Abstract DNA methylation alterations are observed across multiple cancers, occurring early during tumor initiation. However, the evolution of these changes remains poorly understood and difficult to assess at the clonal level. We addressed the clonal evolution of DNA methylation using our previously established mouse organoid model of BRAFV600E oncogene-induced colon cancer (CC) tumorigenesis. We developed a method for profiling genome-wide DNA methylation from low DNA amounts and applied it to that organoid model. 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 but not until 5 months. Whereas BRAFV600E-induced organoids exhibit a step-wise divergence throughout the different time points, likely explained by progressive clonal takeovers. Although there are differences in the clonal dynamics of epigenetic states in the control and BRAFV600E-induced organoids, there is a common basis for the DNA methylation changes in both the lineages explained by the age-in-culture associated methylation changes. This is revealed by the observation that unlike currently proposed models, BRAFV600E did not induce or increase the degree of DNA methylation gains. Furthermore, 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. Additionally, investigation of promoter CpG-island methylation gains revealed that they are decoupled from replication. External validation in other pre-cancerous/CC datasets and experimental cell line datasets, show that methylation gains at regulatory elements are a function of “biological aging” and independent of replication or cell division cycles. The sites that acquire DNA methylation changes in BRAFV600E -induced organoids, compared to control organoids, are enriched for transcription factor motifs important in colorectal cancers, such as Cdx1, Cdx2, and Pparg. In contrast, control organoids show enrichment for transcription factors involved in stress and viral pathways. Our studies provide new insights into the early epigenetic changes involved in BRAFV600Edriven carcinogenesis, which is mainly rooted in the age-dependent drivers of epigenetic alterations, and are potentially subject to clonal selection in the context of uncontrolled growth due to oncogenic signaling. Citation Format: Sara-Jayne Thursby, Zhicheng Jin, Nibedita Patel, Yong Tao, Yuba Bhandari, Daniel Petkovich, Shangzhi Lu, Lijing Yang, Tza-Huei Wang, Thomas Russell Pisanic, Stephen B. Baylin, Hariharan Easwaran, Jacob Blum. Promoter methylation gains in aging and cancer are independent of replication abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1358.
Thursby et al. (Fri,) studied this question.