Hepatocyte Senescence is Initiated through a Klf6-p21 Mechanism which mediates the Production of Cxcl14, a Novel Prognostic Biomarker of Acute Liver Failure

Abstract

<b>Abstract ID 29424</b> <b>Poster Board 226</b> <b>Background:</b> Acute liver failure (ALF) is characterized by hepatic encephalopathy, coagulopathy, peripheral vasodilation and organ failure. The majority of all ALF cases in the United States and UK are due to acetaminophen (APAP) overdose. An extensive body of work has elucidated the molecular mechanisms of hepatocyte necrosis during APAP overdose, but detailed mechanisms involved in the development of ALF have been largely unexplored. Previous work demonstrated that a severe overdose of APAP (600 mg/kg) results in prolonged injury, impaired regeneration and sustained inhibition of cell cycle progression mediated through p21. Cell senescence is a cell fate resulting in cell cycle arrest predominantly controlled by p16 or p21 dependent pathways. Some senescent cells acquire a senescent associated secretory phenotype (SASP) which consists of bioactive signals that can influence the microenvironment. Why some senescent cells develop a SASP, and others do not remains elusive, however the specific molecular program governing senescence is thought to vary depending on cell type and inducing factors. Klf6 is induced during senescence, promotes p21 expression, and when overexpressed decreases proliferation in hepatocellular carcinoma. <b>Aim:</b> We hypothesized that senescent hepatocytes acquire a SASP that directly impedes liver recovery, mediated through the induction of a Klf6-p21 axis, which ultimately drives ALF. <b>Approach and Results:</b> Initial bioinformatic analysis of flow-sorted perinecrotic hepatocytes after a moderate overdose of APAP (300 mg/kg) revealed that these hepatocytes express both Klf6, p21 and Cxcl14 (a SASP constituent). Further, we fractionated hepatocytes and non-parenchymal cells and confirmed that hepatocytes are the primary source of Cxcl14. In both male and female mice, we found a dose-dependent induction of Klf6/p21 and persistent circulating levels of Cxcl14 in the plasma. In parallel experiments, we targeted either the senescent hepatocytes with the senolytic drugs, dasatinib and quercetin, or the SASP constituent Cxcl14 with a neutralizing antibody. We found that targeting Cxcl14 greatly reduced APAP-induced liver injury while targeting the senescent hepatocyte failed to influence liver injury. Experiments analyzing human APAP overdose livers confirmed that Klf6-p21-Cxcl14 axis is upregulated compared to healthy controls. To confirm the link between Klf6, p21 and Cxcl14, we utilized the CRISPRa HepG2 system where we transcriptionally activated Klf6 which strongly induced p21 and Cxcl14 expression. Klf6 is a redox-responsive protein and we have previously demonstrated that APAP-induced inhibition of complex III is an initiating event in the APAP toxicity cascade. Therefore, we conducted further experiments <i>in&nbsp;vitro</i> inhibiting complex III with Antimycin A which resulted in increased Klf6 and p21 expression. <b>Conclusions:</b> Collectively, our data suggests that sustained oxidative stress after a severe APAP overdose leads to continued elevation in Klf6 which promotes p21-mediated hepatocyte senescence and the production of Cxcl14. Importantly, we find that human plasma levels of Cxcl14 can accurately predict patient survival, as it gradually declines in surviving APAP overdose patients but remains consistently elevated in non-surviving APAP overdose patients. This data confirms that the sustained induction of hepatocyte senescence and SASP initiation are critical mechanistic events leading to ALF in human patients.