PROJECT SUMMARY – PROJECT 1 Project 1 is striving to understand the role of transcription factor NRF2 and the gluconeogenic enzyme fructose bisphosphate phosphatase 1 (FBP1) in NASH to HCC progression. Advanced NASH is associated with disruption of hepatocyte autophagy and accumulation of p62 within Mallory-Denk Bodies, which by sequestration of KEAP1 activates NRF2. Although not tumorigenic on its own, NRF2 reprograms hepatocyte metabolism to support HCC initiation. We also found FBP1, previously described as an HCC tumor suppressor, to negatively regulate AKT activation independently of its enzymatic activity. Notably, NRF2 and FBP1 are engaged in a regulatory “tug-of-war,” whereby NRF2 leads to activation of ERK1/2, which phosphorylate FBP1 and induce its ubiquitylation and degradation, and by restraining AKT activation FBP1 enhances GSK3-induced NRF2 degradation. These novel cross-regulatory interactions play a major role in NASH to HCC progression. Although NASH increases HCC risk, it is also accompanied by tumor suppressive hepatocyte senescence. We confirmed abundant hepatocyte senescence in human NASH and using MUP-uPA mice that progress to NASH in response to high fructose diet (HFrD), we found hepatocyte senescence to be triggered by diet-induced DNA damage and activation of the DNA damage response (DDR), which was also detected in human NASH. The DDR results in activation of TP53, which induces FBP1 gene transcription and indirectly leads to NRF2 degradation. The FBP1- NRF2 balance is inverted in favor of FBP1 in mouse and human HCC. By analyzing 349 human HCC specimens, we found that TP53 and FBP1 are downregulated in 75-80% of them, correlating with AKT and NRF2 activation. Single-cell RNA sequencing of diseased human livers led to identification of a novel subpopulation of disease- associated hepatocytes (daHep), the first cells in which TP53 and FBP1 are downregulated and NRF2 is activated. We propose that daHep are the precursors to HCC and equivalent to HCC progenitor cells (HcPC) we detected in mice. Hepatocyte specific FBP1 ablation or NRF2 activation dismantle senescence and greatly accelerate HCC development, preceded by diet-induced DNA damage that leads to accumulation of oncogenic mutations. Despite this major advance in understanding NASH to HCC progression, several major questions remain and will be addressed using HFrD-fed MUP-uPA mice and human NASH and HCC specimens. 1. What causes FBP1 and TP53 downregulation and NRF2 activation in daHep and their mouse equivalents? 2. Do senescent hepatocytes directly convert to HCC progenitors or do HcPC arise from DNA damaged hepatocytes escaping senescence? 3. What causes DNA damage and mutations in HFrD-fed MUP-uPA/FBP1ΔHep or NRASG12V transduced livers? In addition to HFrD feeding, we will use hydrodynamic injection of oncogenic NRASG12V as a faster approach to HCC induction, which our preliminary studies show to be also controlled by the NRF2-FBP1 “tug-of-war,” and will apply ...