Hlady, Ryan A.

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.

Abstract Clear cell renal carcinoma (ccRCC), which arises from the epithelial cells of the proximal tubule represents 70% of all renal cell carcinoma. Mutations that drive ccRCC in conjunction with near ubiquitous and early VHL loss include, PBRM1 (33 - 45%), BAP1 (15%) and SETD2 (13%). Loss of SETD2 leads to diminished occupancy of H3K36me3 in active gene bodies, which is progressively deregulated in primary RCC and metastasis, linking loss of H3K36me to tumor progression and metastasis. N6-Methyladenosine (m6A) is an important post-transcriptional regulator of RNA transcript stability and half-life. Preliminary findings have shown H3K36me3 (deposited by SETD2), targeting the core m6A methyltransferase machinery (METTL3, METTL14 and WTAP) to the mRNA by virtue of its interaction to METTL14 subunit. The goal of this study is to investigate if H3K36me3 depletion as observed in SETD2 mutant ccRCC, leads to a deregulated m6A methylome. We further investigate how deregulated methylome in key pathways leads to cancer initiation and progression in ccRCC. To accomplish our goals, we generated isogenic ccRCC cell lines with H3K36me3 depletion by knocking out SETD2. RNA-immunoprecipitation and sequencing for m6A (MeRIP) showed global deregulation of m6A methylome in SETD2KO isogenic ccRCC cell lines. We also show a number of pro-tumorigenic genes and pathways including cell invasion, inflammation and stemness being differentially methylated and expressed in SETD2KO ccRCC cell lines. Furthermore, we show SETD2KO cells being more sensitive to METTL3 inhibition suggesting the relevance of m6A hypermethylated transcripts in promoting pro-tumorigenic and growth promoting processes in SETD2KO ccRCC cell lines. Citation Format: Shafiq Shaikh, Ryan Wagner, Ryan Hlady, Keith D. Robertson. Perturbed RNA N6-methyladenosine methylome in SETD2 mutant clear cell renal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7074.

There is a paucity of human in vitro models available to study the effects of alcohol in hepatocellular carcinoma (HCC). HCC cell lines are often deficient in either alcohol dehydrogenase (ADH) or cytochrome P450 2E1 (CYP2E1). VL‐17a is currently the most widely studied model available. VL‐17a is derived from a HepG2 cell line engineered to overexpress mouse ADH and human CYP2E1. LH86 is a novel HCC cell line previously developed in our laboratory. Here we compare the LH86 cell line with the current model to identify a novel tool to study the effects of alcohol in HCC. The aim of this study is to evaluate the LH86 cell line as a novel human in vitro model to study the effects of alcohol in HCC. The cell lines were cultured with alcohol and gene expression and epigenomic changes were identified. The LH86 cell line was observed to have unique functional properties indicating that it is a potential novel tool to study the effects of alcohol in HCC. Support or Funding Information R01 AA19976 TO KR AND CL

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 Clear cell renal cell carcinoma (ccRCC) is the most common subtype of renal cell carcinoma (RCC) and accounts for majority of the deaths from renal cancer. SETD2, the sole histone methyltransferase that catalyzes trimethylation of lysine 36 on histone H3 (H3K36me3), is frequently mutated in ccRCC. Studies have shown that SETD2 mutations are often subclonal and the mutation rate dramatically increases from 25% in primary ccRCC to more than 60% in metastatic ccRCC, suggesting SETD2 loss plays an important role in renal cancer metastasis. However, mechanisms underpinning how SETD2 mutation drives invasion and metastasis in ccRCC is largely unknown. Here, by performing RNA-seq and ATAC-seq in a non-tumorigenic renal epithelial cell line isogenic for SETD2 loss-of-function, we found that SETD2 inactivation induces dramatic changes in the transcriptome and drastically enhances chromatin accessibility, which allows binding of a variety of transcription factors. We also discovered that SETD2 loss induces a TGFβ-independent EMT program, which is a major pathway linked to metastasis. Correlation of RNA-seq with ATAC-seq identifies SOX2, OCT2 and PRRX1 as key transcription factors promoting the SETD2 loss-driven EMT program. Furthermore, we found that this distinct EMT program is in part triggered by secreted factors, which suggests that SETD2 mutation promotes a metastasis-permissive tumor microenvironment through paracrine signaling. In summary, our findings reveal that SETD2 is a key regulator of EMT phenotypes that may explain the role of SETD2 mutations in ccRCC metastasis and further uncover potential therapeutic targets for SETD2-mutant ccRCC patients. Citation Format: Tianchu Wang, Ryan Wagner, Ryan Hlady, Sungho Kim, Xiaoyu Pan, Thai Ho, Keith Robertson. SETD2 loss in renal epithelial cells induces epithelial to mesenchymal transition in a TGFβ-independent manner [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1226.

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.

Hepatocellular carcinoma (HCC) is the second most common cause of cancer deaths worldwide. HCC is known to be a CpG Island methylator Phenotype (CIMP) cancer. Tumor development in CIMP cancers is characterized by epigenomic changes including changes in DNA methylation. We have previously shown that overlapping methylation abnormalities are conserved between primary tissue and cell culture models of HCC. Alcohol consumption is known to increase the risk of development of HCC and has also been shown to alter DNA methylation in a manner that contributes to liver disease. Here we study the effect of alcohol as a driver to tumor progression in HCC. We have developed in vitro models of HCC to identify the gene targets altered by ethanol metabolism during tumor progression over an extended period of time.

Hepatocellular Carcinoma (HCC) is a highly fatal liver cancer with no effective treatments. HCC tumors, formed after long‐term viral or alcohol related inflammation and cirrhosis, have a surprisingly low number of genetic mutations. Prior research has found that epigenetic patterns, such as DNA methylation at gene promoter sites, are common in HCC patients. Recent improvements in genetic sequencing and online genetic databases, have allowed us to identify a set of gene targets, including FMO3, for HCC detection or treatment. The hypothesis is that FMO3 is reduced by epigenetic dysregulation, leading to tumor formation. To find the connection between FMO3, epigenetic events and HCC tumor pathways, our lab created novel HCV‐stable and non‐infected HCC cell lines. We also used Huh7 and Huh7.5 cells to show the epigenetic changes in stressed cells and methylation inhibited cells. To mimic inflammation and stress leading to epigenetic changes, Huh7 cells were exposed to heat shock at 40°C for 40 min. Huh7.5 cells were treated with epigenetic inhibitor 5‐Aza‐2 deoxycytidine. RT‐qPCR and Western Blot show that FMO3 is significantly reduced in stressed cells and upregulated when treated with the methylation inhibitor. These results indicate that FMO3 may be used for early cancer risk detection and/or to improve treatment options by identifying the relationship between epigenetic regulation and tumorigenesis. Support or Funding Information NIH NIDDK 1R01DK110024‐01A1

Abstract Background: We previously demonstrated that putative clinical epidemiologic exposures associated with leukemia risk are prevalent among AML patients, and some are associated with unique cytogenetic risk group and with clinical phenotype (Finn, Cancer Epidemiol, 2015). Herein, we studied genome-wide DNA methylation in a cohort of AML patients to evaluate the association of individual hyper- and hypo-methylated CpG sites with epidemiologic exposures and overall survival. Methods: The Mayo Clinic AML Epidemiology Cohort is a highly annotated retrospective case series of 295 consecutive patients (pts) with AML diagnosed and treated at Mayo Clinic Florida and Arizona, with central cytogenetics performed in all cases. The prevalence of clinical epidemiologic exposures, past medical and family history as well as medication use and lifestyle was systematically obtained. After IRB approval, we interrogated the cytogenetic database and successfully obtained leukemia DNA from available remnant diagnostic cytogenetic cell pellets in a cohort of 148 AML patients in the Mayo epidemiology case series and performed an assessment of genome-wide DNA methylation using the Infinium HumanMethylation450K BeadChip. Samples were processed using the R Bioconductor package 'minfi' using Subset Within Array Quantile Normalization (PMID: 22703947). Individual CpGs that did not reach a detection p-value of <0.05 were filtered out. An internal control was included on each array to control for significant batch effects. To determine differential methylation status of 473,864 individual CpG sites (after exclusion of 11,648 that did not pass detection p-value QC), CpG sites with low interpatient variability, a standard deviation <0.05 of methylation values, and an interquartile range <0.05 of methylation values and X/Y chromosome probes were excluded from analysis. Epidemiologic and important clinical variables occurring in at least 10 pts or previously-identified as of unique interest but in fewer than 10 pts were evaluated, including sex, BMI, performance status and comorbidities, tobacco and alcohol use, family history of hematologic malignancy, medications of interest, history of toxin exposure, and history of immunosuppression and/or solid organ transplant or of secondary and therapy-related AML (t-AML). Associations of differential hypo or hypermethylation at 281,259 CpG sites with epidemiologic exposures/clinical variables were evaluated using Spearman's test of correlation (continuous or ordinal exposures), a Wilcoxon rank sum test (dichotomous exposures), or a Kruskal-Wallis rank sum test (multi-category exposures). A Bonferroni correction was applied for multiple testing, after which p <1.8 x 10-7 was considered as significant. To reduce the likelihood of false-negative findings, we additionally considered p <5 x 10-6 as indicating suggestive evidence of an association. CpG sites and associated gene and function was determined using Illumina manifest file & www.ncbi.nlm.nih.gov/gene. Results: Statistically significant associations (p <1.8 x 10-7) with individual epidemiologic and clinical exposures were identified for 109 unique CpG sites, corresponding to differential methylation in the genes listed in Table. Specifically, obesity (predominantly hypomethylation), specific cytogenetic lesions and risk group, and gender were highly significantly associated with unique CpG methylation status, but not smoking, toxin exposure, family history, comorbidity or performance status, secondary or t-AML, immunosuppression, medication use, of family history in the analysis. A further 353 unique CpG sites had defined suggestive associations (ongoing analysis). We also identified 8 additional CpG sites where differential methylation was associated with overall survival (p <5 x 10-6, Table). Conclusion: Obesity, cytogenetic lesions, and sex are associated with differential methylation of unique CpG sites at AML diagnosis, using stringent univariate statistical criteria. Significant CpG sites were identified in genes previously linked to AML biology and prognosis (DOCK6, HOXB3, MIR10A, FOXN3/CHES1, GPX1, MYST2/KAT7, PTPRD), but some represent novel findings in AML. These preliminary results suggest an association of some AML risk factors and clinical variables with unique gene methylation and will undergo validation in a prospective AML epidemiology dataset. Disclosures Cerhan: Jannsen: Other: Scientific Advisory Board; Nanostring: Research Funding; Celgene: Research Funding. Foran:Agios: Research Funding; Xencor, Inc.: Research Funding.