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Drug resistance to nelarabine in leukemia cell lines might be caused by reduced expression of deoxycytidine kinase through epigenetic mechanisms

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Abstract

Purpose

Drug resistance is a serious problem in leukemia therapy. A novel purine nucleoside analogue, nelarabine, is available for the treatment of children with T cell acute lymphoblastic leukemia. We investigated the mechanisms of drug resistance to nelarabine.

Methods

Nelarabine-resistant cells were selected by stepwise and continuous exposure to nelarabine using the limiting dilution method in human B and T cell lymphoblastic leukemia cell lines. Expression analysis was performed using real-time polymerase chain reaction, and epigenetic analysis was performed using methylation-specific polymerase chain reaction and chromatin immunoprecipitation.

Results

The RNA expression level for deoxycytidine kinase (dCK) was decreased in nelarabine-resistant leukemia cells. There were no differences between the parental and nelarabine-resistant leukemia cells in the methylation status of the promoter region of the dCK gene. In the chromatin immune precipitation assay, decreased acetylation of histones H3 and H4 bound to the dCK promoter was seen in the nelarabine-resistant cells when compared to the parental cells. Furthermore, treatment with a novel histone deacetylase inhibitor, vorinostat, promoted the cytotoxic effect of nelarabine along with increased expression of the dCK gene, and it increased acetylation of both histones H3 and H4 bound to the dCK promoter in nelarabine-resistant leukemia cells. The combination index showed that the effect of nelarabine and vorinostat was synergistic.

Conclusion

This study reports that nelarabine with vorinostat can promote cytotoxicity in nelarabine-resistant leukemia cells through epigenetic mechanisms.

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Funding

This study was supported in part by grants from JSPS KAKENHI (Grant-in-Aid for Scientific Research C: 16K10044), the Promotion and Mutual Aid Corporation for Private Schools of Japan and the Science Research Promotion Fund.

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Authors and Affiliations

  1. Department of Pediatrics, Nippon Medical School Chiba Hokusoh Hospital, 1715 Kamagari, Inzai, Chiba, 270-1694, Japan

    Keishi Yoshida, Atsushi Fujita & Takeshi Asano

  2. Department of Pediatrics, Nippon Medical School, Inzai, Chiba, Japan

    Hidehiko Narazaki & Yasuhiko Itoh

Authors
  1. Keishi Yoshida
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  2. Atsushi Fujita
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  3. Hidehiko Narazaki
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  4. Takeshi Asano
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  5. Yasuhiko Itoh
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Contributions

TA and YI designed and conceived the in vitro experiments. HN, AF and KY performed the in vitro experiments, and KY wrote the first draft of the manuscript. TA and KY wrote the paper. The authors analyzed the data, and reviewed, edited and approved the final version of the paper.

Corresponding author

Correspondence to Takeshi Asano.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

The Institutional Review Board of the Nippon Medical School Chiba Hokusoh Hospital approved this study.

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The authors approved the final version of the paper.

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Supplementary Information

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Supplementary file1 (XLSX 10 kb)

Supplementary file2 (XLSX 12 kb)

280_2021_4373_MOESM3_ESM.tif

Supplementary file3. Supplemental Fig. 1. The methylation status of the dCK promoter in BALL, Molt4, NALM6 and SKW3 parental and nelarabine-resistant cells. Methylated and unmethylated dCK promoters were evaluated by methylation-specific PCR using qPCR. Relative values (ratio) were calculated based on the expression level of dCK in BALL parental cells (set as 1). Data are shown as the average from two independent experiments. Identical methylation statuses were observed in the CpG islands of all examined dCK promoters between the parental and nelarabine-resistant cells. (TIF 8270 kb)

280_2021_4373_MOESM4_ESM.tif

Supplementary file4. Supplemental Fig. 2. Combination index (CI)-fraction affected (Fa) curves for BALL/P, BALL/NEL, Molt4/P, Molt4/NEL, NALM6/P, NALM6/NEL, SKW3/P, and SLW3/NEL cells in combination therapy with nelarabine and vorinostat. The drug concentration ratios were as follows: nelarabine+vorinostat (1000:1). CI values less than 1 indicate a synergistic effect, CI values greater than 1 indicate an antagonistic effect and CI values equal to 1 indicate an additive effect. (TIF 8270 kb)

280_2021_4373_MOESM5_ESM.tif

Supplementary file5. Supplemental Fig. 3. Cytotoxicity in BALL, Molt4, NALM6 and SKEW3 parental and nelarabine-resistant cells with or without 5-aza-2′-deoxycytidine treatment. Leukemia cells were incubated with various concentrations of nelarabine for 72 h with or without 0.2 μmol/L of 5-aza-2′-deoxycytidine, and cytotoxicity was evaluated by trypan blue dye exclusion assay. Data are shown as the mean ± standard deviation from three independent experiments. (TIF 33981 kb)

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Yoshida, K., Fujita, A., Narazaki, H. et al. Drug resistance to nelarabine in leukemia cell lines might be caused by reduced expression of deoxycytidine kinase through epigenetic mechanisms. Cancer Chemother Pharmacol 89, 83–91 (2022). https://doi.org/10.1007/s00280-021-04373-4

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  • DOI: https://doi.org/10.1007/s00280-021-04373-4

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