PROJECT SUMMARY/ABSTRACT Hepatocellular carcinoma (HCC) is the 6th leading cause of cancer-associated mortality in the United States, and is rising due to chronic liver disease and its associated sequalae. Currently, the response rates to current therapeutic paradigms consisting of immune checkpoint inhibitors (ICIs) remain low, and there exist an urgent need for novel combinatorial therapies to improve patient mortality. Lack of response to current ICIs is mainly due to a poor understanding of the tumor immune microenvironment (TIME) and how various HCC driver mutations lead to specific immune phenotypes. Additionally, there currently exist no biomarker-driven therapeutics for patient treatment stratification. ꞵ-catenin-active (encoded by mutated CTNNB1 oncogene) HCCs represent approximately 26-35% of HCCs and respond poorly to ICIs due to ꞵ-catenin driving an immunosuppressive TIME and limiting the effector function of lymphocytes important for anti-tumor immunity. We have developed novel ꞵ-catenin-mutated HCC mouse models where mutant CTNNB1 is co-expressed with either the proto-oncogene MET (ꞵ-catenin/hMet) or nuclear factor erythroid 2–related factor 2 (Nrf2) (ꞵ- catenin/Nrf2). These models represent 11% and 10% of all clinical HCC cases, respectively. Our preliminary studies demonstrate that ꞵ-catenin potentiates tumorigenesis in ꞵ-catenin-mutated HCC, and that directly targeting ꞵ-catenin promotes an inflammatory response driving anti-tumor immunity. Based on these observations, our overarching hypothesis is that ꞵ-catenin actively suppresses the adaptive immune response in the TIME and targeting ꞵ-catenin or its downstream immunomodulatory factors may improve susceptibility to ICIs. To investigate, I propose the following specific aims, which will uncover novel mechanisms of ꞵ-catenin signaling in the HCC TIME, aimed at developing precision medicine therapeutics. Specific Aim 1: We will determine the immune cells activated following ꞵ-catenin inhibition, and investigate whether there is in vivo synergy combining ꞵ-catenin inhibition and ICIs through single-cell RNA-sequencing (scRNA-seq) and multiplex immunohistochemistry. Thus, we will identify mechanisms of immunosuppression caused by ꞵ-catenin activation in ꞵ-catenin-mutated HCC. Specific Aim 2: Based on our preliminary data showing interferon regulatory factor- 2 (IRF2) repression in ꞵ-catenin-mutated HCC, we hypothesize that ꞵ-catenin-mutated HCCs may be sensitized to ICIs, or even show spontaneous tumor regression, upon re-expression of IRF2 as a result of enhanced immune response. We will use synthetic biology approaches to selectively induce IRF2 expression at various timepoints in tumorigenesis and monitor tumor burden. We will then use scRNA-seq on the lymphoid population to identify cell types and states regulated by IRF2, and test combination of IFNg (which induces IRF2) + ICI as a therapeutic modality. Contribution to Training: This proposal combines rigorous research trainin...