In the US, approximately 10,000 patients are newly diagnosed annually with cholangiocarcinoma, and their 5-year survival rate is less than 10%. Heterogeneity in cellular origins and molecular signatures of intrahepatic cholangiocarcinoma (ICC) highlight the demand for biomarkers to stratify tumors and generate targeted therapies. Besides biliary epithelial cells (BECs), hepatocytes (HCs) have been considered as a cellular origin of human ICC. This is evident by rapid HC-driven ICC induction through the co-expression of activated AKT (myrAKT) and Notch intracellular domain (NICD) in mouse HCs using sleeping beauty transposon system. Our preliminary analysis using large ICC patient cohorts demonstrates that the genetic signature of AKT-NICD (AN)-driven murine ICC correlates significantly with ~30% of human ICC, supporting the clinical relevance of this ICC model. Deletion of either Sox9 or Yap delayed ICC formation, and instead induced AN-driven SOX9-/YAP+ or SOX9+/YAP- ICC respectively, which also have been identified in human ICC. These data indicate that Notch independently regulates Sox9 and Yap in HC-driven ICC. Importantly, we found that co-repression of Yap and Sox9 completely prevents Notch- dependent ICC formation. However, the mechanisms of tumorigenesis driven by YAP and SOX9, as well as their interactions with AKT signaling, remain poorly understood. Based on our preliminary observations, our central hypothesis is that HC-driven ICC tumor growth depends on transcriptional and epigenetic alterations driven by two major downstream effectors of Notch signaling, SOX9 and YAP, alongside AKT signaling. In aim 1, we aim to test therapeutic effect of co-repression of YAP and SOX9 in advanced ICCs to better address the clinical need for late-stage therapy. We will employ genetic AN-ICC model as well as liver toxin-based ICC model using innovative inducible gene modulation systems to induce simultaneous, conditional and inducible Sox9 and YAP repression in advanced ICC. In aim 2, we are proposing 3 subaims to delineate the molecular mechanisms underlying AN- mediated HC-driven ICC formation. First, using ChIP-seq and bioinformatic tools, we will identify both the unique and the overlapping downstream targets regulated by Sox9 and Yap during HC-derived ICC formation. Second, we will elucidate the pathologic role of the NICD-YAP/TEAD-DNMT1 epigenetic axis in HC-driven ICC development by modulating this axis with pharmacological and genetic tools in the in vivo system and studying its effects on the methylome of ICC tumors. Pursuing these 2 subaims, we will identify downstream effectors of Sox9 and Yap for more selective and safer therapeutic targeting in ICC. Third, we aim to elucidate how AKT mediates HC-to-ICC transformation, which remains poorly understood. The successful execution of this study will 1) provide an essential evidence for considering SOX9 and YAP co- inhibition as an attractive candidate for precision medicine therapy, which is theorized to be the most effective approach to conquer lethal tumor, and 2) reveal the key downstream effectors for AKT, SOX9 and YAP which will help to develop more potent therapeutic options for ICC.

PROJECT NARRATIVE The objective of this proposal is to delineate molecular mechanism underlying hepatocyte-driven intrahepatic cholangiocarcinoma (ICC) development, thereby to contribute for better understanding of the disease pathogenesis as well as identifying novel therapies for this devastating tumor via precision medical approaches. Thus, our proposal addresses a major unmet clinical need. 1