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

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) i...

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Autores principales: Schwartz, Brian E., Rajagopal, Vaishnavi, Smith, Cynthia, Cohick, Evan, Whissell, Gavin, Gamboa, Mario, Pai, Rutuja, Sigova, Alla, Grossman, Iris, Bumcrot, David, Sasidharan, Kavitha, Romeo, Stefano, Sehgal, Alfica, Pingitore, Piero
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600576/
https://www.ncbi.nlm.nih.gov/pubmed/33036387
http://dx.doi.org/10.3390/cells9102247
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author Schwartz, Brian E.
Rajagopal, Vaishnavi
Smith, Cynthia
Cohick, Evan
Whissell, Gavin
Gamboa, Mario
Pai, Rutuja
Sigova, Alla
Grossman, Iris
Bumcrot, David
Sasidharan, Kavitha
Romeo, Stefano
Sehgal, Alfica
Pingitore, Piero
author_facet Schwartz, Brian E.
Rajagopal, Vaishnavi
Smith, Cynthia
Cohick, Evan
Whissell, Gavin
Gamboa, Mario
Pai, Rutuja
Sigova, Alla
Grossman, Iris
Bumcrot, David
Sasidharan, Kavitha
Romeo, Stefano
Sehgal, Alfica
Pingitore, Piero
author_sort Schwartz, Brian E.
collection PubMed
description 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.
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spelling pubmed-76005762020-11-01 Discovery and Targeting of the Signaling Controls of PNPLA3 to Effectively Reduce Transcription, Expression, and Function in Pre-Clinical NAFLD/NASH Settings Schwartz, Brian E. Rajagopal, Vaishnavi Smith, Cynthia Cohick, Evan Whissell, Gavin Gamboa, Mario Pai, Rutuja Sigova, Alla Grossman, Iris Bumcrot, David Sasidharan, Kavitha Romeo, Stefano Sehgal, Alfica Pingitore, Piero Cells Article 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. MDPI 2020-10-07 /pmc/articles/PMC7600576/ /pubmed/33036387 http://dx.doi.org/10.3390/cells9102247 Text en © 2020 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Schwartz, Brian E.
Rajagopal, Vaishnavi
Smith, Cynthia
Cohick, Evan
Whissell, Gavin
Gamboa, Mario
Pai, Rutuja
Sigova, Alla
Grossman, Iris
Bumcrot, David
Sasidharan, Kavitha
Romeo, Stefano
Sehgal, Alfica
Pingitore, Piero
Discovery and Targeting of the Signaling Controls of PNPLA3 to Effectively Reduce Transcription, Expression, and Function in Pre-Clinical NAFLD/NASH Settings
title Discovery and Targeting of the Signaling Controls of PNPLA3 to Effectively Reduce Transcription, Expression, and Function in Pre-Clinical NAFLD/NASH Settings
title_full Discovery and Targeting of the Signaling Controls of PNPLA3 to Effectively Reduce Transcription, Expression, and Function in Pre-Clinical NAFLD/NASH Settings
title_fullStr Discovery and Targeting of the Signaling Controls of PNPLA3 to Effectively Reduce Transcription, Expression, and Function in Pre-Clinical NAFLD/NASH Settings
title_full_unstemmed Discovery and Targeting of the Signaling Controls of PNPLA3 to Effectively Reduce Transcription, Expression, and Function in Pre-Clinical NAFLD/NASH Settings
title_short Discovery and Targeting of the Signaling Controls of PNPLA3 to Effectively Reduce Transcription, Expression, and Function in Pre-Clinical NAFLD/NASH Settings
title_sort discovery and targeting of the signaling controls of pnpla3 to effectively reduce transcription, expression, and function in pre-clinical nafld/nash settings
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7600576/
https://www.ncbi.nlm.nih.gov/pubmed/33036387
http://dx.doi.org/10.3390/cells9102247
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