Zhao, Xia

Background and Aims: Disruption of the epigenome is a hallmark of human disease, including liver cirrhosis and HCC. While genetic heterogeneity is an established effector of pathologic phenotypes, epigenetic heterogeneity is less well understood. Environmental exposures alter the liver-specific DNA methylation landscape and influence the onset of liver cancer. Given that currently available treatments are unable to target frequently mutated genes in HCC, there is an unmet need for novel therapeutics to prevent or reverse liver damage leading to hepatic tumorigenesis, which the epigenome may provide. Approach and Results: We performed genome-wide profiling of DNA methylation, copy number, and gene expression from multiple liver regions from 31 patients with liver disease to examine their crosstalk and define the individual and combinatorial contributions of these processes to liver disease progression. We identified epigenetic heterogeneity hotspots that are conserved across patients. Elevated epigenetic heterogeneity is associated with increased gene expression heterogeneity. Cirrhotic regions comprise 2 distinct cohorts—one exclusively epigenetic, and the other where epigenetic and copy number variations collaborate. Epigenetic heterogeneity hotspots are enriched for genes central to liver function (eg, HNF1A) and known tumor suppressors (eg, RASSF1A). These hotspots encompass genes including ACSL1, ACSL5, MAT1A, and ELFN1, which have phenotypic effects in functional screens, supporting their relevance to hepatocarcinogenesis. Moreover, epigenetic heterogeneity hotspots are linked to clinical measures of outcome. Conclusions: Substantial epigenetic heterogeneity arises early in liver disease development, targeting key pathways in the progression and initiation of both cirrhosis and HCC. Integration of epigenetic and transcriptional heterogeneity unveils putative epigenetic regulators of hepatocarcinogenesis.

Histone‐lysine N‐methyltransferase SETD2 (SETD2), the sole histone methyltransferase that catalyzes trimethylation of lysine 36 on histone H3 (H3K36me3), is often mutated in clear cell renal cell carcinoma (ccRCC). SETD2 mutation and/or loss of H3K36me3 is linked to metastasis and poor outcome in ccRCC patients. Epithelial‐to‐mesenchymal transition (EMT) is a major pathway that drives invasion and metastasis in various cancer types. Here, using novel kidney epithelial cell lines isogenic for SETD2, we discovered that SETD2 inactivation drives EMT and promotes migration, invasion, and stemness in a transforming growth factor‐beta‐independent manner. This newly identified EMT program is triggered in part through secreted factors, including cytokines and growth factors, and through transcriptional reprogramming. RNA‐seq and assay for transposase‐accessible chromatin sequencing uncovered key transcription factors upregulated upon SETD2 loss, including SOX2, POU2F2 (OCT2), and PRRX1, that could individually drive EMT and stemness phenotypes in SETD2 wild‐type (WT) cells. Public expression data from SETD2 WT/mutant ccRCC support the EMT transcriptional signatures derived from cell line models. In summary, our studies reveal that SETD2 is a key regulator of EMT phenotypes through cell‐intrinsic and cell‐extrinsic mechanisms that help explain the association between SETD2 loss and ccRCC metastasis.

Abstract Background and Aims Chronic HCV infection is a leading etiologic driver of cirrhosis and ultimately HCC. Of the approximately 71 million individuals chronically infected with HCV, 10%‐20% are expected to develop severe liver complications in their lifetime. Epigenetic mechanisms including DNA methylation and histone modifications become profoundly disrupted in disease processes including liver disease. Approach and Results To understand how HCV infection influences the epigenome and whether these events remain as “scars” following cure of chronic HCV infection, we mapped genome‐wide DNA methylation, four key regulatory histone modifications (H3K4me3, H3K4me1, H3K27ac, and H3K27me3), and open chromatin in parental and HCV‐infected immortalized hepatocytes and the Huh7.5 HCC cell line, along with DNA methylation and gene‐expression analyses following elimination of HCV in these models through treatment with interferon‐α (IFN‐α) or a direct‐acting antiviral (DAA). Our data demonstrate that HCV infection profoundly affects the epigenome (particularly enhancers); HCV shares epigenetic targets with interferon‐α targets; and an overwhelming majority of epigenetic changes induced by HCV remain as “scars” on the epigenome following viral cure. Similar findings are observed in primary human patient samples cured of chronic HCV infection. Supplementation of IFN‐α/DAA antiviral regimens with DNA methyltransferase inhibitor 5‐aza‐2’‐deoxycytidine synergizes in reverting aberrant DNA methylation induced by HCV. Finally, both HCV‐infected and cured cells displayed a blunted immune response, demonstrating a functional effect of epigenetic scarring. Conclusions Integration of epigenetic and transcriptional data elucidate key gene deregulation events driven by HCV infection and how this may underpin the long‐term elevated risk for HCC in patients cured of HCV due to epigenome scarring.

Alpha‐1 antitrypsin deficiency (AATD) liver disease is characterized by marked heterogeneity in presentation and progression, despite a common underlying gene mutation, strongly suggesting the involvement of other genetic and/or epigenetic modifiers. Variation in clinical phenotype has added to the challenge of detection, diagnosis, and testing of new therapies in patients with AATD. We examined the contribution of DNA methylation (5‐methylcytosine [5mC]) to AATD liver disease heterogeneity because 5mC responds to environmental and genetic cues and its deregulation is a major driver of liver disease. Using liver biopsies from adults with early‐stage AATD and the ZZ genotype, genome‐wide 5mC patterns were interrogated. We compared DNA methylation among patients with early AATD, and among patients with normal liver, cirrhosis, and hepatocellular carcinoma derived from multiple etiologic exposures, and linked patient clinical/demographic features. Global analysis revealed significant genomic hypomethylation in AATD liver–impacting genes related to liver cancer, cell cycle, and fibrosis, as well as key regulatory molecules influencing growth, migration, and immune function. Further analysis indicated that 5mC changes are localized, with hypermethylation occurring within a background of genome‐wide 5mC loss and with patients with AATD manifesting distinct epigenetic landscapes despite their mutational homogeneity. By integrating clinical data with 5mC landscapes, we observed that CpGs differentially methylated among patients with AATD disease are linked to hallmark clinical features of AATD (e.g., hepatocyte degeneration and polymer accumulation) and further reveal links to well‐known sex‐specific effects of liver disease progression. Conclusion: Our data reveal molecular epigenetic signatures within this mutationally homogeneous group that point to ways to stratify patients for liver disease risk.