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NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity

Nature Genetics volume 48, pages 367–373 (2016)Cite this article

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Abstract

Widely used as anticancer and immunosuppressive agents, thiopurines have narrow therapeutic indices owing to frequent toxicities, partly explained by TPMT genetic polymorphisms. Recent studies identified germline NUDT15 variation as another critical determinant of thiopurine intolerance, but the underlying molecular mechanisms and the clinical implications of this pharmacogenetic association remain unknown. In 270 children enrolled in clinical trials for acute lymphoblastic leukemia in Guatemala, Singapore and Japan, we identified four NUDT15 coding variants (p.Arg139Cys, p.Arg139His, p.Val18Ile and p.Val18_Val19insGlyVal) that resulted in 74.4–100% loss of nucleotide diphosphatase activity. Loss-of-function NUDT15 diplotypes were consistently associated with thiopurine intolerance across the three cohorts (P = 0.021, 2.1 × 10−5 and 0.0054, respectively; meta-analysis P = 4.45 × 10−8, allelic effect size = −11.5). Mechanistically, NUDT15 inactivated thiopurine metabolites and decreased thiopurine cytotoxicity in vitro, and patients with defective NUDT15 alleles showed excessive levels of thiopurine active metabolites and toxicity. Taken together, these results indicate that a comprehensive pharmacogenetic model integrating NUDT15 variants may inform personalized thiopurine therapy.

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Figure 1: NUDT15 genetic variants and their effects on nucleotide diphosphatase activity.
Figure 2: Association of NUDT15 diplotype with mercaptopurine tolerance during ALL therapy in Guatemala, Singapore and Japan.
Figure 3: Effects of NUDT15 on thiopurine metabolism and cytotoxicity.
Figure 4: NUDT15 variants and mercaptopurine metabolism in children during ALL therapy.

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Acknowledgements

We thank the patients and parents who participated in the clinical trials included in this study, H. Toyoda at Mie University for his assistance in processing the Japanese Pediatric Leukemia/Lymphoma Study Group samples and C. Smith at St. Jude Children's Research Hospital for querying the 1000 Genomes Project data. This work was supported by the US National Institutes of Health (CA021765 and GM115279), the American Lebanese Syrian Associated Charities of St. Jude Children's Research Hospital, the Order of St. Francis Foundation, the V Foundation for Cancer Research and the Danish Childhood Cancer Foundation. The Japanese Pediatric Leukemia/Lymphoma Study Group ALL-B12 study is supported by the Japanese Ministry of Health, and the MaSpore ALL studies are supported by the National Medical Research Council (Singapore), Children's Cancer Foundation and the Viva Foundation for Children with Cancer. J.J.Y. is an American Society of Hematology Scholar. T.M. is supported by the Mie Prefecture Study-Abroad Scholarship (Mie, Japan). U.H. and M.S. are supported by the Robert Bosch Foundation (Stuttgart, Germany). K.H. is supported by the Pediatric Oncology Education Program grant (CA23944). T.I. is supported by Alex's Lemonade Stand Foundation's pediatric oncology student training (POST) program.

Author information

Author notes

  1. Rina Nishii and Virginia Perez-Andreu: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Pharmaceutical Sciences, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Takaya Moriyama, Rina Nishii, Virginia Perez-Andreu, Wenjian Yang, Xujie Zhao, Ting-Nien Lin, Keito Hoshitsuki, Robert McCorkle, Lie Li, Mary V Relling, William E Evans & Jun J Yang

  2. Department of Pediatrics, Mie University Graduate School of Medicine, Mie, Japan

    Takaya Moriyama, Kentaro Kihira, Yoshihiro Komada & Hiroki Hori

  3. Department of Pediatrics and Developmental Biology, Tokyo Medical and Dental University Graduate School of Medicine, Tokyo, Japan

    Rina Nishii

  4. Unidad Nacional de Oncología Pediátrica, Guatemala City, Guatemala

    Federico Antillon Klussmann & Cesar Rolando Najera

  5. Francisco Marroquin Medical School, Guatemala City, Guatemala

    Federico Antillon Klussmann

  6. School of Pharmacy, University of Pittsburgh, Pittsburgh, Pennsylvania, USA

    Keito Hoshitsuki

  7. Department of Paediatrics and Adolescent Medicine, University Hospital Rigshospitalet, Copenhagen, Denmark

    Jacob Nersting & Kjeld Schmiegelow

  8. Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany

    Ute Hofmann & Matthias Schwab

  9. Department of Clinical Pharmacology, University Hospital Tübingen, Tübingen, Germany

    Ute Hofmann & Matthias Schwab

  10. Department of Pediatric Hematology and Oncology Research, National Center for Child Health and Development, Tokyo, Japan

    Motohiro Kato

  11. Department of Hematology/Oncology, Saitama Children's Medical Center, Saitama, Japan

    Katsuyoshi Koh

  12. National University Cancer Institute, National University Health System, Singapore

    Shirley Kow-Yin Kham, Zhiwei Chen, Edwynn Kean-Hui Chiew & Allen E J Yeoh

  13. Graduate School of Medicine and Faculty of Medicine, The University of Tokyo, Tokyo, Japan

    Tomoya Isobe

  14. Department of Pediatrics, Children's Hospital of Los Angeles, Los Angeles, California, USA

    Deepa Bhojwani

  15. Department of Chemical Biology and Therapeutics, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Cynthia Jeffries & Yan Lu

  16. German Cancer Consortium, German Cancer Research Center (DKFZ), Heidelberg, Germany

    Matthias Schwab

  17. Department of Pharmacy and Biochemistry, University of Tübingen, Tübingen, Germany

    Matthias Schwab

  18. Department of Oncology, St. Jude Children's Research Hospital, Memphis, Tennessee, USA

    Hiroto Inaba & Ching-Hon Pui

  19. Department of Pediatrics, St. Luke's International Hospital, Tokyo, Japan

    Atsushi Manabe

  20. Institute of Clinical Medicine, University of Copenhagen, Copenhagen, Denmark

    Kjeld Schmiegelow

  21. Viva University Children's Cancer Centre, Yong Loo Lin School of Medicine, National University of Singapore, Singapore

    Allen E J Yeoh

Authors
  1. Takaya Moriyama
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  2. Rina Nishii
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  3. Virginia Perez-Andreu
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  5. Federico Antillon Klussmann
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  15. Lie Li
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  34. Jun J Yang
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Contributions

Supervised research: J.J.Y. Conceived and designed the experiments: T.M., R.N., H.H., K.S., A.E.J.Y., W.E.E. and J.J.Y. Performed the experiments: T.M., R.N., V.P.-A., X.Z., T.-N.L., K.H., J.N., K. Kihira, U.H., R.M., L.L., C.R.N., T.I., Z.C., E.K.-H.C., C.J., Y.L. and M.S. Performed statistical analysis: V.P.-A. and W.Y. Analyzed the data: T.M., R.N., V.P.-A., W.Y., J.N., U.H., R.M., L.L., T.I., C.J., Y.L., M.S., H.H., K.S. and J.J.Y. Contributed reagents, materials and analysis tools: F.A.K., K. Kihira, Y.K., M.K., K. Koh, C.R.N., S.K.-Y.K., Z.C., E.K.-H.C., D.B., H.I., C.-H.P., M.V.R., A.M., H.H., K.S. and A.E.J.Y. Wrote the manuscript: T.M., W.E.E. and J.J.Y.

Corresponding author

Correspondence to Jun J Yang.

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Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 NUDT15 diplotypes in worldwide populations.

(a) NUDT15 diplotype frequency was based on phased data from the 1000 Genomes Project. (b) Diplotype nomenclatures. Populations include European (CEU, TSI, FIN, GBR and IBS), African (YRI, LWK, GWD, MSL, ESN and ASW), American (MXL, PUR, CLM and PEL), South Asian (GIH, PJL, BEB, STU and ITU) and East Asian (CHB, JPT, CHS, CDX and KHV), as described at http://www.1000genomes.org/category/frequently-asked-questions/population.

Supplementary Figure 2 Enzymatic activity of wild-type and variant NUDT15 with TdGTP as the substrate.

(a) Each variant NUDT15 was expressed in E. coli, and purified protein was subjected to diphosphatase activity measurement with TdGTP as the substrate. (b) Variant or wild-type proteins were combined to determine the level of NUDT15 activity in patients with different diplotypes. Each experiment was performed in triplicate and was repeated at least three times. Center values (dots) represent the means of triplicate experiments; error bars, s.d.

Supplementary Figure 3 Thermostability assay for wild-type and variant NUDT15.

(a–f) Purified NUDT15 was incubated with SyproOrange, and the mixture was heated from 20 ºC to 95 ºC in increments of 0.2 ºC in the Quant Studio 12K Flex Real-Time PCR system. The temperature midpoint for protein unfolding transition, Tm (indicated by the red arrows), was calculated on the basis of the Boltzmann model. Experiments were performed in triplicate to estimate standard deviation.

Supplementary Figure 4 Association of NUDT15 diplotype with mercaptopurine tolerance during ALL therapy.

(a) Guatemalan cohort. (b) Singaporean cohort. (c) Japanese cohort. Mercaptopurine (MP) dose was adjusted during maintenance therapy to avoid excessive host toxicities (myelosuppression and infections), and the tolerated MP dosage was defined as the stable dose for at least 14 d. There were no significant differences in tolerated MP dosage between NUDT15 diplotypes within the intermediate-activity group (*1/*2, *1/*3, *1/*4 and *1/*5; P = 0.44) or within the low-activity group (*2/*3, *3/*3 and *3/*5; P = 0.41) in the combined cohorts using the Kruskal-Wallis test. Cases with TPMT variants (rs1800462, rs1800460 and rs1142345) were excluded from the analysis. Each box includes data between the 25th and 75th percentiles, with the horizontal line indicating the median.

Supplementary Figure 5 Effects of NUDT15 and TPMT genotypes on mercaptopurine tolerance during ALL therapy.

(a) Guatemalan cohort. (b) Singaporean cohort. Mercaptopurine (MP) dose was adjusted during maintenance therapy to avoid host toxicities (myelosuppression and infections), and the tolerated MP dosage was defined as the stable dose for at least 14 d. TPMT diplotypes were based on rs1800462, rs1800460 and rs1145345. No TPMT variants were observed in the Japanese cohort. Each box includes data between the 25th and 75th percentiles, with the horizontal line indicating the median. P value was estimated for the association of NUDT15 diplotype (as normal-, intermediate- and low-activity groups) with the tolerated MP dosage by using linear regression model with (*) or without (**) adjusting for TPMT genotype (as wild type or heterozygous).

Supplementary Figure 6 NUDT15 knockdown in a lymphoid cell line.

Nalm6 cells were transfected with lentiviral particles of shRNA specific to human NUDT15 (NUDT15 KD) or scrambled sequence (control), and stable clones were established by puromycin selection. (a,b) NUDT15 expression was determined in knockdown and control cells by RT-PCR (a) and immunoblotting (b). Experiments were performed in triplicate and were repeated at least three times.

Supplementary Figure 7 Effects of NUDT15 expression on mercaptopurine and thioguanine cytotoxicity.

(a,b) NUDT15-knockdown (NUDT15 KD; red) cells were established by lentiviral transduction of NUDT15-specific shRNA, and control cells (black) were transduced with non-targeted vectors. Cytotoxicity was determined by MTT assay following incubation for 72 h with increasing concentrations of MP (a) and TG (b). Mean values are plotted in each panel; error bars, s.d. from triplicate experiments.

Supplementary Figure 8 Effects of NUDT15 expression on cytotoxicity and metabolism of azathioprine.

(a,b) NUDT15-knockdown (NUDT15 KD; red) Nalm6 cells were established by lentiviral transduction of NUDT15-specific shRNA, and control cells (black) were transduced with non-targeted vectors. Azathioprine cytotoxicity was determined by MTT assay following drug exposure for 72 h (a). DNA-TG level was quantified after exposure for 48 h to azathioprine (b). Mean values are plotted in each panel; error bars, s.d. from triplicate experiments for cytotoxicity and duplicate experiments for DNA-TG level.

Supplementary Figure 9 NUDT15 variants and mercaptopurine metabolism in children during ALL therapy.

(a,b) DNA-TG levels were analyzed in the Singaporean (a) and Japanese (b) cohorts. Sixty-three and 44 samples were successfully measured from 32 cases with wild-type TPMT in the Singaporean and Japanese cohorts, respectively. An average DNA-TG level was estimated for each patient and then normalized on the basis of the tolerated MP dosage. The ratio of DNA-TG/MP dosage was plotted against the NUDT15 diplotypes. There were no significant differences in normalized DNA-TG between diplotypes within the intermediate-activity group (*1/*2, *1/*3 and *1/*5; P = 0.29) or within the low-activity group (*3/*5, *3/*3 and *2/*3; P = 0.37) in the combined cohorts using the Kruskal-Wallis test.

Supplementary Figure 10 NUDT15 genotype was associated with the thioguanine sensitivity of primary ALL blasts in vitro.

In 285 children with newly diagnosed ALL treated at St. Jude Children’s Research Hospital, primary leukemia cells at diagnosis were evaluated for sensitivity to TG using MTT assay as previously described (N. Engl. J. Med. 351, 533, 2004). NUDT15 genotype was summarized as diplotype (Online Methods), and its association with TG sensitivity (with LC50 as a continuous variable) was determined by a linear regression model (Online Methods).

Supplementary Figure 11 NUDT15 genotype and its protein stability in vitro.

Variant or wild-type NUDT15 cDNA encoding an N-terminal FLAG tag was transiently expressed in HEK293T cells using polyethylenimine reagent. Cycloheximide (CHX; 50 mg/ml) was added 48 h after transfection, and NUDT15 protein level was monitored after 0, 24 and 48 h by immunoblotting with β-actin as the loading control. Immunoblot images are shown (top), and protein level was quantified using ImageJ software (bottom).

Supplementary Figure 12 Expression and purification of wild-type and variant human NUDT15 proteins.

N-terminally His-tagged human NUDT15 was expressed in E. coli BL21 with IPTG induction and purified by affinity chromatography. One microgram of protein was loaded onto a 4–15% SDS-PAGE gel for electrophoresis, and the gel was stained with Coomassie Brilliant Blue.

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Moriyama, T., Nishii, R., Perez-Andreu, V. et al. NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity. Nat Genet 48, 367–373 (2016). https://doi.org/10.1038/ng.3508

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