Abstract: The systemic treatment of urothelial cancer (UC) has advanced over the last several years to include the use of immune checkpoint inhibitors for the treatment of metastatic disease. While helpful in many cases, the majority of patients do not respond to immune checkpoint inhibitor treatment. These patients’ tumors have or acquire the ability to suppress an active immune response despite immune checkpoint inhibition. Therefore, it is critical to develop agents that can modulate the anti-tumor immune response toward a less suppressive microenvironment and can combine with immune checkpoint inhibitors for more effective treatment. Entinostat is a selective class I histone deacetylase (HDAC) inhibitor that leads to histone hyperacetylation, chromatin remodeling, and gene expression alterations. We have preliminary data that entinostat can increase immune gene signature expression, alter predicted neoantigen expression, and synergize with immune checkpoint inhibition in murine models of UC. Based on these findings, a study evaluating the ability of entinostat to promote an anti-tumor immune response in human UC tissues is warranted. Specifically, this proposal will use a window of opportunity study of entinostat in patients with muscle-invasive UC to test the hypothesis that entinostat can augment the antigen-driven immune response induced by immune checkpoint inhibition alone. This hypothesis will be tested through characterization of immune gene expression, tumor neoantigens, and T cell receptor repertoires in patients treated with entinostat plus pembrolizumab compared to patients treated with pembrolizumab alone and compared to untreated patients. The research strategy proposed herein will simultaneously evaluate entinostat as a promising new agent in development for UC and fulfill my short-germ career goal of strengthening my scientific training in immunogenomic research methods. After completion of this proposal, I will be well positioned for research independence to achieve my long-term career goal to become a leader in genitourinary oncology with a focus on mechanism-based translational studies of novel combinations using epigenetic and immunomodulatory agents in combination with immunotherapy.