| Summary: | Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are emerging worldwide epidemics, projected to become the leading cause of liver transplants. The strongest genetic risk factor for NAFLD/NASH susceptibility and progression is a single-nucleotide polymorphism (SNP) in the patatin-like phospholipase domain-containing 3 gene (<i>PNPLA3</i>), rs738409, encoding the missense mutation I148M. This aminoacidic substitution interferes with the normal remodeling of lipid droplets in hepatocytes. It is also thought to play a key role in promoting liver fibrosis by inhibiting the release of retinol from hepatic stellate cells. Reducing <i>PNPLA3</i> levels in individuals homozygous for 148M may be an effective treatment for the entire spectrum of NAFLD, based on gene dosage analysis in the human population, as well as the protective effect of another naturally occurring SNP (rs2294918) in <i>PNPLA3</i> which, when co-inherited, reduces <i>PNPLA3</i> mRNA levels to 50% and counteracts disease risk. By screening a clinical compound library targeting specific signaling pathways active in primary human hepatocytes, we identified momelotinib, a drug evaluated in clinical trials to treat myelofibrosis, as a potent down-regulator of <i>PNPLA3</i> expression, across all genotypes. We found that momelotinib treatment yielded >80% reduction in <i>PNPLA3</i> mRNA in human primary hepatocytes and stellate cells, as well as in vivo via acute and chronic treatment of WT mice. Using a human multilineage 3D spheroid model of NASH homozygous for the PNPLA3 mutant protein, we additionally show that it decreases <i>PNPLA3</i> mRNA as well as intracellular lipid content. Furthermore, we show that the effects on <i>PNPLA3</i> coincide with changes in chromatin accessibility within regulatory regions of the <i>PNPLA3</i> locus, consistent with inhibition occurring at the level of transcription. In addition to its primary reported targets, the JAK kinases, momelotinib inhibits several non-JAK kinases, including ACVR1. Using a combination of targeted siRNA knockdowns and signaling pathway perturbations, we show that momelotinib reduces the expression of the <i>PNPLA3</i> gene largely through the inhibition of BMP signaling rather than the JAK/STAT pathway. Overall, our work identified momelotinib as a potential NASH therapeutic and uncovered previously unrecognized connections between signaling pathways and <i>PNPLA3.</i> These pathways may be exploited by drug modalities to “tune down” the level of gene expression, and therefore offer a potential therapeutic benefit to a high at-risk subset of NAFLD/NASH patients.
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