OVERALL – PROJECT SUMMARY This is a renewal of the NCI-supported Mayo Clinic SPORE in Hepatobiliary Cancers (HBC), with a foundation built upon innovative translational science and utilizing state-of-the-art approaches to understand and treat liver cancers; these are the second leading cause of cancer deaths in the country. The breadth of the projects spans hepatocellular carcinoma (HCC), cholangiocarcinoma (CCA), and pediatric/young adult fibrolamellar (FLC). Three unique translational projects promise significant steps forward, supported by three well-constructed and respon- sive cores. Additionally, robust Developmental Research and Career Enhancement Programs continue to identify and support talented HBC-investigators, many from underrepresented groups. Ongoing support from the Mayo Foundation and Mayo Clinic Comprehensive Cancer Center benefit these components by enabling integrated resources, skilled staff, and leadership guidance to ensure success. The approach remains comprehensive, milestone focused, and patient centered. For example, the completion of former Project 3 has led to a novel first-in- class compound that has advanced to clinical trial. Three projects have developed successfully into innovative renewal pro- jects that utilize novel approaches of immunotherapeutics, computational biology, and chemical genomics. The translational projects in this renewal proposal investigate HBC biology to enhance therapeutic response, building on recent, promising clinical and preclinical advances, summarized below. Diagnostics and drug repurposing for FLC: Pro- ject 1 demonstrates 1) differential therapeutic sensitivity of FLC but with 2) variability in patient responsiveness, and now extends into a novel functional approach to precision medicine by 3) defining therapeutics that affect FLC cell viability, while also 4) developing blood- and urine-based biomarkers for tumor burden. Targeting yes- associated protein (YAP), transcriptional enhanced associate domain (TEAD) oncogenic signaling, and thera- peutic resistance in CCA: developed from studies defining alternative mechanisms activating this “undruggable” target (YAP) in CCA, Project 2 will 1) leverage a novel inhibitor in preclinical models and 2) test clinical efficacy in a clinical trial. Reversal of the immune suppressive HCC microenvironment through appropriately timed and optimally delivered combination viroimmunotherapies: advancing the use of oncolytic viruses to augment the killing of tumor cells for HCC, new Project 3 now will 1) test the clinical efficacy of a novel virotherapy combined with standard of care immunotherapy, 2) develop new combination viroimmunotherapies with increased potency in pre-clinical Sleeping Beauty models, and 3) develop a novel standard of care, virotherapy, CAR T-cell therapy combination. Collectively, this innovative, interactive, and highly collaborative translational research program is expected to advance prevention, early detection, diagnosis, tr...