen metabolism in cancer and I believe this foundation will help answer key outstanding questions in the utilization of key metabolites to control gene expression and protein function. Cumulatively, this proposal provides time in both my predoctoral and postdoctoral training to learn skills necessary to achieve my goal of an independent investigator in cancer metabolism.