NUDT15 Hydrolyzes 6-Thio-Deoxygtp to Mediate the Anticancer Efficacy of 6-Thioguanine Nicholas C.K
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Published OnlineFirst August 16, 2016; DOI: 10.1158/0008-5472.CAN-16-0584 Cancer Therapeutics, Targets, and Chemical Biology Research NUDT15 Hydrolyzes 6-Thio-DeoxyGTP to Mediate the Anticancer Efficacy of 6-Thioguanine Nicholas C.K. Valerie1, Anna Hagenkort1, Brent D.G. Page1, Geoffrey Masuyer2, Daniel Rehling2, Megan Carter2, Luka Bevc1, Patrick Herr1, Evert Homan1, Nina G. Sheppard1, Pa l Stenmark2, Ann-Sofie Jemth1, and Thomas Helleday1 Abstract Thiopurines are a standard treatment for childhood leuke- for NUDT15, a finding supported by a crystallographic deter- mia, but like all chemotherapeutics, their use is limited by mination of NUDT15 in complex with 6-thio-GMP. We found inherent or acquired resistance in patients. Recently, the nucle- that NUDT15 R139C mutation did not affect enzymatic activity oside diphosphate hydrolase NUDT15 has received attention on but instead negatively influenced protein stability, likely due to the basis of its ability to hydrolyze the thiopurine effector a loss of supportive intramolecular bonds that caused rapid metabolites 6-thio-deoxyGTP (6-thio-dGTP) and 6-thio-GTP, proteasomal degradation in cells. Mechanistic investigations in thereby limiting the efficacy of thiopurines. In particular, cells indicated that NUDT15 ablation potentiated induction of increasing evidence suggests an association between the the DNA damage checkpoint and cancer cell death by 6-thio- NUDT15 missense variant, R139C, and thiopurine sensitivity. guanine. Taken together, our results defined how NUDT15 In this study, we elucidated the role of NUDT15 and NUDT15 limits thiopurine efficacy and how genetic ablation via the R139C in thiopurine metabolism. In vitro and cellular results R139C missense mutation confers sensitivity to thiopurine argued that 6-thio-dGTP and 6-thio-GTP are favored substrates treatment in patients. Cancer Res; 76(18); 5501–11. Ó2016 AACR. Introduction thiobases (5–8). Incorporation of 6-thio-dGTP is neither par- ticularly toxic nor mutagenic (6, 9–11). However, methylation, Interfering with nucleotide metabolism is one of the most most likely by S-adenosylmethionine (SAM; ref. 12), and a successful treatment strategies against cancer. Nucleoside analo- second round of replication generate the Me-6-thio-dG:T mis- gues target rapidly proliferating cancer cells by disrupting pro- pair, which is detected by the mismatch repair (MMR) machin- cesses related to RNA and DNA synthesis. Even after half a century ery (12, 13). Processing of this lesion presumably leads to futile of clinical research (1), thiopurines remain one of the most attempts of repair, irreparable DNA damage, and cell death effective maintenance therapies against childhood leukemia (14–16). Because this cascade requires 2 rounds of DNA rep- [acute lymphoblastic leukemia (ALL); ref. 2] and are also used lication, the antiproliferative effects of thiopurines are notice- as anti-inflammatory and immunosuppressant drugs (3). ably delayed (14). MMR-deficient cells are significantly less Three thiopurines are used in the clinic: 6-thioguanine sensitive to thiopurines (14), yet residual sensitivity in these (6-TG), azathioprine (AZA-T), and 6-mercaptopurine (6-MP). cells indicates that thiopurines may act via additional mechan- The metabolism of thiopurines is complex and involves a isms. The complexity of thiopurine metabolism complicates number of enzymes and intermediates that may improve or treatment regimens and imposes a restrictive therapeutic win- impair effective treatment (4). The nucleoside triphosphates, dow that requires close monitoring (2, 3, 10). 6-thio-GTP and 6-thio-dGTP, are the primary active metabo- In searching for novel thiopurine sensitivity factors, several lites. 6-Thio-dGTP is a substrate for DNA polymerases leading research groups have identified a missense variant in NUDT15 to substitution of 0.01% to 0.1% of canonical guanine bases for (rs116855232) that results in an arginine to cysteine substitution at position 139 (R139C) and thiopurine intolerance. Patients fl 1Science for Life Laboratory, Division of Translational Medicine and with in ammatory bowel disease (IBD) having this mutation Chemical Biology, Department of Medical Biochemistry and Biophys- showed increased side effects after thiopurine treatment (17, 18). ics, Karolinska Institutet, Stockholm, Sweden. 2Department of Bio- Accordingly, this NUDT15 variant was found to influence the chemistry and Biophysics, Stockholm University, Stockholm, Sweden. sensitivity to 6-MP in children with ALL (19–21). NUDT15 [nudix Note: Supplementary data for this article are available at Cancer Research hydrolase 15, also known as MutT homolog 2 (MTH2)] belongs Online (http://cancerres.aacrjournals.org/). to the NUDIX hydrolase family and was initially described as an N.C.K. Valerie, A. Hagenkort, and A.-S. Jemth contributed equally to this work. oxidized nucleotide sanitation enzyme, similar to MTH1 (NUDT1 Corresponding Authors: Thomas Helleday, Karolinska Institutet, Science for Life or nudix hydrolase 1; ref. 22). A comparison of MTH1 and Laboratory, Stockholm S-171 21, Sweden. Phone: 46-0-852480000; Fax: 44- NUDT15 revealed that NUDT15 had minimal activity toward 2086613107; E-mail: [email protected]; Pa l Stenmark, oxidized nucleotides in vitro and in cells. However, a substrate [email protected]; and Ann-Sofie Jemth, annsofi[email protected] screen identified that NUDT15 could hydrolyze 6-thio-dGTP and doi: 10.1158/0008-5472.CAN-16-0584 6-thio-GTP, which, like the pharmacogenetic studies, suggested a Ó2016 American Association for Cancer Research. possible role for NUDT15 in thiopurine metabolism (23). www.aacrjournals.org 5501 Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 2016 American Association for Cancer Research. Published OnlineFirst August 16, 2016; DOI: 10.1158/0008-5472.CAN-16-0584 Valerie et al. Herein, we present that NUDT15 is a key enzyme in thiopurine Determination of kinetic parameters of WT and R139C metabolism and tempers the activity of thiopurine drugs by NUDT15 catalyzing the hydrolysis of 6-thio-dGTP and 6-thio-GTP. We NUDT15-mediated hydrolysis of 5 mmol/L 6-thio-dGTP, resolve the first crystal structure of NUDT15 in complex with a 6-thio-GTP, dGTP, and GTP was monitored after 10, 20, and reaction product, 6-thio-GMP, thereby elucidating key determi- 30 minutes of incubation at 22C in assay buffer (100 mmol/L nants for the observed substrate selectivity. With in vitro and Tris-acetate, pH 7.5, 40 mmol/L NaCl, 10 mmol/L magnesium cellular experiments, we explore the activity of NUDT15 R139C acetate, 1 mmol/L DTT) using 4 nmol/L NUDT15 WT. For and find that the protein maintains catalytic activity but is unsta- determination of kinetic parameters, initial rates were determined ble under physiologic conditions. In cells, NUDT15 R139C is using reaction buffer, 4 nmol/L NUDT15 WT or R139C and rapidly degraded, likely causing thiopurine sensitivity in patients concentrations of the substrates ranging from 0 to 40 mmol/L for with this mutation. In line with this, depletion of the protein with 6-thio-dGTP and 6-thio-GTP and 0 to 50 mmol/L for dGTP and NUDT15-specific RNAi in cancer cells increases the sensitivity to 6- GTP (or 0–400 mmol/L for R139C). NUDT15 R139C reactions TG. Altogether, our data suggest that NUDT15 is integral to were performed in the absence or presence of 1 mmol/L DTT. thiopurine metabolism and acts as a barrier to therapeutic efficacy. Formed pyrophosphate (PPi) was detected using PPiLight Inor- ganic Pyrophosphate Assay (Lonza) as described previously (23). Materials and Methods Crystallization and structure determination Protein production Full-length NUDT15 (15 mg/mL) was crystallized with 2 The construct pNIC28hNUDT15 for bacterial expression of mmol/L 6-thio-GTP in 15 mmol/L HEPES, 225 mmol/L NaCl, NUDT15 was a gift from the Structural Genome Consortium 7.5% glycerol, and 1.5 mmol/L TCEP (pH 7.5). Hanging drop (Stockholm, Sweden). NUDT15 wild-type (WT) was expressed vapor diffusion experiments at 4C were performed, and NUDT15 and purified as earlier described (23). Site-directed mutagenesis was mixed with reservoir solution (30% PEG3350, 0.1 mol/L Tris, for the R139C, R139S, R139A, and R139K mutants was performed pH 8.5, and 0.24 mol/L MgCl ) in a 1:1 ratio. Diffraction quality as described by Li and colleagues (24). The mutants were 2 crystals appeared overnight and grew to full size within 3 days. The expressed from pNIC28 in BL21DE3 at 37 C and grown for 4 crystals were extracted quickly without additional cryoprotectant hours after induction by 1 mmol/L isopropyl b-D-1-thiogalacto- and flash-frozen in liquid nitrogen. Data collection was per- pyranoside (IPTG) before harvesting by centrifugation. Bacteria formed at beam line ID29 at ESRF, Grenoble, at 100 K and were lysed using Bugbuster (Merck-Millipore), fortified by ben- wavelength 1.072 Å equipped with a Pilatus 3 6M detector zonase (2.5 U/mL, Merck-Millipore), and cOmplete Mini, EDTA- (Dectris). Data reduction and processing were carried out using free protease inhibitor (Roche) was added to the cleared lysate. Mosflm (25) and Aimless (26) program from the CCP4 suite (27). NUDT15 R139C was purified using HisTrap HP (GE Healthcare) The structure was solved by molecular replacement of the tem- and 100 mmol/L HEPES (pH 7.5), 500 mmol/L NaCl, 10 mmol/L plate structure file with PDB ID 5BON using Phaser (28). The imidazole, and 10% glycerol as starting buffer. Proteins were resultant models were refined using Refmac5 (29) and Arp/wARP eluted using an imidazole gradient (0–500 mmol/L), and the (30) was used for initial addition of the water molecules. Manual His-tag was removed by using TEV protease and passing the adjustments of the model were carried out using Coot (31). protein over a HisTrap HP column. Following dialysis with ion Validation was conducted with Molprobity (32). Relevant statis- exchange chromatography starting buffer [20 mmol/L HEPES tics can be found in Supplementary Table S1.