Comprehensive analysis of epigenetic and epitranscriptomic genes’ expression in human NAFLD

Non-alcoholic fatty liver disease (NAFLD) is a multifactorial condition with a complex etiology. Its incidence is increasing globally in parallel with the obesity epidemic, and it is now considered the most common liver disease in Western countries. The precise mechanisms underlying the development and progression of NAFLD are complex and still poorly understood. The dysregulation of epigenetic and epitranscriptomic mechanisms is increasingly recognized to play pathogenic roles in multiple conditions, including chronic liver diseases. Here, we have performed a comprehensive analysis of the expression of epigenetic and epitranscriptomic genes in a total of 903 liver tissue samples corresponding to patients with normal liver, obese patients, and patients with non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), advancing stages in NAFLD progression. We integrated ten transcriptomic datasets in an unbiased manner, enabling their robust analysis and comparison. We describe the complete landscape of epigenetic and epitranscriptomic genes’ expression along the course of the disease. We identify signatures of genes significantly dysregulated in association with disease progression, particularly with liver fibrosis development. Most of these epigenetic and epitranscriptomic effectors have not been previously described in human NAFLD, and their altered expression may have pathogenic implications. We also performed a comprehensive analysis of the expression of enzymes involved in the metabolism of the substrates and cofactors of epigenetic and epitranscriptomic effectors. This study provides novel information on NAFLD pathogenesis and may also guide the identification of drug targets to treat this condition and its progression towards hepatocellular carcinoma. (AU)

Comprehensive analysis of epigenetic and epitranscriptomic genes' expression in human NAFLD.

Non-alcoholic fatty liver disease (NAFLD) is a multifactorial condition with a complex etiology. Its incidence is increasing globally in parallel with the obesity epidemic, and it is now considered the most common liver disease in Western countries. The precise mechanisms underlying the development and progression of NAFLD are complex and still poorly understood. The dysregulation of epigenetic and epitranscriptomic mechanisms is increasingly recognized to play pathogenic roles in multiple conditions, including chronic liver diseases. Here, we have performed a comprehensive analysis of the expression of epigenetic and epitranscriptomic genes in a total of 903 liver tissue samples corresponding to patients with normal liver, obese patients, and patients with non-alcoholic fatty liver (NAFL) and non-alcoholic steatohepatitis (NASH), advancing stages in NAFLD progression. We integrated ten transcriptomic datasets in an unbiased manner, enabling their robust analysis and comparison. We describe the complete landscape of epigenetic and epitranscriptomic genes' expression along the course of the disease. We identify signatures of genes significantly dysregulated in association with disease progression, particularly with liver fibrosis development. Most of these epigenetic and epitranscriptomic effectors have not been previously described in human NAFLD, and their altered expression may have pathogenic implications. We also performed a comprehensive analysis of the expression of enzymes involved in the metabolism of the substrates and cofactors of epigenetic and epitranscriptomic effectors. This study provides novel information on NAFLD pathogenesis and may also guide the identification of drug targets to treat this condition and its progression towards hepatocellular carcinoma.

Identification and experimental validation of druggable epigenetic targets in hepatoblastoma.

BACKGROUND & AIMS: Hepatoblastoma (HB) is the most frequent childhood liver cancer. Patients with aggressive tumors have limited therapeutic options; therefore, a better understanding of HB pathogenesis is needed to improve treatment. HBs have a very low mutational burden; however, epigenetic alterations are increasingly recognized. We aimed to identify epigenetic regulators consistently dysregulated in HB and to evaluate the therapeutic efficacy of their targeting in clinically relevant models. METHODS: We performed a comprehensive transcriptomic analysis of 180 epigenetic genes. Data from fetal, pediatric, adult, peritumoral (n = 72) and tumoral (n = 91) tissues were integrated. Selected epigenetic drugs were tested in HB cells. The most relevant epigenetic target identified was validated in primary HB cells, HB organoids, a patient-derived xenograft model, and a genetic mouse model. Transcriptomic, proteomic and metabolomic mechanistic analyses were performed. RESULTS: Altered expression of genes regulating DNA methylation and histone modifications was consistently observed in association with molecular and clinical features of poor prognosis. The histone methyltransferase G9a was markedly upregulated in tumors with epigenetic and transcriptomic traits of increased malignancy. Pharmacological targeting of G9a significantly inhibited growth of HB cells, organoids and patient-derived xenografts. Development of HB induced by oncogenic forms of ß-catenin and YAP1 was ablated in mice with hepatocyte-specific deletion of G9a. We observed that HBs undergo significant transcriptional rewiring in genes involved in amino acid metabolism and ribosomal biogenesis. G9a inhibition counteracted these pro-tumorigenic adaptations. Mechanistically, G9a targeting potently repressed the expression of c-MYC and ATF4, master regulators of HB metabolic reprogramming. CONCLUSIONS: HBs display a profound dysregulation of the epigenetic machinery. Pharmacological targeting of key epigenetic effectors exposes metabolic vulnerabilities that can be leveraged to improve the treatment of these patients. IMPACT AND IMPLICATIONS: In spite of recent advances in the management of hepatoblastoma (HB), treatment resistance and drug toxicity are still major concerns. This systematic study reveals the remarkable dysregulation in the expression of epigenetic genes in HB tissues. Through pharmacological and genetic experimental approaches, we demonstrate that the histone-lysine-methyltransferase G9a is an excellent drug target in HB, which can also be harnessed to enhance the efficacy of chemotherapy. Furthermore, our study highlights the profound pro-tumorigenic metabolic rewiring of HB cells orchestrated by G9a in coordination with the c-MYC oncogene. From a broader perspective, our findings suggest that anti-G9a therapies may also be effective in other c-MYC-dependent tumors.

Dual Pharmacological Targeting of HDACs and PDE5 Inhibits Liver Disease Progression in a Mouse Model of Biliary Inflammation and Fibrosis.

Liver fibrosis, a common hallmark of chronic liver disease (CLD), is characterized by the accumulation of extracellular matrix secreted by activated hepatic fibroblasts and stellate cells (HSC). Fibrogenesis involves multiple cellular and molecular processes and is intimately linked to chronic hepatic inflammation. Importantly, it has been shown to promote the loss of liver function and liver carcinogenesis. No effective therapies for liver fibrosis are currently available. We examined the anti-fibrogenic potential of a new drug (CM414) that simultaneously inhibits histone deacetylases (HDACs), more precisely HDAC1, 2, and 3 (Class I) and HDAC6 (Class II) and stimulates the cyclic guanosine monophosphate (cGMP)-protein kinase G (PKG) pathway activity through phosphodiesterase 5 (PDE5) inhibition, two mechanisms independently involved in liver fibrosis. To this end, we treated Mdr2-KO mice, a clinically relevant model of liver inflammation and fibrosis, with our dual HDAC/PDE5 inhibitor CM414. We observed a decrease in the expression of fibrogenic markers and collagen deposition, together with a marked reduction in inflammation. No signs of hepatic or systemic toxicity were recorded. Mechanistic studies in cultured human HSC and cholangiocytes (LX2 and H69 cell lines, respectively) demonstrated that CM414 inhibited pro-fibrogenic and inflammatory responses, including those triggered by transforming growth factor ß (TGFß). Our study supports the notion that simultaneous targeting of pro-inflammatory and fibrogenic mechanisms controlled by HDACs and PDE5 with a single molecule, such as CM414, can be a new disease-modifying strategy.

Epigenetics in Liver Fibrosis: Could HDACs be a Therapeutic Target?

Chronic liver diseases (CLD) represent a worldwide health problem. While CLDs may have diverse etiologies, a common pathogenic denominator is the presence of liver fibrosis. Cirrhosis, the end-stage of CLD, is characterized by extensive fibrosis and is markedly associated with the development of hepatocellular carcinoma. The most important event in hepatic fibrogenesis is the activation of hepatic stellate cells (HSC) following liver injury. Activated HSCs acquire a myofibroblast-like phenotype becoming proliferative, fibrogenic, and contractile cells. While transient activation of HSCs is part of the physiological mechanisms of tissue repair, protracted activation of a wound healing reaction leads to organ fibrosis. The phenotypic changes of activated HSCs involve epigenetic mechanisms mediated by non-coding RNAs (ncRNA) as well as by changes in DNA methylation and histone modifications. During CLD these epigenetic mechanisms become deregulated, with alterations in the expression and activity of epigenetic modulators. Here we provide an overview of the epigenetic alterations involved in fibrogenic HSCs transdifferentiation with particular focus on histones acetylation changes. We also discuss recent studies supporting the promising therapeutic potential of histone deacetylase inhibitors in liver fibrosis.