Abstract
Thiopurines are a standard treatment for childhood leukemia, but like all chemotherapeutics, their use is limited by inherent or acquired resistance in patients. Recently, the nucleoside diphosphate hydrolase NUDT15 has received attention on the basis of its ability to hydrolyze the thiopurine effector metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP, thereby limiting the efficacy of thiopurines. In particular, increasing evidence suggests an association between the NUDT15 missense variant, R139C, and thiopurine sensitivity. In this study, we elucidated the role of NUDT15 and NUDT15 R139C in thiopurine metabolism. In vitro and cellular results argued that 6-thio-dGTP and 6-thio-GTP are favored substrates for NUDT15, a finding supported by a crystallographic determination of NUDT15 in complex with 6-thio-GMP. We found that NUDT15 R139C mutation did not affect enzymatic activity but instead negatively influenced protein stability, likely due to a loss of supportive intramolecular bonds that caused rapid proteasomal degradation in cells. Mechanistic investigations in cells indicated that NUDT15 ablation potentiated induction of the DNA damage checkpoint and cancer cell death by 6-thioguanine. Taken together, our results defined how NUDT15 limits thiopurine efficacy and how genetic ablation via the R139C missense mutation confers sensitivity to thiopurine treatment in patients.
| Original language | English |
|---|---|
| Pages (from-to) | 5501-5511 |
| Number of pages | 11 |
| Journal | Cancer Research |
| Volume | 76 |
| Issue number | 18 |
| DOIs | |
| Publication status | Published - 15 Sept 2016 |
Funding
We thank Prof. Astrid Gr?slund and Axel Abelein for help and advice regarding the circular dichroism analysis. We thank the beamline scientists at BESSY, Berlin, ESRF, Grenoble for their support in data collection, PSF for protein purification and Biostruct-X for support. We also thank Kristina Edfeldt and Sabina Eriksson for their support in the Helleday Lab, Elis?e Wiita for help with DSF experiments and Saeed Eshtad for providing us with the non-targeting shRNA construct. This project is primarily supported by The Knut and Alice Wallenberg Foundation (T. Helleday, P. Stenmark). Further support was received from the Swedish Research Council (T. Helleday, P. Stenmark), the European Research Council (T. Helleday), G?ran Gustafsson Foundation (T. Helleday), Swedish Cancer Society (T. Helleday, P. Stenmark), the Swedish Children's Cancer Foundation (N.G. Sheppard, T. Helleday), the Swedish Pain Relief Foundation (T. Helleday), the Wenner-Gren Foundation (P. Herr, P. Stenmark), the ?ke Wiberg Foundation (P. Stenmark), the Torsten and Ragnar S?derberg Foundation (T. Helleday), the Canadian Institutes of Health Research and the Breast Cancer Society of Canada (B.D.G. Page), and the David and Astrid Hagel?n Foundation (B.D.G. Page). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
ASJC Scopus subject areas
- Oncology
- Cancer Research
