AbstractThe 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 DNA methyltransferase 1 (DNMT1)-specific inhibitor (GSK3685032/GSK5032) over several months to remove as much non-essential CpG methylation as possible. Profiling of the remaining DNA methylome revealed an unexpected, enriched retention of DNA methylation on the X-chromosome. Strikingly, the identified retained X-chromosome DNA methylation patterns accurately predicted de novo DNA hypermethylation in colon cancer patient methylomes in the TCGA COAD/READ cohort. 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.