Project Summary Ewing sarcoma (EWS) is the second most common tumor involving bone in children and young adults and is fatal in most patients with metastatic or relapsed disease. Patients who survive are left with a lifetime of late effects from the toxic therapy they receive. There have been no new, successful targeted drugs developed to treat EWS for nearly forty years, and we have reached the limit on how much we can intensify chemotherapy treatments. New therapeutic approaches are necessary to prevent relapse and cure more patients. Immunotherapy has altered the treatment landscape for many adult solid tumors but has not yet mediated substantial benefit for children with EWS. Chimeric antigen receptor (CAR) T cells have revolutionized the treatment of children with relapsed leukemia and lymphoma. Recently, we found that CAR T cells targeting GD2, a sugar expressed on the surface of many pediatric cancers, are active in children with incurable brain tumors. This proposal focuses on applying GD2 CAR T cells to EWS. Because GD2 is heterogeneously expressed on EWS, we will explore multiple mechanisms to effectively apply CAR T cells in this disease: 1) targeting a closely related ganglioside that is highly expressed when GD2 is low and 2) utilizing epigenetic inhibitors to significantly increase GD2, making CAR T cells better able to recognize tumor cells. In Aim 1, we will define the expression of gangliosides and their related synthase enzymes on patient tissues and test CARs against an alternative ganglioside. In Aim 2, we will utilize epigenetic inhibitors to increase GD2 on EWS tumors in vitro and in vivo. In Aim 3, we will test combinatorial approaches of CAR T cells and epigenetic inhibitors. Successful completion of these studies will result in new therapeutic options for children with Ewing sarcoma.