Abstract B025: Role of clock and clock-controlled gene methylation in colorectal adenoma risk and regulation of global DNA methylation: Pilot study
Authors: Burch James B. , Wells Jonathan , Mercadel Joshua , Krupa Morgan , Kothandaraman Venkat , Hurley Thomas , Murphy Angela , Lloyd Stephen , Strayer Scott , Sathe Gauri , Hébert James 
Published: December 2022

Dysregulation of the clock and clock-controlled genes, which have epigenetic and tumor suppressor properties, may drive colorectal adenoma and tumor formation. Global DNA hypomethylation (measured by LINE-1) is an early event in colorectal carcinogenesis. The potential role of clock genes in regulating LINE-1 methylation has not been described. The goals of this exploratory pilot were to: identify DNA methylation sites (CpGs) in candidate clock and clock-controlled gene promotors to target in an adenoma case-control study among average risk screening colonoscopy patients; and evaluate possible relationships between the candidate gene methylation and global DNA methylation (LINE-1). Initially, in silico analyses were performed on candidate clock (PER1, PER2, PER3, RORA) and clock-controlled (IL-6, TNF-alpha, MTNR1A, MLH1) gene sequences. Methylation data in the Cancer Genome Atlas (TCGA) were queried with the MEXPRESS and Wanderer search engines to identify CpGs with differential methylation between colon adenocarcinoma and control tissue (p&lt;0.05). These CpGs were further evaluated for differential methylation in leukocyte DNA from 22 histologically confirmed adenoma cases relative to 22 controls (no polyp or normal biopsy), as well as 12 adenoma tissue DNA samples relative to 12 adjacent normal gastrointestinal (GI) tissue biopsies. All participants provided informed consent. Next generation sequencing (NGS) quantified percent methylation at target CpGs (Ion S5™ sequencer with Ion Torrent S5 server for sequence alignment). LINE-1 was quantified via pyrosequencing. Statistical comparisons of methylation at each CpG in leukocyte DNA (cases versus controls) were performed via Wilcoxon Rank-Sum tests. CpG methylation in adenomas versus normal GI tissue was assessed via Wilcoxon signed rank tests. A total of 826 CpGs were interrogated, and 155 differentially methylated sites were identified; RORA was the most prevalent gene represented (34% of CpGs identified), followed by MLH1 (19%) and TNF-alpha (12%). An additional 442 CpGs in proximity to the original 155 sites, but without in silico TCGA data, were also sequenced, and another 170 CpGs were identified. Frequently represented genes included RORA (35%), MTNR1A (18%), TNF-alpha (11%) and PER2 (11%). LINE-1 hypomethylation was observed in colorectal adenomas (66±5%) relative to normal GI tissue (71±2%, n=23, p&lt;0.001); no differences were noted in leukocyte DNA. Serial linear regression with a gene-level false discovery rate was used to identify target CpGs that predict LINE-1 methylation. Seven target CpGs in adenoma (IL-6, PER2), and 2 CpGs in leukocyte DNA (RORA), were associated with LINE-1. This pilot study yielded a set of evidence-based target CpGs (N=325) in candidate clock and clock-controlled genes with differential methylation in colorectal adenoma or cancer patients, and 9 of those were related to LINE-1 methylation. Understanding the role of clock and clock-controlled genes in colorectal adenoma formation may lead to novel strategies for colorectal cancer prevention or treatment.

Citation Format: James B. Burch, Jonathan Wells, Joshua Mercadel, Morgan Krupa, Venkat Kothandaraman, Thomas Hurley, Angela Murphy, Stephen Lloyd, Scott Strayer, Gauri Sathe, James Hébert. Role of clock and clock-controlled gene methylation in colorectal adenoma risk and regulation of global DNA methylation: Pilot study [abstract]. In: Proceedings of the AACR Special Conference on Colorectal Cancer; 2022 Oct 1-4; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;82(23 Suppl_1):Abstract nr B025.