Abstract B027: Modeling age-related DNA methylation changes with intestinal organoids

Abstract Intestinal organoids, three-dimensional cultures from intestinal stem cells, are a potential model for studying aging. DNA methylation can serve as biological clock of aging, and we hypothesized that organoid DNA methylation could be a valuable tool for aging research. We compared DNA methylation in organoids to that seen in primary aging intestinal epithelium. Genome wide, we found very substantial DNA methylation changes during organoid establishment and culture, with 27% of total CpG sites undergoing loss of methylation (hypomethylation), and 11% gaining methylation (hypermethylation). The observed hypermethylation primarily affected CpG islands and corresponded to CpG sites susceptible to age-related methylation in-vivo (p < 0.001 compared to unchanged sites). Early-passage (P0 and P2) organoids derived from 4- and 24-month-old mice, preserved aging-specific methylation patterns, with a correlation coefficient of 0.48 (p < 0.001) between methylation differences in-vivo and in-vitro. Age-related hypermethylation continued to change with passage, and linear modeling indicated that organoids age at an average rate of 0.46 months per week in culture. By contrast, hypomethylation occurred predominantly in non-aging-associated genomic regions such as non-promoter, non-CpG island regions (including transposable elements), and correlated neither with differentiation nor aging in-vivo. Thus, methylation changes in organoids reflect at least two different biological processes – aging, as measured by CpG island hypermethylation, and an unexplained stochastic hypomethylation defect. Treatment with the DNA methylation inhibitor decitabine lowered the methylation age of organoids from 22 months to 10 months, showing the potential of organoids to model extrinsic effects on age-related DNA hypermethylation. Citation Format: Himani Vaidya, Gennaro Calendo, Anthomy Pompetti, Curt Balch, Kelsey Keith, Jozef Mazdo, Jaroslav Jelinek, Jean-Pierre Issa. Modeling age-related DNA methylation changes with intestinal organoids [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 B027.