PROJECT SUMMARY As a ubiquitous HSP90 paralog in the endoplasmic reticulum (ER), GRP94 plays important roles in protein quality control in the secretory pathway by participating in both the unfolded protein response and the ER-associated protein degradation pathway. My laboratory has demonstrated that GRP94 is a strategically important target for cancer, because it controls multiple key molecular pathways in cell growth, migration, immune tolerance and differentiation, including integrins, TLRs, IGF-II, Wnt co-receptor LRP6, and GARP (or LRRC32). GARP (Glycoprotein A Repetitions Predominant) is responsible for surface docking and activation of latent TGFb and a focus of this proposal We have made significant contributions to this area through immunological and biochemical studies, including: 1) that GARP is an important molecule for cancer immune evasion via regulating multiple cell types (e.g., cancer cells, platelets, regulatory T cells, B cells). 2) We discovered a novel mechanism of TGFb activation from cell surface GARP-TGFb complex via proteolytic cleavage of GARP. 3) GARP has been found to be aberrantly expressed in multiple human cancers to promote oncogenesis via both cancer cell-intrinsic and -extrinsic mechanisms. 4) Preclinical studies suggest that GARP is a novel therapeutic target for cancer immunotherapy. These accomplishments have deepened our conviction that the study of GRP94 and its client network will lead to better understanding of this chaperone biology in cancer and to development of novel cancer therapeutics, alone or in combination with approved immunotherapeutic agents. In the next phase of the study, we will address the hypothesis that GRP94/GARP-targeted therapy applied to multiple vulnerable cancers will overcome immune resistance to checkpoint inhibitors. First, we will determine the roles and molecular mechanism involved in GRP94 regulation of TGFb biogenesis, activation and signaling. This aim will focus on structural analysis of the GRP94-GARP complex, and on resolving mechanisms of GRP94 in folding two other molecules important in regulating TGFb signaling: LRRC33 and LRG1. Second, we will develop novel cancer immunotherapeutic strategies targeting GRP94 and GARP. The goal is to advance the top first-in-class agent(s) among several pre-clinical leads through a milestone-driven strategy. This includes agents to inhibit GARP cleavage, GARP-specific antibodies, drug-like GRP94-selective inhibitors, antibodies against the cell surface GRP94 (ectoGRP94) preferentially expressed on cancer cells, and T cells engineered to express chimeric antigen receptor (CAR) composed of a single-chain antibody against ectoGRP94 fused with T cell signaling motifs (GRP94-CAR-T). Overall, the impact of this study lies in fundamental understanding of GRP94 in regulating the TGFb pathway and in developing promising next generation immunotherapeutic agents.