Project Summary/Abstract The research objective is to identify the source of chronic inflammation in obesity-mediated hepatocellular carcinoma (HCC). Hepatocellular carcinoma (HCC), a major form of liver cancer, is the fourth leading cause of cancer-related deaths worldwide. In recent years, obesity has emerged as the major and independent risk factor for HCC, and HCC is predicted to the third leading cause of cancer-related deaths in the United States by 2030 due to obesity epidemic. Obesity-induced nonalcoholic fatty liver disease (NAFLD), which affects nearly 25% of the US population, is a key driver of HCC in obese individuals. Despite this strong association between obesity and HCC, the mechanisms that drive HCC development in obesity is not clearly understood. Non-resolving chronic inflammation is a major contributor to the development and progression of HCC in obesity and damage associated molecular patterns (DAMPs) are one of the proposed mediators of HCC. Necroptosis is a programmed cell death that has been shown to play a major role in inflammation through the release of DAMPs, which bind to cell surface receptors of immune cells to induce inflammation. In this application, we propose that hepatocyte necroptosis in NAFLD is a key factor in HCC progression. Our preliminary data support the role of necroptosis in HCC, e.g., blocking necroptosis using Ripk3-/- or Mlkl-/- mice reduced hepatic inflammation, infiltration of innate immune cells, and HCC incidence in mice fed a choline deficient-high fat diet (CD-HFD) that do not induce obesity. In a genetic mouse model of spontaneous HCC (Sod1-/- mice) inhibiting necroptosis using necroptosis inhibitor, necrostatin-1s, reduce hepatic necroptosis, inflammation, and pathways mediating HCC development. Based on this, we propose that hepatocyte necroptosis is the major source of hepatic inflammation in the microenvironment where HCC is initiated. We hypothesize that progression of NAFLD to HCC is due to increased inflammation that arises from hepatocyte necroptosis and that preventing necroptosis will reduce inflammation and progression of NAFLD to HCC. This hypothesis will be tested using genetic approaches to block or activate necroptosis in hepatocytes by targeting RIPK3 or MLKL, kinases in the necroptosis pathway, and assessing its effect on inflammation and HCC. In Aim 1, necroptosis will be specifically reduced/blocked in hepatocytes using hepatocyte specific Ripk3 or Mlkl knockout mice, and determine if blocking hepatocyte necroptosis reduces inflammation and delays/reduces HCC development in response to an HCC- inducing diet; In Aim 2, necroptosis will be induced specifically in hepatocytes by overexpressing RIPK3 or MLKL using a novel Ripk3 or Mlkl knockin mouse model we have developed to determine if inducing necroptosis in hepatocytes leads to an increase in liver inflammation and increased progression and incidence of HCC.