Reduced Maintenance DNA Methylation Thresholds Enable Sensitive Reporter Assays for UHRF1 and DNMT1 Inhibition

ABSTRACT Aberrant DNA methylation and consequent silencing of tumor suppressor gene (TSGs) are maintained by UHRF1‐mediated recruitment of DNMT1, making the UHRF1‐DNMT1 axis as an attractive target for hypomethylating agents (HMAs). However, fully reversing DNA methylation abnormalities requires lowering DNMT1 below a deep threshold, posing major drug discovery challenges. Here, we demonstrated that a similarly stringent threshold applied to UHRF1 in maintaining hypermethylated TSG suppression. Genetically reducing redundancy in DNMT1 or UHRF1 markedly lowered these thresholds, sensitizing cells to their inhibition. These findings were translated into engineered recombinant reporter systems tied to endogenous hypermethylated promoters in hypomorphic cells, substantially improving sensitivity and dynamic range for monitoring HMA‐induced TSG reactivation. Pilot high‐throughput screening validated assay robustness and specificity for DNMT1 and UHRF1 inhibitors. Additionally, these platforms enabled optimization of timing and combination strategies pairing DNMT1/UHRF1 inhibitors with other epigenetic agents to maximize TSG re‐expression. Our study supports the concept of maintenance methylation thresholds and provides versatile tools for discovering novel HMAs and designing epigenetic combination therapies.