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Original research
Epigenetic mechanisms and metabolic reprogramming in fibrogenesis: dual targeting of G9a and DNMT1 for the inhibition of liver fibrosis
  1. Marina Barcena-Varela1,
  2. Hannah Paish2,
  3. Laura Alvarez1,
  4. Iker Uriarte1,3,
  5. Maria U Latasa1,
  6. Eva Santamaria1,3,
  7. Miriam Recalde1,
  8. Maria Garate1,
  9. Alex Claveria1,
  10. Leticia Colyn1,
  11. Maria Arechederra1,
  12. Maria J Iraburu4,
  13. Malgorzata Milkiewicz5,
  14. Piotr Milkiewicz6,
  15. Bruno Sangro3,7,
  16. Stuart M Robinson8,
  17. Jeremy French8,
  18. Ana Pardo-Saganta9,
  19. Julen Oyarzabal10,
  20. Felipe Prosper11,
  21. Krista Rombouts12,
  22. Fiona Oakley2,
  23. Jelena Mann2,
  24. Carmen Berasain1,3,
  25. Matias A Avila1,3,
  26. Maite G Fernandez-Barrena13,14
  1. 1Hepatology Program, CIMA, University of Navarra, IdiSNA, Pamplona, Spain
  2. 2Newcastle Fibrosis Research Group, Institute of Cellular Medicine, Newcastle University Faculty of Medical Sciences, Newcastle upon Tyne, UK
  3. 3Clinica Universidad de Navarra, CIBERehd, Pamplona, Spain
  4. 4Department of Biochemistry and Genetics, University of Navarra, Pamplona, Navarra, Spain
  5. 5Department of Medical Biology, Pomeranian Medical University, Szczecin, Poland
  6. 6Department of General, Transplant and Liver Surgery, Warsaw Medical University, Szczecin, Poland
  7. 7Liver Unit. Department of Internal Medicine, Clinica Universidad de Navarra, IdisNA, Pamplona, Spain
  8. 8North East's Hepato-Pancreato-Biliary (HPB) Centre, Newcatle upon Tyne Hospitals NHS Foundation Trust, Newcastle, UK
  9. 9Cell Therapy Program, Cima, University of Navarra, Pamplona, Spain
  10. 10Molecular Therapies Program, Cima, University of Navarra, Pamplona, Spain
  11. 11Oncohematology and Cell Therapy Programs, CIMA, University of Navarra, IdiSNA, Pamplona, Spain
  12. 12Institute for Liver and Digestive Health, Royal Free, University College London, UCL, London, UK
  13. 13CIBEREHD, Madrid, Spain
  14. 14Hepatology Program, Centro de Investigacion Medica Aplicada, Pamplona, Navarra, Spain
  1. Correspondence to Professor Matias A Avila, Hepatology, CIMA-University of Navarra, Pamplona, Spain; maavila{at}unav.es; Dr Maite G Fernandez-Barrena, Hepatology, CIMA-University of Navarra, Pamplona, Spain; magarfer{at}unav.es

Abstract

Objective Hepatic stellate cells (HSC) transdifferentiation into myofibroblasts is central to fibrogenesis. Epigenetic mechanisms, including histone and DNA methylation, play a key role in this process. Concerted action between histone and DNA-mehyltransferases like G9a and DNMT1 is a common theme in gene expression regulation. We aimed to study the efficacy of CM272, a first-in-class dual and reversible G9a/DNMT1 inhibitor, in halting fibrogenesis.

Design G9a and DNMT1 were analysed in cirrhotic human livers, mouse models of liver fibrosis and cultured mouse HSC. G9a and DNMT1 expression was knocked down or inhibited with CM272 in human HSC (hHSC), and transcriptomic responses to transforming growth factor-β1 (TGFβ1) were examined. Glycolytic metabolism and mitochondrial function were analysed with Seahorse-XF technology. Gene expression regulation was analysed by chromatin immunoprecipitation and methylation-specific PCR. Antifibrogenic activity and safety of CM272 were studied in mouse chronic CCl4 administration and bile duct ligation (BDL), and in human precision-cut liver slices (PCLSs) in a new bioreactor technology.

Results G9a and DNMT1 were detected in stromal cells in areas of active fibrosis in human and mouse livers. G9a and DNMT1 expression was induced during mouse HSC activation, and TGFβ1 triggered their chromatin recruitment in hHSC. G9a/DNMT1 knockdown and CM272 inhibited TGFβ1 fibrogenic responses in hHSC. TGFβ1-mediated profibrogenic metabolic reprogramming was abrogated by CM272, which restored gluconeogenic gene expression and mitochondrial function through on-target epigenetic effects. CM272 inhibited fibrogenesis in mice and PCLSs without toxicity.

Conclusions Dual G9a/DNMT1 inhibition by compounds like CM272 may be a novel therapeutic strategy for treating liver fibrosis.

  • fibrogenesis
  • glucose metabolism
  • gene regulation
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Footnotes

  • CB, MAA and MGF-B are co-senior authors.

  • Twitter @Maite G Fernandez-Barrena

  • Contributors Performed experiments and data interpretation: MB-V, HP, LA, IU, MUL, ES, MR, MG, AC, LC, MA, MJI, MM, AP-S. Provided key biological samples and materials: PM, BS, SMR, JF, JO, FP, KR, FO, JM. Critical revision of the manuscript: JO, FP, KR, FO, JM, MJI, CB. Study design, data interpretation, manuscript writing and submission: CB, MGF-B, MAA.

  • Funding We thank the financial support of CIBERehd; grant PI16/01126 from Instituto de Salud Carlos III (ISCIII) co-financed by “Fondo Europeo de Desarrollo Regional” (FEDER) “Una manera de hacer Europa”; grant 58/17 from Gobierno de Navarra; grants SAF2014-54191-R, SAF2017-88933-R and SAF2019-104878RB-100 from FEDER/Ministerio de Ciencia, Innovación y Universidades-Agencia Estatal de Investigación; grant BIO15/CA/011 from Bio-Eusko Fundazioa (Eitb maratoia); grant from Asociación Española Contra el Cáncer (AECC) Scientific Foundation Rare Cancers grant 2017; HEPACARE Project from Fundación La Caixa; Fundación Eugenio Rodríguez Pascual; Fundación Echébano; Fundación Mario Losantos and Fundación M Torres. We thank Mr Eduardo Ávila and Mr Sergio Durá for their generous contribution. FPI fellowships from Ministerio de Educación, Cultura y Deporte to MB-V, MG and MR; FIMA-CIMA fellowship to AC; Gobierno de Navarra fellowship to LC; AECC post-doctoral fellowship to MA and Ramón y Cajal Program contract to MGF-B.This work was also funded by a UK Medical Research Council programme grants to JM, FO and others (MR/K10019494/1, MK/K001949/1, MR/R023026/1); National Institute on Alcohol Abuse and Alcoholism (NIAAA) (grant UO1AA018663). The research was further supported by the National Institute for Health Research Newcastle Biomedical Research Centre based at Newcastle Hospitals NHS Foundation Trust and Newcastle University.

  • Competing interests None declared.

  • Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.

  • Patient consent for publication Not required.

  • Ethics approval This study was approved by the Newcastle & North Tyneside Research Ethics Committee. Human samples were processed following standard operating procedures approved by the Ethical and Scientific Committees of the University of Navarra and the Medical University of Warsaw. Animal care and procedures were approved by the Animal Care Committee of the University of Navarra or the Newcastle Animal Welfare and Ethical Review Board and performed under a UK Home Office license.

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Data availability statement Data are available in a public, open access repository. GEO repository (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE139504).

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