RP2 – Nuclear PDH1 drives epigenetic TIC reprograming in alcohol-associated HCC ABSTRACT Hepatocellular carcinoma (HCC) is the third leading cause of malignancy-related mortality in the world. Metabolic liver disease such as alcohol-associated liver disease (AALD), metabolic dysfunction associated fatty liver disease (MAFLD) or a combination of both sometimes referred to both alcoholic and non- alcoholic steatohepatitis (BASH), are becoming a major etiologic background of HCC. Therapeutic efficacy for HCC is limited due largely to therapy-resistance and recurrence in which tumor-initiating stem-like cells (TICs) are implicated via their self-renewal activity driven by the pluripotency factor NANOG. Proposed research addresses a novel epigenetic regulation of NANOG mediated by mitochondrion (mito)-nucleus crosstalk in TICs which we hypothesize as a potential therapeutic target. The mito pyruvate dehydrogenase (PDH1) is recently shown to translocate to the nucleus to supply acetyl-CoA from pyruvate to stimulate H3K27 acetylation (H3K27ac) in stem cells. We identified the RAB7 GTPase-activating protein TBC1D15 as a novel oncoprotein responsible for p53 degradation and NOTCH activation. TBC1D15 is now suggested to mobilize mitochondria (mito) by untethering mito-endosome interactions. Our preliminary results reveal: 1) NOTCH Intracellular Domain (NICD), TBC1D15 and mito fission protein 1 (FIS1) form a tripartite complex at the mito outer membrane in TICs; 2) mito are closely associated with nuclei in TICs and this spatial feature is diminished by TBC1D15 knockdown (KD) which disrupts the tripartite interaction; 3) increased nuclear PDH1 in TICs is blunted by TBC1D15 KD; and 4) PDH1 KD or inhibitor treatment reduces H3K27ac at the NANOG distal enhancer, NANOG expression and self-renewal in TICs. These results collectively suggest a novel epigenetic mechanism of NANOG upregulation mediated by PDH1 which is translocated to nuclei via a mito-nuclear proximity facilitated by the NICD- TBC1D15-FIS1 interaction. Hypothesis: Mito-nucleus transfer of PDH1 generates pyruvate-derived acetyl-CoA in nucleus and drives sustained NANOG upregulation via H3K27ac of its enhancer. The NICD1-TBC1D15-FIS1 interaction initiates this PDH1 nuclear translocation and is a plausible therapeutic target for alcohol-associated HCC. Aim 1. To dissect molecular mechanisms of the mito-nucleus transfer of PDH1: 1.1. To test if NOTCH1-TBC1D15-FIS1 interaction facilitates perinuclear mito localization and PDH1 accessibility to the nucleus; 1.2: To test the proposed PDH1 transfer mechanism in human HCC organoids; 1-3: To perform in silico analysis to establish the 3-D structural basis for the NICD- TBC1D15-FIS1 interaction and to screen for small molecule inhibitors which target the interaction and characterize their activities by fluorescence polarization and microscale thermophoresis assays. Aim 2. To determine if nuclear PDH1 generates acetyl-CoA to increase H3K27ac at the NANOG enha...