NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity

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.

Figure 1. NUDT15 genetic variants and their effects on nucleotide diphosphatase activity

Four coding variants were identified representing 5 haplotypes (*1 to *5, panel A). Each variant NUDT15 was expressed in E. Coli and purified protein was subjected to diphosphatase activity measurement with TGTP as the substrate (panel B). Variant or wildtype proteins were combined to determine the level of NUDT15 activity in patients with different diplotypes (panel C). There were no significant differences in nucleotide diphosphatase activity between diplotypes within the intermediate group (green shade, P = 0.73) or within the low activity group (red shade, P = 0.19), as determined using Kruskal-wallis test. Center values (dots) represent mean of triplicates and error bars indicate standard deviation.

Figure 2. Association of NUDT15 diplotype with MP tolerance during ALL therapy in Guatemala, Singapore, and Japan

Patients were classified as “normal”, “intermediate”, or “low” NUDT15 activity on the basis of their diplotype at 4 coding variants (panels A, B, and C for the Guatemalan, Singaporean, and Japanese cohorts, respectively). MP dosage was adjusted during maintenance therapy to avoid host toxicities and tolerated MP dosage was defined as the average over at least 14 daily dosages after at least 9 weeks of maintenance therapy. Cases with TPMT variants (rs1800462, rs1800460, and rs1142345) were excluded from the analysis. P value was calculated by using linear regression test, after adjusting for co-variates when applicable. In panels A–C, each box includes data between 25^th and 75^th percentiles, with horizontal line indicating median. Similar association analyses were performed to compare tolerated MP dosage between normal and intermediate NUDT15 groups (P = 0.04, 0.00049, and 0.0033 for Panels A, B, and C, respectively). Meta-analysis combining test statistics from three cohorts indicated consistent association (Forest plot, Panel D, P = 4.45 × 10⁻⁸), with no significant heterogeneity across cohorts (P = 0.34). Allelic effect size indicates the change in MP dosage for every copy of the NUDT15 risk allele. The length of each horizontal line represents the range of 95% confidence interval of allelic effect size with the tick indicating median (gray boxes are proportional to weights of each cohort used in meta-analysis). Dashed vertical line denotes allelic effect size in the meta-analysis with the lateral tips of diamond representing 95% confidence interval.

Figure 3. Effects of NUDT15 on thiopurine metabolism and cytotoxicity

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. Thiopurine metabolites (cytosolic TGMP and TGTP, and DNA-incorporated thioguanine, [DNA-TG]) were analyzed after treatment with 1.25 μM of MP (A and B) or TG (E and F) for 48 hours. DNA-TG was measured in cells exposed to increasing concentrations of MP (D) and TG (H). Cytotoxicity was determined by MTT assay following 72-hour incubation with MP (C, 1.25 μM) or TG (G, 1.25 μM). Mean values are plotted in each panel with error bars indicating standard deviation from triplicates.

Figure 4. NUDT15 variants and MP metabolism in children during ALL therapy

DNA-TG levels were analyzed in Singaporean (A and C) and Japanese (B and D) cohorts. Sixty-three and 44 samples were successfully measured from 32 cases with wildtype TPMT in Singapore and Japanese cohorts, respectively. An average DNA-TG level was estimated for each patient. The associations between MP dosage and DNA-TG were evaluated by two-sided Spearman rank test (A and B). DNA-TG level was normalized based on actual MP dosage (the average of 14 days) prior to metabolite measurements and then correlated with NUDT15 diplotype (as normal, intermediate, or low NUDT15 activity) using a linear regression model (two-sided, C and D). Similar association analyses were also performed to evaluate the difference in normalized DNA-TG between normal and intermediate NUDT15 groups (P = 0.14 and 0.039 for Panels C and D, respectively). Meta-analysis combining test statistics from two cohorts indicated consistent association of NUDT15 diplotype with normalized DNA-TG (P = 2.2 × 10⁻⁸), without significant heterogeneity across cohorts (P = 0.63). Each box includes data between 25^th and 75^th percentiles, with horizontal line indicating median.