PROJECT SUMMARY / ABSTRACT Hepatocellular carcinoma (HCC) incidence and mortality is increasing in the US and worldwide. Around ~50- 60% of HCC patients will receive systemic therapies. After a decade of primacy of sorafenib, the combination regime of immune checkpoint inhibitor (ICI) atezolizumab (anti-PDL1) with bevacizumab (anti-VEGFA) demonstrated superior clinical benefits (median survival of ~19 months) and has become the standard of care. However, the rate of objective response remains at ~30%. In parallel, our group demonstrated that 1) HCC etiology differentially impacts outcome with patients with HCC deriving from non-alcoholic steatohepatitis (NASH) benefitting significantly less from immunotherapy; 2) Dysfunctional CD8 cells are implicated in the underlying mechanism of resistance in NASH-HCC; 3) Newly generated gene signatures predict response to ICI; and 4) The immune excluded class is driven by Wnt signaling/CTNNB1 mutations in HCC and, thus discovered KIT/MAPK/Wnt inhibitors combined with ICI are adequate strategies to rescue this mechanism of immune- evasion. Our central hypothesis is that such decreased response to ICIs in patients with NASH-HCC can be reverted therapeutically using combinations of ICI and KIT/MAPK/Wnt signaling blockers; and candidate biomarkers of response can be identified and validated. Thus, the overarching goal of this proposal is to gain further insight into mechanisms of both NASH-HCC immune response and resistance via state-of-the-art single cell technologies, so as to identify biomarkers and overcome resistance through the rational testing of combinatorial immunotherapeutic strategies, which could eventually increase the number of HCC patients deriving clinical benefit from immunotherapy. To accomplish this goal, we seek to achieve the following specific aims: 1) To map the immune cell microenvironment in human NASH-HCC by using single cell-based approaches and high-resolution spatial transcriptomics; 2) To identify biomarkers predicting response and resistance to the combination of atezolizumab plus bevacizumab in human NASH-HCC by testing identified gene signatures and molecular markers of response (using transcriptomics and mutational profiling), as well as spatial transcriptomics; 3) To develop therapeutic strategies to overcome ICI resistance in NASH-HCC using specific patient-derived organoids with immune component and mouse models that recapitulate the human NASH-HCC microenvironment. These hypothesis-driven strategies include testing drugs blocking key pathways of immune evasion (Wnt) in combination with ICI. The pursuit of these aims will be coupled with our expertise in NASH- related hepatocarcinogenesis, genomics and transcriptomics, single-cell based technologies, immuno-oncology, generation of organoids reconstituted with TILs and mouse modeling. We expect that our proposal will bring precision immune-oncology closer to the clinics, will promote clinical trials including KIT/MAPK/WNT inhib...