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3952 Mitotic Perturbation Is a Key Mechanism of Action of Decitabine in Myeloid Tumor Treatment

Program: Oral and Poster Abstracts
Session: 604. Molecular Pharmacology and Drug Resistance: Myeloid Neoplasms: Poster III
Hematology Disease Topics & Pathways:
Research, Fundamental Science, Acute Myeloid Malignancies, AML, Non-Biological therapies, Chemotherapy, Diseases, Therapies, Myeloid Malignancies, Biological Processes, molecular biology
Monday, December 12, 2022, 6:00 PM-8:00 PM

Tomohiro Yabushita, MD PhD1,2*, Takumi Chinen, PhD3*, Atsuya Nishiyama, PhD4*, Shuhei Asada, MD PhD1*, Keita Yamamoto, MD PhD2*, Naru Sato, MD PhD1*, Yutaka Enomoto, PhD1*, Yosuke Tanaka, PhD1*, Keiko Katoh, PhD5*, Kaori Saitoh, PhD5*, Takamasa Ishikawa, PhD5*, Hitoshi Sato, PhD6*, Tomoyoshi Soga, PhD5*, Yasuhito Nannya, M.D., Ph.D.7, Makoto Nakanishi, MD PhD4*, Daiju Kitagawa, PhD3*, Toshio Kitamura, MD PhD1 and Susumu Goyama, MD PhD2*

1Division of Cellular Therapy, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan
2Division of Molecular Oncology, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan
3Department of Physiological Chemistry, Graduate School of Pharmaceutical Science, The University of Tokyo, Tokyo, Japan
4Division of Cancer Cell Biology, Institute of Medical Science, The University of Tokyo, Tokyo, Japan
5Institute for Advanced Biosciences, Keio University, Yamagata, Japan
6Division of Medical Genome Sciences, Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Tokyo, Japan
7Department of Hematology/Oncology, IMSUT Hospital, The University of Tokyo, Tokyo, Japan

Background: Decitabine (DAC) is one of the first generation of epigenetic drugs used clinically to treat myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Historically, DAC was considered as a cytotoxic agent when it was first developed in the 1960s. Subsequent studies in 1980s showed that DAC can induce DNMT1 degradation and DNA hypomethylation at non-cytotoxic concentration. Since then, DNA demethylation has been considered as the main mechanism of action of DAC. The DAC-induced DNA hypomethylation was shown to induce upregulation of tumor suppressor genes or endogenous retroviruses, which could contribute to the antitumor effect of DAC. Recently, DAC was also shown to be effective even in MDS and AML with TP53 mutations. However, it remains unclear why myeloid tumors, especially those with TP53 mutations, are particularly susceptible to DAC treatment.

Methods: Cancer Dependency Map (DepMap) database was used to evaluate in vitro sensitivity of human cancer cell lines to DAC. 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, cSAM cells were cultured with DMSO or DAC for 1 week. We extracted genomic DNA from the cells at the start and end of our screen and quantified the relative representation of each sgRNA. To assess the effect of DAC on mitosis, we established the time-lapse system optimized for non-adherent MDS-L-2007 cells (a human secondary AML (sAML) cell line derived from an MDS cell line, MDS-92).

Results: We first explored DepMap database and found that myeloid tumors are susceptible to DAC at clinically achievable concentration, while most solid tumor cell lines are not. The CRISPR/dCas9-activation screen revealed that genes involved in chromosome segregation, including Cdk1, Cdc20, Cdca8 and Dsn1, were significantly enriched in DAC-resistant cells, indicating that overexpression of mitotic regulators conferred resistance to DAC in cSAM cells. We also found that DAC strongly induced aneuploidy and polyploidy (>4n) in myeloid tumors, especially in those with TP53 mutations and sAML cells. Furthermore, the time-lapse imaging revealed that DAC treatment prolonged the duration of metaphase and increased multipolar mitosis and abscission failure in MDS-L-2007 cells (Figure). Thus, DAC perturbs mitosis and has cytotoxic effects at low concentrations in myeloid tumors.

Next, we assessed the role of DNMT1 in the DAC-induced mitotic defects in myeloid tumors. To our surprise, the DAC-induced aneuploidy and apoptosis were markedly attenuated in DNMT1-depleted myeloid tumors. In contrast, overexpression of Dnmt1 increased sensitivity 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, chromatin-bound DNMT1 protein was retained even with DAC, whereas DAC induced rapid degradation of DNMT1 in the nucleoplasm fraction. Importantly, the Dnmt1-C1229A mutant, which loses the ability to induce DNMT1-DNA covalent bond formation, did not enhance the DAC-driven mitotic defects. In addition, DAC treatment increased frequency of cohesion loss between sister chromatids in MDS-L-2007 cells, as evidenced by the absence of centromere connection of sister chromatids at metaphase. Moreover, the in vitro Xenopus cell-free system which reproduces replication-dependent DNA methylation maintenance revealed that addition of 5-aza-dCTP strongly promoted chromatin binding of DNMT1, while it inhibited that of cohesion complex members (SMC3 and SCC1). Taken together, these results strongly indicate that the aberrant DNMT1 retention on chromatin induced by DAC leads to cohesion loss and mitotic defects in myeloid tumors.

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 and cohesion loss of sister chromatids. Our study also suggests that DAC will be most effective on tumors with impaired mitotic fidelity, such as AML and MDS with TP53 mutations.

Disclosures: Nannya: Takeda Pharmaceutical Company: Speakers Bureau; Pfizer: Speakers Bureau; Astrazeneca: Speakers Bureau; Nippon Shinyaku: Speakers Bureau; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Kyowa-Kirin: Speakers Bureau; Janssen Pharmaceutical: Speakers Bureau; Filgen: Speakers Bureau; Sumitomo Pharma: Speakers Bureau; Daiichi Sankyo Co., Ltd: Research Funding; Otsuka Pharmaceutical: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Bristol Myers Squibb: Speakers Bureau; Chugai Pharmaceutical: Speakers Bureau; Fuji Pharma: Honoraria; Asahi Kasei Pharma: Speakers Bureau; Daiichi Sankyo RD Novare: Research Funding.

*signifies non-member of ASH