Mitotic perturbation is a key mechanism of action of decitabine in myeloid tumor treatment

Decitabine (DAC) is clinically used to treat myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML). Our genome-wide CRISPR-dCas9 activation screen using MDS-derived AML cells indicates that mitotic regulation is critical for DAC resistance. DAC strongly induces abnormal mitosis (abscission failure or tripolar mitosis) in human myeloid tumors at clinical concentrations, especially in those with TP53 mutations or antecedent hematological disorders. This DAC-induced mitotic disruption and apoptosis are significantly attenuated in DNMT1-depleted cells. In contrast, overexpression of Dnmt1, but not the catalytically inactive mutant, enhances DAC-induced mitotic defects in myeloid tumors. We also demonstrate that DAC-induced mitotic disruption is enhanced by pharmacological inhibition of the ATR-CLSPN-CHK1 pathway. These data challenge the current assumption that DAC inhibits leukemogenesis through DNMT1 inhibition and subsequent DNA hypomethylation and highlight the potent activity of DAC to disrupt mitosis through aberrant DNMT1-DNA covalent bonds. [Display omitted] •Genomic screen identifies mitotic regulation as a key decitabine resistance pathway•Clinical doses of decitabine induce extensive mitotic disruption in myeloid tumors•Decitabine-induced covalent binding of DNMT1 to DNA causes mitotic disruption•Inhibition of ATR-CHK1 synergizes with decitabine by impairing mitotic fidelity Yabushita et al. perform a genome-wide CRISPR activation screen to identify core regulators of resistance to decitabine in myeloid tumors. This screen reveals the potent activity of clinical-dose decitabine to disrupt mitosis via DNMT1-DNA adducts, challenging the prevailing notion that DNA demethylation is the primary mechanism of action of decitabine.