Using synthetic biology to understand DNA methylation maintenance in cancer

Abstract

Loss of DNA methylation is common to the vast majority of human cancers and is considered a hallmark of cancer epigenomes. Current models propose that improper maintenance of DNA methylation could underpin this hypomethylation. However, these models are based on evidence derived from static snapshots of cancer methylomes. In addition, DNA methylation represents an important therapeutic target in cancer treatment. DNA hypomethylating agents (HMAs) are evidenced to promote antitumour immunity, however, existing HMAs have limited applications due to poor pharmacometrics and cytotoxicity. Thus, advancing our understanding of DNA hypomethylation in cancer, alongside identifying more effective drugs to target DNA methylation, is crucial for enhancing therapeutic strategies in cancer treatment. To gain insight into the dynamics of DNA methylation maintenance in colorectal cancer cells, I developed a methylation-sensitive reporter system. This piggyBac transposon-based system allows stable integration of a methylated eGFP transgene (eGFPme) at distinct locations across the genome. I show that methylation of this reporter construct represses its expression in colorectal cancer cells. Reporter integrations were identified across the whole genome, enabling tracking of genome-wide DNA methylation maintenance. To test whether HCT116 cells are able to faithfully propagate DNA methylation, cells containing premethylated reporters were maintained in culture for up to 8 weeks, equivalent to ~75 cell divisions. The majority of methylated reporters remained repressed and maintained their methylated status over this time course. These findings demonstrate that methylation patterns remain consistently preserved at synthetic reporters in HCT116 colorectal cancer cells. My results highlight the functionality of the DNA methylation maintenance machinery in colorectal cancer cells, despite the presence of extensive hypomethylation. Having demonstrated that DNA methylation is maintained in colorectal cancer cells, I next investigated the contribution of different components of the DNA methylation maintenance machinery to methylation maintenance. To do this, I assessed reporter activity in knock-out (KO) and degron cell lines. DNMT1 is the primary maintenance methyltransferase, responsible for maintaining DNA methylation following replication. As expected, reporter repression and methylation were not maintained in HCT116 cells lacking DNMT1 function. It has been previously reported that cooperation between DNMT1 and the de novo methyltransferase DNMT3B is required to maintain DNA methylation in mouse embryonic stem cells. However, this has not previously been tested in human cancer cells. To investigate the contribution of DNMT3B to DNA methylation maintenance, I assessed reporter activity in DNMT3BKO cells. No significant differences in reporter expression or methylation were detected in DNMT3BKO cells compared to HCT116. My results indicate that DNMT3B does not have a detectable contribution, in this experimental system, to methylation maintenance in human HCT116 cells. Next, I investigated the contribution of UHRF1 to DNA methylation maintenance. UHRF1 is an emerging therapeutic target of interest due to its essential role in DNA methylation maintenance, and its overexpression in some cancers. I integrated the eGFPme reporter into DNMT1 and UHRF1 degron cell lines. This degron system enables rapid depletion of the degron-tagged proteins, and I monitored reporter activity in response to protein depletion. Following DNMT1 or UHRF1 depletion, rapid upregulation of the reporter gene was detected. These results support that targeting UHRF1 is an efficient method to deplete DNA methylation colorectal cancer cells. Furthermore, these findings demonstrate that the reporter is activated in response to acute loss of DNA methylation. Finally, I sought to test whether the reporter system developed in this study could be utilised as a drug screening platform, to investigate compounds that modify DNA methylation. My previous results provided evidence for therapeutic targeting of DNMT1 and UHRF1. Therefore, I screened several novel inhibitors of DNMT1 and UHRF1. Removal of DNA methylation maintenance using the DNMT1 inhibitor GSK-3484862 resulted in a ~60% reduction of genome-wide methylation and rapid reporter activation. Importantly, I showed that GSK-3484862 treatment led to the activation of HERV-1 elements and IFN-1 signalling, which is thought to be important for the immunostimulatory applications of hypomethylating agents in cancer treatment. Together, my findings provide proof-of-concept for the use of this reporter system in large-scale compound library screen, to identify novel hypomethylating agents. The methylation-sensitive reporter system developed in this study was utilised to provide novel insights into the mechanisms underlying DNA methylation maintenance in cancer. This reporter system has future applications as a screening platform, to identify genetic factors involved in DNA methylation, and novel hypomethylating compounds with therapeutic potential