PROJECT SUMMARY/ABSTRACT Mutations in telomerase and telomere attrition are major risk factors for liver fibrosis and its progression to hepatocellular carcinoma (HCC). However, due to a lack of adequate models and intrinsic difficulties in studying human telomerase in physiologically relevant cells, the molecular mechanisms responsible for liver fibrosis and cancer in settings of DNA damage arising from short telomeres remain elusive. While telomerase knockout mice corroborate the importance of telomere maintenance and DNA repair for liver function, the molecular mechanisms that govern liver abnormalities in patients with damaged telomeres are still unknown. Likewise, the specific signaling pathways that trigger failure of hepatic cells following telomere shortening and accumulation of DNA damage remain to be determined. In addition, mutations in the promoter region of the telomerase reverse transcriptase component (TERT) have been described as the initial and most prevalent mutation in HCC. While these mutations have been shown to reactivate telomerase, the functional relevance of this process during failure and transformation of hepatic cells has yet to be interrogated. The focus of this proposal is to use human pluripotent stem cells as a novel platform to understand the detrimental effects of mutant telomerase, telomere shortening and accumulation of DNA damage in different hepatic cell lineages. We have previously generated isogenic hPSC lines harboring several disease-specific mutations in telomerase and have successfully derived telomerase-mutant human hepatocytes and hepatic stellate cells in vitro, following established protocols that recapitulate the in vivo development of these lineages. Here, two specific aims are proposed that utilize this platform to understand the molecular consequences of telomere erosion, DNA damage, and telomerase impairment for the function of hepatic cells, and to determine their role during early stages of transformation. In Aim 1 we will determine the role of telomere shortening and DNA damage accumulation during fibrotic failure of different hepatic cell lineages with impaired telomerase. We will determine the extent to which mitigation of DNA damage, reactivation of HNF4α, and modulation p53 prevent fibrotic triggering in telomerase-mutant hepatocytes with variable telomere lengths. As liver fibrosis and its progression to HCC are multicellular responses we will determine the role of progressive telomere shortening during the direct and the paracrine fibrotic activation of hepatic stellate cells. In Aim 2, we will investigate the molecular consequences of mutations in the TERT promoter region during progression of HCC, in settings of exacerbated DNA damage due to eroded telomeres. Specifically, we will analyze the biochemical and functional consequences of mutations in the TERT promoter region for hepatocyte function and immortalization. These studies will determine the molecular mechanisms of liver f...