P407: mitotic fidelity is a resistance mechanism to decitabine in myeloid tumors.

Topic: 3. Acute myeloid leukemia - Biology & Translational Research Background: Decitabine (DAC) is an epigenetic drug clinically used for the treatment of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). However, its exact mechanism of action is unclear. The epigenetic mechanism of action does not fully explain the high rates of remission induction achieved in TP53-mutated myeloid malignancies or the high efficacy in high-risk MDS compared with low-risk MDS. Aims: The purpose of our study is to uncover the exact mechanism of action of DAC against myeloid tumors. Methods: To identify essential genes that regulate sensitivity of myeloid tumors to DAC, we performed a genome-wide CRISPR-dCas9 activation screen using murine MDS/AML cells expressing ASXL1 and SETBP1 mutants (cSAM cells). After transduction of mouse genome-wide CRISPRa-v2 library, they were cultured with DMSO or DAC for 1 week. We extracted genomic DNA at the start and end of our screen and quantified the relative representation of each sgRNA. For assessment of the mitotic process, we established the time-lapse high-content confocal imaging system optimized for non-adherent MDS-L-2007 cells. Results: Our CRISPR/dCas9-activation screen revealed that genes involved in chromosome segregation, including Cdk1, Cdc20, Cdca8 and Dsn1, were related to DAC resistance (Figure). We also found that DAC strongly induced aneuploidy and polyploidy (>4n) in myeloid tumors, especially in those with TP53 mutations and secondary AML cells. Furthermore, the time-lapse imaging revealed that DAC prolonged the duration of metaphase and increased multipolar mitosis and abscission failure in MDS-L-2007 cells. Thus, DAC perturbs mitosis and has cytotoxic effects at clinically achievable low concentrations in myeloid tumors. Next, we assessed the role of DNMT1 in the DAC-induced mitotic defects in myeloid tumors. The DAC-induced aneuploidy and apoptosis were markedly attenuated in DNMT1-depleted myeloid tumors. In contrast, overexpression of Dnmt1 increased mitotic abnormalities of myeloid tumors to DAC. These results indicate that DAC perturbs mitosis not through DNMT1 degradation and subsequent DNA demethylation, but through DNMT1 itself in myeloid tumors. In line with this, DAC induced rapid degradation of DNMT1 in the nucleoplasm fraction, whereas chromatin-bound DNMT1 protein was retained even with DAC. The Dnmt1-C1229A mutant, which loses the ability to induce DNMT1-DNA covalent bond formation, did not enhance the DAC-driven mitotic defects. Furthermore, the cell-free Xenopus system showed that the DNMT1 adducts on DNA physically inhibit chromatin binding of the cohesin complex to DNA. These data suggested that the historically overlooked role of DAC in disrupting the mitotic process by the DNMT1 adducts in myeloid malignancies. In addition to mitotic regulators, transcriptome and metabolome analyses combined with our screen revealed the involvement of cholesterol metabolism in DAC resistance. Inhibition of cholesterol synthesis using statins delayed mitotic progression, suggesting that cholesterol is also important for proper mitosis. Moreover, co-treatment with DAC and statin caused mitotic catastrophe and showed synergistic growth-inhibitory effects in leukemia cells. Summary/Conclusion: These findings challenge the current assumption that DAC inhibits leukemogenesis by inducing DNA hypomethylation. Rather, the anti-leukemia effect of DAC is likely mediated mainly by the non-epigenetic mechanism: mitotic perturbation through aberrant DNMT1-DNA covalent bonds. Our study also suggests that perturbation of faithful mitotic processes by inhibition of cholesterol synthesis provides a strategy to overcome DAC resistance in myeloid tumors. Fig.) CRSIPR dCas9-activation screen identified mitosis as important targets of DAC.Keywords: Myelodysplastic syndrome, decitabine, Acute myeloid leukemia, Azacitidine