Abstract: Immunotherapy is becoming a pillar of cancer treatment, and many responsive patients have experienced durable, or even curative, outcomes. For reasons that remain unclear, however, only a minority of cancer patients benefit from immune checkpoint blockade (ICB) therapies. Expanding this remarkable achievement to most cancer patients is being actively sought through multiple avenues, including combined immunotherapy, such as blocking both PD-1 and CTLA-4. In addition, promising results have been observed when ICB therapies were combined with available chemotherapies that potentiate the immune-mediated anti-tumor response. This opens the possibility that the use of small molecules could restore the immune system?s recognition of cancer cells as a foreign entity and thus would potentiate immunotherapeutic progress. In this context, we have identified a cyclic peptide, EnnA, as a novel inhibitor of heat shock protein 90 (Hsp90), with a potent ability to unleash the immune system against tumor cells. This discovery stemmed from our effort to find new Hsp90 inhibitors that circumvent known side effects that have hampered the clinical progress of first-generation inhibitors. In particular, this compound does not induce a heat shock response, which had reduced the efficacy of early inhibitors through activation of pro-survival mechanisms. EnnA induces immunogenic cancer cell death, promotes tumor immune cell infiltration, and unleashes a powerful T cell-mediated immune response, resulting in highly efficacious tumor killing in a syngeneic mouse model. Molecularly, EnnA interferes with several oncogenic pathways and reduces the protein level of the programmed cell death ligand-1 (PD-L1), a key mediator of tumor- induced immune tolerance. We therefore propose that EnnA is a promising anti-tumor agent targeting Hsp90 through a novel mechanism of action involving cancer cell toxicity that increases its immunogenicity and modulation of the tumor microenvironment to reduce immunotolerance. In Aim 1, we will perform preclinical development of EnnA as a drug to determine its toxicity and the potential impact of EnnA on the immune system of mice. We will also characterize the EnnA-Hsp90 interaction through mutational and biochemical analyses. In Aim 2, we will determine how inhibition of Hsp90 by EnnA interferes with PD-L1 chaperoning and function. In Aim 3, we will define immune cell mechanisms underlying EnnA?s anti-tumor effect. Combining EnnA with anti-CTLA-4 will be tested, and comprehensive profiling of immune cells involved in the anti-tumor activity will be implemented. In Aim 4, we will test the importance of EnnA- induced cancer cell autophagy and Hsp90? cell surface exposure in immune-dependent tumor eradication. If successful, these studies will shed light on the role of the Hsp90 in promoting immune tolerance and will provide an innovative approach to potentiate immunotherapy using a novel Hsp90 inhibitor.