Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Pharmacology

Thiopurine-mediated impairment of hematopoietic stem and leukemia cells in Nudt15R138C knock-in mice

Leukemia volume 34, pages 882–894 (2020)Cite this article

Subjects

Abstract

Thiopurines are widely used as antileukemia agents and immunosuppressants. Recent large-scale clinical studies revealed a strong association between the NUDT15 p.Arg139Cys (NUDT15R139C) polymorphism and severe thiopurine-induced leukocytopenia. We established knock-in mice harboring p.Arg138Cys (Nudt15R138C), which corresponds to the human polymorphism. A clinically relevant dose of mercaptopurine (MP) induced lethal cytopenia in Nudt15R138C-harboring mice. MP dose reduction attenuated the hematopoietic toxicity, phenocopying clinical observations and providing Nudt15 genotype-based tolerable doses of MP. High-dose MP induced acute damage to hematopoietic stem and progenitor cells (HSPCs) in Nudt15R138C/R138C mice. A competitive transplantation assay revealed that not only Nudt15R138C/R138C HSPCs, but also Nudt15+/R138C HSPCs suffered stronger damage than Nudt15+/+ HSPCs, even by lower-dose MP, after long-term administration. In a Nudt15 genotype-based posttransplantation leukemia recurrence model generated by bone marrow replacement with congenic wild-type cells and a small number of leukemia stem cells, MP prolonged the survival of mice with posttransplantation Nudt15R138C/R138C leukemia recurrence. In conclusion, our model will facilitate NUDT15 genotype-based precision medicine by providing safer estimates for MP dosing, and our findings highlighted the high susceptibility of hematopoietic stem cells to MP and suggested that exploiting thiopurine toxicity might be a novel treatment approach for leukemia in NUDT15R139C-harboring patients.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

References

  1. Elion GB. The purine path to chemotherapy. Science. 1989;244:41–7.

    Article  CAS  Google Scholar 

  2. Karran P, Attard N. Thiopurines in current medical practice: molecular mechanisms and contributions to therapy-related cancer. Nat Rev Cancer. 2008;8:24–36.

    Article  CAS  Google Scholar 

  3. Goldberg R, Irving PM. Toxicity and response to thiopurines in patients with inflammatory bowel disease. Expert Rev Gastroenterol Hepatol. 2015;9:891–900.

    Article  CAS  Google Scholar 

  4. Koren G, Ferrazini G, Sulh H, Langevin AM, Kapelushnik J, Klein J, et al. Systemic exposure to mercaptopurine as a prognostic factor in acute lymphocytic leukemia in children. N Engl J Med. 1990;323:17–21.

    Article  CAS  Google Scholar 

  5. Thomas DA, O’Brien S, Cortes J, Giles FJ, Faderl S, Verstovsek S, et al. Outcome with the hyper-CVAD regimens in lymphoblastic lymphoma. Blood. 2004;104:1624–30.

    Article  CAS  Google Scholar 

  6. Reinisch W, Angelberger S, Petritsch W, Shonova O, Lukas M, Bar-Meir S, et al. Azathioprine versus mesalazine for prevention of postoperative clinical recurrence in patients with Crohn’s disease with endoscopic recurrence: efficacy and safety results of a randomised, double-blind, double-dummy, multicentre trial. Gut. 2010;59:752–9.

    Article  CAS  Google Scholar 

  7. Fraser AG, Orchard TR, Jewell DP. The efficacy of azathioprine for the treatment of inflammatory bowel disease: a 30 year review. Gut. 2002;50:485–9.

    Article  CAS  Google Scholar 

  8. Su Y, Hon YY, Chu Y, Van de Poll ME, Relling MV. Assay of 6-mercaptopurine and its metabolites in patient plasma by high-performance liquid chromatography with diode-array detection. J Chromatogr B Biomed Sci Appl. 1999;732:459–68.

    Article  CAS  Google Scholar 

  9. Brem R, Karran P. Oxidation-mediated DNA cross-linking contributes to the toxicity of 6-thioguanine in human cells. Cancer Res. 2012;72:4787–95.

    Article  CAS  Google Scholar 

  10. Valerie NC, Hagenkort A, Page BD, Masuyer G, Rehling D, Carter M, et al. NUDT15 Hydrolyzes 6-Thio-DeoxyGTP to mediate the anticancer efficacy of 6-thioguanine. Cancer Res. 2016;76:5501–11.

    Article  CAS  Google Scholar 

  11. Tzoneva G, Perez-Garcia A, Carpenter Z, Khiabanian H, Tosello V, Allegretta M, et al. Activating mutations in the NT5C2 nucleotidase gene drive chemotherapy resistance in relapsed ALL. Nat Med. 2013;19:368–71.

    Article  CAS  Google Scholar 

  12. Meyer JA, Wang J, Hogan LE, Yang JJ, Dandekar S, Patel JP, et al. Relapse-specific mutations in NT5C2 in childhood acute lymphoblastic leukemia. Nat Genet. 2013;45:290–4.

    Article  CAS  Google Scholar 

  13. Evans WE, Hon YY, Bomgaars L, Coutre S, Holdsworth M, Janco R, et al. Preponderance of thiopurine S-methyltransferase deficiency and heterozygosity among patients intolerant to mercaptopurine or azathioprine. J Clin Oncol. 2001;19:2293–301.

    Article  CAS  Google Scholar 

  14. Schmiegelow K, Forestier E, Kristinsson J, Soderhall S, Vettenranta K, Weinshilboum R, et al. Thiopurine methyltransferase activity is related to the risk of relapse of childhood acute lymphoblastic leukemia: results from the NOPHO ALL-92 study. Leukemia. 2009;23:557–64.

    Article  CAS  Google Scholar 

  15. Relling MV, Altman RB, Goetz MP, Evans WE. Clinical implementation of pharmacogenomics: overcoming genetic exceptionalism. Lancet Oncol. 2010;11:507–9.

    Article  Google Scholar 

  16. Ban H, Andoh A, Tanaka A, Tsujikawa T, Sasaki M, Saito Y, et al. Analysis of thiopurine S-methyltransferase genotypes in Japanese patients with inflammatory bowel disease. Intern Med. 2008;47:1645–8.

    Article  Google Scholar 

  17. Asada A, Nishida A, Shioya M, Imaeda H, Inatomi O, Bamba S, et al. NUDT15 R139C-related thiopurine leukocytopenia is mediated by 6-thioguanine nucleotide-independent mechanism in Japanese patients with inflammatory bowel disease. J Gastroenterol. 2016;51:22–9.

    Article  CAS  Google Scholar 

  18. Kakuta Y, Kawai Y, Okamoto D, Takagawa T, Ikeya K, Sakuraba H, et al. NUDT15 codon 139 is the best pharmacogenetic marker for predicting thiopurine-induced severe adverse events in Japanese patients with inflammatory bowel disease: a multicenter study. J Gastroenterol. 2018;53:1065–78.

  19. Yang SK, Hong M, Baek J, Choi H, Zhao W, Jung Y, et al. A common missense variant in NUDT15 confers susceptibility to thiopurine-induced leukopenia. Nat Genet. 2014;46(Sep):1017–20.

    Article  CAS  Google Scholar 

  20. Moriyama T, Nishii R, Perez-Andreu V, Yang W, Klussmann FA, Zhao X, et al. NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity. Nat Genet. 2016;48(Apr):367–73.

    Article  CAS  Google Scholar 

  21. Kakuta Y, Naito T, Onodera M, Kuroha M, Kimura T, Shiga H, et al. NUDT15 R139C causes thiopurine-induced early severe hair loss and leukopenia in Japanese patients with IBD. Pharmacogenomics J. 2016;16(Jun):280–5.

    Article  CAS  Google Scholar 

  22. Yang JJ, Landier W, Yang W, Liu C, Hageman L, Cheng C, et al. Inherited NUDT15 variant is a genetic determinant of mercaptopurine intolerance in children with acute lymphoblastic leukemia. J Clin Oncol. 2015;33(Apr):1235–42.

    Article  CAS  Google Scholar 

  23. Nishii R, Moriyama T, Janke LJ, Yang W, Suiter C, Lin TN, et al. Preclinical evaluation of NUDT15-guided thiopurine therapy and its effects on toxicity and anti-leukemic efficacy. Blood. 2018;131:2466–74.

  24. Akiyama S, Matsuoka K, Fukuda K, Hamada S, Shimizu M, Nanki K, et al. Long-term effect of NUDT15 R139C on hematologic indices in inflammatory bowel disease patients treated with thiopurine. J Gastroenterol Hepatol. 2019. https://doi.org/10.1111/jgh.14693. [Epub ahead of print].

  25. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protoc. 2013;8:2281–308.

    Article  CAS  Google Scholar 

  26. Kawahara M, Pandolfi A, Bartholdy B, Barreyro L, Will B, Roth M, et al. H2.0-like homeobox regulates early hematopoiesis and promotes acute myeloid leukemia. Cancer Cell. 2012;22:194–208.

    Article  CAS  Google Scholar 

  27. Yamamoto R, Wilkinson AC, Ooehara J, Lan X, Lai CY, Nakauchi Y, et al. Large-scale clonal analysis resolves aging of the mouse hematopoietic stem cell compartment. Cell Stem Cell. 2018;22:600–7. e604

    Article  CAS  Google Scholar 

  28. Hartford C, Vasquez E, Schwab M, Edick MJ, Rehg JE, Grosveld G, et al. Differential effects of targeted disruption of thiopurine methyltransferase on mercaptopurine and thioguanine pharmacodynamics. Cancer Res. 2007;67:4965–72.

    Article  CAS  Google Scholar 

  29. Ramsey LB, Janke LJ, Edick MJ, Cheng C, Williams RT, Sherr CJ, et al. Host thiopurine methyltransferase status affects mercaptopurine antileukemic effectiveness in a murine model. Pharmacogenet Genomics. 2014;24:263–71.

    Article  CAS  Google Scholar 

  30. Yang L, Boyd K, Kaste SC, Kamdem Kamdem L, Rahija RJ, Relling MV. A mouse model for glucocorticoid-induced osteonecrosis: effect of a steroid holiday. J Orthop Res. 2009;27:169–75.

    Article  CAS  Google Scholar 

  31. Okuda H, Stanojevic B, Kanai A, Kawamura T, Takahashi S, Matsui H, et al. Cooperative gene activation by AF4 and DOT1L drives MLL-rearranged leukemia. J Clin Invest. 2017;127:1918–31.

    Article  Google Scholar 

  32. Krivtsov AV, Twomey D, Feng Z, Stubbs MC, Wang Y, Faber J, et al. Transformation from committed progenitor to leukaemia stem cell initiated by MLL-AF9. Nature. 2006;442:818–22.

    Article  CAS  Google Scholar 

  33. Ono R, Masuya M, Nakajima H, Enomoto Y, Miyata E, Nakamura A, et al. Plzf drives MLL-fusion-mediated leukemogenesis specifically in long-term hematopoietic stem cells. Blood. 2013;122:1271–83.

    Article  CAS  Google Scholar 

  34. Kubota T, Nishida A, Takeuchi K, Iida T, Yokota H, Higashi K, et al. Frequency distribution of thiopurine S-methyltransferase activity in red blood cells of a healthy Japanese population. Ther Drug Monit. 2004;26:319–21.

    Article  CAS  Google Scholar 

  35. Lowenthal A, Meyerstein N, Ben-Zvi Z. Thiopurine methyltransferase activity in the Jewish population of Israel. Eur J Clin Pharm. 2001;57:43–6.

    Article  CAS  Google Scholar 

  36. Hanai H, Iida T, Takeuchi K, Arai O, Watanabe F, Abe J, et al. Thiopurine maintenance therapy for ulcerative colitis: the clinical significance of monitoring 6-thioguanine nucleotide. Inflamm Bowel Dis. 2010;16:1376–81.

    Article  Google Scholar 

  37. McLeod HL, Lin JS, Scott EP, Pui CH, Evans WE. Thiopurine methyltransferase activity in American white subjects and black subjects. Clin Pharm Ther. 1994;55:15–20.

    Article  CAS  Google Scholar 

  38. Otterness DM, Keith RA, Weinshilboum RM. Thiopurine methyltransferase: mouse kidney and liver assay conditions, biochemical properties and strain variation. Biochem Pharm. 1985;34:3823–30.

    Article  CAS  Google Scholar 

  39. Watters JW, Zhang W, Meucci MA, Hou W, Ma MK, McLeod HL. Analysis of variation in mouse TPMT genotype, expression and activity. Pharmacogenetics. 2004;14:247–54.

    Article  CAS  Google Scholar 

  40. U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER). Estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers. Rockville, MD: U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER); 2005.

  41. Takizawa H, Regoes RR, Boddupalli CS, Bonhoeffer S, Manz MG. Dynamic variation in cycling of hematopoietic stem cells in steady state and inflammation. J Exp Med. 2011;208:273–84.

    Article  CAS  Google Scholar 

  42. Wilson A, Laurenti E, Oser G, van der Wath RC, Blanco-Bose W, Jaworski M, et al. Hematopoietic stem cells reversibly switch from dormancy to self-renewal during homeostasis and repair. Cell. 2008;135:1118–29.

    Article  CAS  Google Scholar 

  43. Tsujimoto S, Osumi T, Uchiyama M, Shirai R, Moriyama T, Nishii R, et al. Diplotype analysis of NUDT15 variants and 6-mercaptopurine sensitivity in pediatric lymphoid neoplasms. Leukemia. 2018;32:2710–4.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We thank the Central Research Laboratory of Shiga University of Medical Science, particularly Yasuhiro Mori for technical support with FACS analyses and sorting and Takefumi Yamamoto for technical support with mouse irradiation. This work was supported by JSPS KAKENHI Grant Number JP16K09846 (MK) and the Takeda Science Foundation (MK).

Author information

Author notes

  1. These authors contributed equally: Goichi Tatsumi, Masahiro Kawahara, Takayuki Imai

Authors and Affiliations

  1. Division of Gastroenterology and Hematology, Department of Medicine, Shiga University of Medical Science, Shiga, Japan

    Goichi Tatsumi, Masahiro Kawahara, Takayuki Imai, Ai Nishishita-Asai, Atsushi Nishida, Osamu Inatomi, Katsuyuki Kito & Akira Andoh

  2. Department of Hematology and Oncology, Graduate School of Medicine, Kyoto University, Kyoto, Japan

    Goichi Tatsumi

  3. Tsuruoka Metabolomics Laboratory, National Cancer Center, Yamagata, Japan

    Akihiko Yokoyama

  4. Division of Gastroenterology, Tohoku University Graduate School of Medicine, Miyagi, Japan

    Yoichi Kakuta

Authors
  1. Goichi Tatsumi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Masahiro Kawahara
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Takayuki Imai
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ai Nishishita-Asai
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Atsushi Nishida
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Osamu Inatomi
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Akihiko Yokoyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Yoichi Kakuta
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Katsuyuki Kito
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Akira Andoh
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

GT maintained the mouse strains and performed transplantation and FACS sorting and analyses. MK designed the mouse model, conducted all experiments, analyzed data, provided funding and wrote the paper. TI maintained the mouse strains and performed histology and cytology experiments. AN designed the mouse model. AA-N performed some cytology experiments and FACS analyses. OI edited the paper. AY provided the MLL-AF9 vector and helped the establishment of leukemia model. YK, KK, and AA wrote and edited the paper.

Corresponding author

Correspondence to Masahiro Kawahara.

Ethics declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Tatsumi, G., Kawahara, M., Imai, T. et al. Thiopurine-mediated impairment of hematopoietic stem and leukemia cells in Nudt15R138C knock-in mice. Leukemia 34, 882–894 (2020). https://doi.org/10.1038/s41375-019-0583-9

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41375-019-0583-9

This article is cited by

Search

Advanced search

Quick links