Chromosome-specific retention of cancer-associated DNA hypermethylation following pharmacological inhibition of DNMT1

The DNA methylation status of the X-chromosome in cancer cells is often overlooked because of computational difficulties. Most of the CpG islands on the X-chromosome are mono-allelically methylated in normal female cells and only present as a single copy in male cells. We treated two colorectal cancer cell lines from a male (HCT116) and a female (RKO) with increasing doses of a novel DNA methyltransferase 1 (DNMT1)-specific inhibitor (GSK3685032/GSK5032) over a long time period to remove as much non-essential CpG methylation as possible. Analysis of the residual methylation patterns in the heavily treated cells showed a very surprising non-uniform distribution of retained methylation on specific chromosomes. For example, probes on the X-chromosome preferentially retained methylation 2-3-fold more frequently compared to those on autosomes. This was true for probes methylated on the active X chromosome in normal cells such as XIST and also for genes which acquired methylation in cancer such as ARMCX2 and MAGEH1. The maintenance of high DNA methylation on a subset of probes after such exhaustive treatment suggests that it might have functional significance for cell survival. Importantly, the same X-linked probes we found in the treated cells were also methylated in a large number of colorectal/rectal cancers in the TCGA data set and to a lesser extent in other cancer types. These results suggest that a re-examination of tumors for X-linked DNA methylation changes may enable greater understanding of the importance of epigenetic silencing of cancer related genes. Genomic DNA was isolated from parental HCT116, HCT116 DKO1, or RKO cells and HCT116 and RKO cells that had been treated for >100 days with >IC90 of the DNMT1-specific inhibitor GSK5032.