Abstract LB-150: Genetic depletion of DNMT1 reveals a DNMT1 threshold controlling DNA methylation in human cells

Abstract

Abstract Altered DNA methylation patterns are a central feature of most types of human cancer cells. However, the exact roles of DNA methyltransferases (DNMTs) in human DNA methylation maintenance are not well characterized. The long-established model that DNA methyltransferase DNMT1 maintains most DNA methylation in human cells has been challenged by multiple DNMT1 knockdown/knockout studies in the past decade. A potential role for de novo DNA methyltransferases DNMT3A and DNMT3B in maintaining DNA methylation in cancer cells has been suggested but is also not precisely known. To investigate the above issues, we have utilized both genetic recombination and CRISPR technology to deplete DNMTs individually or in combination in human colorectal cancer cell line HCT116. Previously we created a strong HCT116 DNMT1 hypomorph (1KO) by genetic recombination. Although 1KO cells lost about 85% of DNMT1 protein, they still maintained the epigenetic silencing of tumor suppressor genes and showed only 20% decrease in overall DNA methylation. We now show that further depletion of DNMT1 in 1KO cells, to less than 1% of wild type HCT116 cells, resulted in elimination of most DNA methylation and reactivation of key tumor suppressor genes including p16. In addition, we found that DNA methylation in HCT116 cells is controlled by different DNMT1 thresholds. Surprisingly, when we disrupted DNMT3A or DNMT3B with CRISPR in HCT116 cells, we see altered DNA methylation patterns involving both gains and losses of DNA methylation. Since abnormal DNA hypermethylation in cancer often associates with epigenetic gene silencing, this observation is especially relevant for the function of DNMT3A mutations in acute myeloid leukemia. Finally, we demonstrated that depletion of DNMT1-targeting protein UHRF1 efficiently reduced DNA methylation in human colon cancer cells. Our results not only defined the distinct roles of individual DNMTs in human cancer cell DNA methylation maintenance but also suggested UHRF1 inhibition as an alternative approach to reverse abnormal DNA hypermethylation in human cancer cells. Citation Format: Yi Cai, Hsing-chen Tsai, Ray-whay Yen, Limin Xia, Yang Zhang, Stephen Baylin. Genetic depletion of DNMT1 reveals a DNMT1 threshold controlling DNA methylation in human cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-150. doi:10.1158/1538-7445.AM2015-LB-150