Inhibition of TOPORS E3 ubiquitin ligase augments the efficacy of DNA hypomethylating agents in myeloid malignancies via DNMT1 stabilization

DNA hypomethylating agents (HMAs) are commonly used to treat myeloid malignancies including myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML), but its therapeutic effect is not sufficient in most cases. Elucidating the molecular targets that enhance the efficacy of HMAs may lead to better treatment strategies. CRISPR/Cas9 knockout screening in the presence of HMAs revealed that knockout of TOPORS, encoding a ubiquitin/SUMO E3 ligase, augments the efficacy of HMAs on MDS/AML cells, whereas it has little effect on the growth in the absence of HMAs. TOPORS knockout in MDS/AML cells intensified apoptosis and mitotic defect upon HMA treatment. The synergistic effect was also confirmed in the xenograft models. Analyses of Topors-deficient mice indicated that Topors is dispensable for normal hematopoiesis but its loss sensitizes murine MLL-AF9 leukemic cells to HMAs. A second CRISPR/Cas9 screening using TOPORS knockout MDS/AML cells revealed that the UHRF1-DNMT1 axis is involved in increased sensitivity to HMAs in the absence of TOPORS. HMAs incorporated into DNA trap DNMT1 and form DNA-DNMT1 cross-links, which causes mitotic defect and apoptosis. Proteomics and biochemical analyses demonstrated that TOPORS loss causes inefficient ubiquitination of SUMOylated DNMT1 in DNA-DNMT1 cross-links, resulting in insufficient proteasomal degradation of trapped DNMT1 These results suggest that TOPORS functions as a SUMO-targeted ubiquitin ligase of SUMOylated DNMT1 in DNA-DNMT1 cross-links. Our findings provide a novel attractive therapeutic strategy for myeloid malignancies that interferes with resolution of DNA-DNMT1 cross-links by targeting DNMT1 post-translational modifications.