PROJECT 2 - PROJECT SUMMARY The long-term objective of this project is to define molecular and cellular mechanisms that drive cholangiocarcinoma (CCA) carcinogenesis, therapeutic resistance, and evasion of immune surveillance through preclinical studies and a first-in-human clinical trial. The project is focused on the mechanisms by which YAP- TEAD signaling can promote CCA development and survival and proposing TEAD inhibition as a novel therapeutic approach. The overall objectives are to examine: (a) the dependence of CCA carcinogenesis on YAP-TEAD signaling and the immunomodulatory mechanisms of YAP-TEAD signaling in preclinical CCA models, (b) the effects of therapeutic targeting of TEAD in preclinical models of cholangiocarcinoma both as a single agent and in combination with standard of care chemotherapy or immunotherapy, and (c) the safety and efficacy of a novel TEAD inhibitor, CTX1009685, in a first-in-human clinical trial for advanced CCA. In preliminary studies, there were several pivotal observations. First, inhibition of YAP-TEAD signaling decreased CCA cell viability in vitro and sensitized CCA cells to the combinatorial chemotherapy of gemcitabine and cisplatin, a standard of care treatment for advanced CCA. These in vitro effects translated to decreases in tumor size in vivo utilizing patient-derived xenograft models. Similarly, extended targeting to include YAP, it’s paralog TAZ, and the YAP-activating molecule LCK was associated with increased CCA cell death, decreases in tumor sizes in vivo, and sensitization to gemcitabine and cisplatin. Mechanistically, it was observed that this extended targeting approach was associated with increases in infiltrating cytotoxic T cells and decreases in granulocytic myeloid- derived suppressor (G-MDSC) cells. Based on these data, the CENTRAL HYPOTHESIS is that YAP signaling provides therapeutic resistance in CCA, which can be overcome utilizing selective YAP-TEAD inhibition. The approach will employ complementary in vitro and in vivo experimental approaches with mouse and human cell lines, patient-derived xenografts and mouse models to explore the hypothesis in three integrated SPECIFIC AIMS. First, the team will utilize doxycycline-inducible in vivo models and spatial transcriptomic approaches to understand the YAP-TEAD mediated initiating events in CCA carcinogenesis and utilize a novel TEAD inhibitor to evaluate efficacy of TEAD targeting as a therapeutic approach in multiple, fully characterized in vivo CCA models. Second, the investigators will utilize sophisticated syngeneic CCA models to explore the mechanisms underlying YAP-mediated immune evasion and explore therapeutic targeting utilizing TEAD inhibition and clinically approved immune checkpoint inhibitors. Third, a first-in-human clinical trial will be conducted evaluating safety and efficacy of the TEAD inhibitor CTX1009685 in patients with advanced CCA. This technically and conceptually innovative application is also significant b...