Project Summary Liver cancer is the fourth leading cause of cancer-related death in the world and mortality rates are rising, which, in part, reflects historical drug development campaigns focused on single agent therapies (multi- targeted kinase inhibitors and immunotherapy) that have limited efficacy in patients. Additionally, there are a lack of targeted, personalized therapeutic options for this cancer type. Thus, there is an urgent need to identify novel tailored therapeutic strategies. Recent advances in the therapeutic targeting of hepatocellular carcinoma (HCC) have discovered superiority in efficacy for either small molecule inhibitors (for example, lenvatinib) or biologics (for example, bevacizumab) used in combination with immunotherapy, though less than 30% of patients respond to these new frontline therapies. A major gap in the field is the lack of appreciation for inter- patient heterogeneity and a failure to identify drugs that (I) uniquely target driver-specific vulnerabilities, or (II) improve immunotherapy. This proposal focuses on an unexplored strategy for therapy design in liver cancer, which leverages the usage of in vivo CRISPR screens and a novel patient-derived ex vivo autologous tumor organoid-T cell co-culture platform to provide new biological insight to mechanisms of drug potency on two critical components of the tumor microenvironment (TME), and allow assessment of combination therapies for the improvement of immunotherapy. I hypothesize that drug advancement in HCC, particularly in combination with immunotherapy, is dependent on drug-mediated modulation of tumor-T cell interactions. This hypothesis will be tested in two aims: (1) dissection of the mechanism of action of a novel candidate drug, WNTinib, in immunologically tractable models of HCC using in vivo CRISPR screens, and (2) identification of novel immunotherapy combinations for the personalized treatment of HCC. For Aim 1, WNTinib’s key targets (or mechanism’s contributing to activity or resistance) will be investigated using two separate genome-scale CRISPR sgRNA screening strategies: (I) transducing genetically-defined murine HCC organoids transplanted orthotopically into immunocompetent and immunocompromised mice, and (II) transducing T cells from OT-I;Cas9 and OT-II;Cas9 mice and transplanting them into tumor models expressing the OVA-derived peptide SIINFEKL. Analyses done in the presence of WNTinib will identify WNTinib’s tumor cell intrinsic, TME-dependent, and T cell-dependent targets or mechanisms. For Aim 2, I will use HCC patient tumor biopsies to establish organoids and tumor infiltrating lymphocyte (TIL) cultures. This approach will allow the study of tumor and T cells as separate components and in co-culture together. I will expand this platform to include drug screening, alone or in combination with immunotherapy, using a panel of drugs representing various stages of HCC clinical advancement. Results from this proposal will represent a major s...