Abstract B004: Promoter methylation gains in aging and cancer are independent of replication

Abstract DNA methylation alterations are observed across multiple cancers, occurring early during tumor initiation. However, evolution and drivers of these changes remain poorly understood and difficult to assess at the clonal level. Consequently, we developed a method for profiling genome-wide DNA methylation from low DNA amounts and applied it to the organoid model of BRAFV600E oncogene-induced colon cancer (CC) tumorigenesis. Single organoid clones, derived from single stem cells, were profiled every month for 5 months to map nucleotide-level dynamic heterogeneity in methylation changes. Uninduced control organoids exhibit a pattern of epigenetic drift leading towards clonal convergence not until 5 months. Whereas BRAFV600E-induced organoids exhibit a step-wise divergence throughout the different time points, likely explained by progressive clonal takeovers. The methylation changes in BRAFV600E induced organoids are explained by the age-in-culture associated methylation changes; especially BRAFV600E did not induce epigenetic changes, as previously thought. The CpG sites affected in the control and BRAFV600E comparison were not shared but mapped to the same genomic elements, indicating common drivers of methylation changes regardless of oncogene. Investigation of promoter CpG-island methylation gains revealed that they are decoupled from replication. External validation in other pre-cancerous and CC datasets, and experimental cell line data sets, show that methylation gains at regulatory elements are a function of “biological aging” and independent of replication or cell division cycles. Our studies provide new insights into the early epigenetic changes involved in BRAFV600E driven carcinogenesis rooted in the age-dependent drivers of epigenetic alterations. Citation Format: Sara-Jayne Thursby, Zhicheng Jin, Nibedita Patel, Yong Tao, Yuba Bhandari, Daniel Petkovich, Shangzhi Lu, Lijing Yang, Tza-Huei Wang, Thomas R. Pisanic II, Stephen B Baylin, Hariharan Easwaran. Promoter methylation gains in aging and cancer are independent of replication [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Methylation, Clonal Hematopoiesis, and Cancer; 2025 Feb 1-4; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2025;85(3 Suppl):Abstract nr B004.