Discovery and Targeting of the Signaling Controls of PNPLA3 to Effectively Reduce Transcription, Expression, and Function in Pre-Clinical NAFLD/NASH Settings

Abstract

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 (PNPLA3), 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 PNPLA3 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 PNPLA3 which, when co-inherited, reduces PNPLA3 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 PNPLA3 expression, across all genotypes. We found that momelotinib treatment yielded >80% reduction in PNPLA3 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 PNPLA3 mRNA as well as intracellular lipid content. Furthermore, we show that the effects on PNPLA3 coincide with changes in chromatin accessibility within regulatory regions of the PNPLA3 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 PNPLA3 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 PNPLA3. 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.

Genetics Is of the Essence to Face NAFLD

“…In keeping with these findings, tissue expression of PNPLA3 is significantly enhanced in biopsies of patients carrying the p.I148M polymorphism, whereas the rare rs2294918 PNPLA3 (p.E434K) variant attenuates the impact of the p.I148M on steatosis and circulating liver enzymes in NAFLD patients, whereby down-modulating PNPLA3 expression on the LDs (up to 50%) [57,60]. In addition, Schwartz and collaborators demonstrated that momelotinib, a drug used in clinical trials to treat myelofibrosis, may represent an effective modulator of PNPLA3 expression, yielding >80% reduction in PNPLA3 mRNA levels and hampering intracellular lipid content in human primary hepatocytes and stellate cells [60]. Another possible mechanism underlying the TG engulfment in hepatocytes carrying the p.I148M variant is linked to the impairment of lipophagy in hepatocytes, thus dampening autophagic fluxes and LD degradation [61].…”

“…In addition, in mice overexpressing Pnpla3 p.I148M the softening of Pnpla3 expression by shRNA or by or proteolysis-targeting chimera (PROTAC)-mediated degradation reduced TG storages [59]. In keeping with these findings, tissue expression of PNPLA3 is significantly enhanced in biopsies of patients carrying the p.I148M polymorphism, whereas the rare rs2294918 PNPLA3 (p.E434K) variant attenuates the impact of the p.I148M on steatosis and circulating liver enzymes in NAFLD patients, whereby down-modulating PNPLA3 expression on the LDs (up to 50%) [57,60]. In addition, Schwartz and collaborators demonstrated that momelotinib, a drug used in clinical trials to treat myelofibrosis, may represent an effective modulator of PNPLA3 expression, yielding >80% reduction in PNPLA3 mRNA levels and hampering intracellular lipid content in human primary hepatocytes and stellate cells [60].…”

Genetic predisposition similarities between NASH and ASH: Identification of new therapeutic targets

“…Injection of antisense oligonucleotides against hepatic PNPLA3 in mice fed steatogenic and steatohepatitisinducing diets reduced fat, inflammation and fibrosis, with a more pronounced benefit in animals bearing the I148M protein variant [25]. The beneficial effect of PNPLA3 downregulation on J o u r n a l P r e -p r o o f hepatocellular fat and HSC transactivation could also be achieved by small molecules, such as momelonib, active on the JAK 1/2 and TGF-β/SMAD pathways [77]. Even if this compound could possibly suffer from off target effects and could not result suitable for chronic administration for a non-neoplastic condition, the aforementioned study provides a proof-of-principle that small molecules may achieve the goal of suppressing PNPLA3.…”

Separating the apples from the oranges: from NAFLD heterogeneity to personalized medicine

“…Considering that the leading causes of mortality include cardiovascular events and cancer [18], NAFLD patients should be comprehensively classified based also on their prominent extrahepatic features rather than on their "intra-hepatic" manifestations alone [23][24][25]. Table 2 [26][27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42] summarizes some selected topics of future research. In conclusion, in 1997, the distinguished liver immunologist Albert J Czaja wrote that "NASH is currently a generic diagnosis that encompasses multiple diseases.…”