DNMT1 is required for efficient DSB repair and maintenance of replication fork stability, and its loss reverses resistance to PARP inhibitors in cancer cells.

Cancer cells with breast cancer susceptibility gene (BRCA) mutations inevitably acquire resistance to PARP inhibitors (PARPi), and new strategies to maximize the efficacy of PARPi are urgently needed for the treatment of patients with BRCA1/2-mutant cancers. Here, we provide evidence that DNMT1 plays essential roles in DNA repair and the maintenance of replication fork stability by associating with the RPA complex and the SFPQ/NONO/FUS complex. DNMT1 depletion impairs RPA1 recruitment to stalled replication forks and inhibits DNA‒RNA hybrid (R-loop) resolution as well as the retention of RPA1 and SFPQ/NONO/FUS complexes at double-stranded DNA breaks (DSBs). Moreover, PARP1 activity is required for DNMT1 retention at DSB sites by modulating its protein stability, which is tightly and dynamically regulated by PARP1-mediated PARylation and PARG- and NUDT16-mediated dePARylation. DNMT1 PARylation further recruits the E3 ubiquitin ligase CHFR to enhance its ubiquitination and target it for proteasome-dependent degradation. Notably, DNMT1 is also required for irradiation (IR)-mediated and PARPi-induced activation of the G2 arrest checkpoint. The combination of DNMT1i with PARPi significantly attenuates PARPi-induced ATR-Chk1 signaling and enhances the degradation of the stalled replication fork mediated by PARPi, resulting in increased chromosomal aberrations and cell death in BRCA-proficient and BRCA-deficient cancer cells. Therefore, our findings provide novel insights into the mechanism by which DNMT1 inhibitors (DNMT1i) reverse PARPi resistance and indicate that targeting the PARP-DNMT1 pathway is a promising strategy for cancer therapy.