Abstract This multiple-PI application proposes investigations of cellular and molecular mechanisms of Fibrolamellar Hepatocellular Carcinoma, FLC. FLC is a disease that occurs in children, adolescents and in young adults without a history of other liver disorders such as fibrosis or cirrhosis. Numerous studies in FLC patients and in animal models clearly showed that FLC is caused by a fusion protein kinase DNAJB1-PKAc which is sufficient to induce liver cancer resembling FLC in mouse models. We found that the fusion kinase DNAJB1-PKAc phosphorylates b-catenin at Ser675 in FLC patients and in FLC-derived spheroid cells. This phosphorylation leads to elevation of the b-catenin-TCF4 complex which might activate multiple cancer genes via two pathways: interaction with and activation of unique chromosomal regions (Cancer-Enhancing Genomic Regions or Aggressive Liver Cancer Domains, CEGRs/ALCDs) and via direct activation of the promoters of genes involved in metabolic rewiring. The main hypothesis of this application is that the DNAJB1-PKAc-b-catenin axis is a critical mediator of FLC pathology via activation of CEGRs/ALCDs-dependent neuronal genes, oncogenes and collagens as well as genes involved in pathological metabolic changes. Three Specific Aims are designed to test this hypothesis. Specific Aim 1 will examine the role of the DNAJB1-PKAc-b-catenin pathway in activation of CEGRs/ALCDs-dependent cancer genes in FLC. The focus of these studies is on the FLC patients who have relapsed tumor and lung metastases which are potentially caused by activation of DNAJB1-PKAc-b-catenin- dependent neuronal pathways. Specific Aim 2 will investigate the role of the DNAJB1-PKAc-b-catenin-TCF4 axis in FLC using a mouse model of FLC. We have generated an FLC mouse model, in which DNAJB1-PKAc protein is created and expressed at high levels at 4 months after injections of gRNAs. We will examine development of pathological changes in the liver and investigate the DNAJB1-PKAc-b-catenin-TCF4 pathway at different stages of FLC. We will inhibit b-catenin by PRI-724 in the FLC mouse model, examine development of FLC, and determine downstream molecular mediators. Specific Am 3 will determine the role of the DNAJB1- PKAc-b-catenin-TCF4 axis in rewiring of metabolic pathways in FLC. Our preliminary data suggest that the urea cycle and associated metabolic processes are rewired toward increased collagen synthesis and maturation in FLC, and the treatment of FLC spheroids with PRI-724 reverts these changes. Therefore, we will test the hypothesis that the DNAJB1-PKAc-β-catenin-TCF4 axis is a critical event for metabolic rewiring and will also determine the impact of metabolic rewiring on FLC proliferation and survival. Thus, this project will elucidate mechanisms of FLC and develop a background for b-catenin-based therapy.