Rina Nishii

NUDT15 polymorphisms alter thiopurine metabolism and hematopoietic toxicity

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.

Germline genetic variation in ETV6 and risk of childhood acute lymphoblastic leukaemia: a systematic genetic study

Background Hereditary predisposition is rarely suspected for childhood acute lymphoblastic leukemia (ALL). Recent studies identified germline ETV6 variations associated with marked familial clustering of hematologic malignancies, pointing to this gene as a potentially important genetic determinant for ALL susceptibility. The aims of the current study are to comprehensively identify ALL predisposition variants in ETV6 and to determine the extent to which they contribute to the overall risk of childhood ALL. Methods Whole-exome sequencing of an index family with multiple cases of ALL was performed to identify causal variants for ALL predisposition. Targeted sequencing of ETV6 was done in 4,405 children from the Childrens Oncology Group (COG) and St. Jude Childrens Research Hospital frontline ALL trials. Patients were included in this study on the basis of their enrollment in these clinical trials and the availability of germline DNA. ETV6 variant genotypes were compared with non-ALL controls to define ALL-related germline risk variants. ETV6 variant function was characterized bioinformatically and correlated with clinical and demographic features in 2,021 children with ALL. Findings We identified a novel nonsense ETV6 variant (p.R359X) with a high penetrance of familial ALL. Subsequent targeted sequencing of ETV6 in 4,405 childhood ALL cases discovered 31 exonic variants (4 nonsense, 21 missense, 1 splice site, and 5 frame shift variants) that are potentially related to ALL risk in 35 cases (0.79%). Fifteen (48%) of the 31 ALL-related ETV6 variants clustered in the ETS domain and predicted to be highly deleterious. Children with ALL-related ETV6 variants were significantly older at leukemia diagnosis than others (10.2 years [IQR 5.3-13.8] vs 4.7 years [IQR 3.0-8.7], P=0.017). The hyperdiploid leukemia karyotype was strikingly overrepresented in ALL cases harboring germline ETV6 risk variants compared to the wildtype group (9 of 14 cases [64.3%] vs 538 of 2,007 cases [26.8%]; P=0.0050). Interpretation Our findings indicated germline ETV6 variations as the basis of a novel genetic syndrome associated with predisposition to childhood ALL. Funding This study was supported by the National Institutes of Health and by the American Lebanese Syrian Associated Charities.

Novel variants in NUDT15 and thiopurine intolerance in children with acute lymphoblastic leukemia from diverse ancestry

Prolonged exposure to thiopurines (eg, mercaptopurine [MP]) is essential for curative therapy in acute lymphoblastic leukemia (ALL), but is also associated with frequent dose-limiting hematopoietic toxicities, which is partly explained by inherited genetic polymorphisms in drug metabolizing enzymes (eg, TPMT). Recently, our group and others identified germ line genetic variants in NUDT15 as another major cause of thiopurine-related myelosuppression, particularly in Asian and Hispanic people. In this article, we describe 3 novel NUDT15 coding variants (p.R34T, p.K35E, and p.G17_V18del) in 5 children with ALL enrolled in frontline protocols in Singapore, Taiwan, and at St. Jude Childrens Research Hospital. Patients carrying these variants experienced significant toxicity and reduced tolerance to MP across treatment protocols. Functionally, all 3 variants led to partial to complete loss of NUDT15 nucleotide diphosphatase activity and negatively influenced protein stability. In particular, the p.G17_V18del variant protein showed extremely low thermostability and was completely void of catalytic activity, thus likely to confer a high risk of thiopurine intolerance. This in-frame deletion was only seen in African and European patients, and is the first NUDT15 risk variant identified in non-Asian, non-Hispanic populations. In conclusion, we discovered 3 novel loss-of-function variants in NUDT15 associated with MP toxicity, enabling more comprehensive pharmacogenetics-based thiopurine dose adjustments across diverse populations.

The effects of inherited NUDT15 polymorphisms on thiopurine active metabolites in Japanese children with acute lymphoblastic leukemia

Thiopurines [e.g. mercaptopurine (MP)] are widely used as chemotherapeutic agents in the treatment of pediatric acute lymphoblastic leukemia with dose-limiting hematopoietic toxicity. Recently, germline variants in NUDT15 have been identified as a major genetic cause for MP-related bone marrow suppression, and there is increasing interest in the clinical implementation of NUDT15 genotype-guided MP dose individualization. Therefore, we sought to evaluate the effects of NUDT15 on thiopurine metabolism and identify pharmacologic markers to inform NUDT15 genotype-guided MP dosing. In 55 Japanese children with acute lymphoblastic leukemia, we simultaneously measured both thioguanine nucleotides (TGN) in red blood cells and DNA-incorporated thioguanine (DNA-TG) in white blood cells. TGN levels were significantly lower in patients with NUDT15 deficiency, likely because of toxicity-related MP dose reduction. In contrast, when exposed to the same dose of MP, DNA-TG accumulated more efficiently in vivo with increasing number of risk alleles in NUDT15 (P=4.0×10−9). Cytosolic TGN and nuclear DNA-TG were correlated positively with each other across genotype groups (P=6.5×10−4), but the ratio of DNA-TG to TGN was significantly higher in NUDT15-deficient patients (P=3.6×10−9), consistent with excessive MP activation. In conclusion, our results suggest that DNA-TG is a more relevant MP metabolite than TGN to inform NUDT15 genotype-guided dose adjustments.

Preclinical evaluation of NUDT15-guided thiopurine therapy and its effects on toxicity and antileukemic efficacy

Thiopurines (eg, 6-mercaptopurine [MP]) are highly efficacious antileukemic agents, but they are also associated with dose-limiting toxicities. Recent studies by us and others have identified inherited NUDT15 deficiency as a novel genetic cause of thiopurine toxicity, and there is a strong rationale for NUDT15-guided dose individualization to preemptively mitigate adverse effects of these drugs. Using CRISPR-Cas9 genome editing, we established a Nudt15-/- mouse model to evaluate the effectiveness of this strategy in vivo. Across MP dosages, Nudt15-/- mice experienced severe leukopenia, rapid weight loss, earlier death resulting from toxicity, and more bone marrow hypocellularity compared with wild-type mice. Nudt15-/- mice also showed excessive accumulation of a thiopurine active metabolite (ie, DNA-incorporated thioguanine nucleotides [DNA-TG]) in an MP dose-dependent fashion, as a plausible cause of increased toxicity. MP dose reduction effectively normalized systemic exposure to DNA-TG in Nudt15-/- mice and largely eliminated Nudt15 deficiency-mediated toxicity. In 95 children with acute lymphoblastic leukemia, MP dose adjustment also directly led to alteration in DNA-TG levels, the effects of which were proportional to the degree of NUDT15 deficiency. Using leukemia-bearing mice with concordant Nudt15 genotype in leukemia and host, we also confirmed that therapeutic efficacy was preserved in Nudt15-/- mice receiving a reduced MP dose compared with Nudt15+/+ counterparts exposed to a standard dose. In conclusion, we demonstrated that NUDT15 genotype-guided MP dose individualization can preemptively mitigate toxicity without compromising therapeutic efficacy.

Combination of common and novel rare NUDT15 variants improves predictive sensitivity of thiopurine-induced leukopenia in children with acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer in the world, with treatment outcomes improving dramatically in the past several decades due to the combined usage of multiple drugs and sophisticated therapeutic protocols. Thiopurine (e.g., 6-mercaptopurine) is primarily used in ALL treatment, but can induce severe adverse drug reactions (ADR), including leukopenia and hepatotoxicity. Inherited predispositions to both ALL susceptibility and treatment outcomes have been noticed. Pharmacogenetics studies have indicated that cases of leukopenia can be largely explained by single nucleotide polymorphisms (SNPs) in TPMT and NUDT15 with varied variant allele frequencies among different ethnicities. In particular, the common missense variant in NUDT15 (i.e., rs116855232) has been reported to be consistently associated with thiopurineinduced leukopenia, either through genome-wide association studies or candidate replications in several independent patient cohorts, made up for the most part by East Asians and Hispanics. Subsequently, several less frequent and rare variants have also been identified that negatively impact on NUDT15 function, and which can further increase the predictive sensitivity of the eventual dosage of thiopurine. As a consequence, it is necessary to sequence the full length of NUDT15 in order to determine the initial clinical dosage of thiopurine, which, unlike TPMT, has had a limited number of functional variants identified thus far. In the study herein, which is a CCCG-ALL-2015 protocol based trial of Chinese pediatric ALL patients aged from one to fifteen years old (N = 188), we systematically investigated the association of thiopurine-induced ADRs with all NUDT15 variants in the West China Second Hospital between 2015 and 2016. 6-mercaptopurine (6MP) was used in multiple phases of ALL therapy: remission induction (standard dosage of 60mg/m in the last two weeks), consolidation (25mg/m), and maintenance (50mg/m). Forty-eight patients in total experienced thiopurine-induced leukopenia, requiring an obligatory reduction of the dosage of 6-MP, an interruption of the therapy, and/or the prescription of human granulocyte colony-stimulating factor (G-CSF). Thirty-seven patients experienced thiopurine-induced hepatotoxicity, which was characterized by an increase in aspartate transaminase and/or alanine transaminase by more than five times following the introduction of thiopurine. Firstly, we estimated the correlation of thiopurineinduced leukopenia/hepatotoxicity events with clinical characteristics, and found no significant association (Table 1). Next, the impact of the reported NUDT15 and TPMT SNPs on leukopenia was evaluated, including rs116855232 (p. R139C), rs186364861 (p. V18I), and rs554405994 (p. V18_V19insGV) in NUDT15, and rs1142345 (inducing p. Y240C) in TPMT. Not surprisingly, the genotypes of rs116855232, rs554405994 and rs1142345 were significantly associated with 6-MPinduced leukopenia (Table 1). However, rs554405994 completely lost its significance in a multivariate model after adjusting for rs116855232, due to the linkage dise-

Loss of DNA Damage Response in Neuroblastoma and Utility of a PARP Inhibitor

Background Neuroblastoma (NB) is the most common solid tumor found in children, and deletions within the 11q region are observed in 11% to 48% of these tumors. Notably, such tumors are associated with poor prognosis; however, little is known regarding the molecular targets located in 11q. Methods Genomic alterations of ATM , DNA damage response (DDR)-associated genes located in 11q ( MRE11A, H2AFX , and CHEK1 ), and BRCA1, BARD1, CHEK2, MDM2 , and TP53 were investigated in 45 NB-derived cell lines and 237 fresh tumor samples. PARP (poly [ADP-ribose] polymerase) inhibitor sensitivity of NB was investigated in in vitro and invivo xenograft models. All statistical tests were two-sided. Results Among 237 fresh tumor samples, ATM, MRE11A, H2AFX , and/or CHEK1 loss or imbalance in 11q was detected in 20.7% of NBs, 89.8% of which were stage III or IV. An additional 7.2% contained ATM rare single nucleotide variants (SNVs). Rare SNVs in DDR-associated genes other than ATM were detected in 26.4% and were mutually exclusive. Overall, samples with SNVs and/or copy number alterations in these genes accounted for 48.4%. ATM-defective cells are known to exhibit dysfunctions in homologous recombination repair, suggesting a potential for synthetic lethality by PARP inhibition. Indeed, 83.3% NB-derived cell lines exhibited sensitivity to PARP inhibition. In addition, NB growth was markedly attenuated in the xenograft group receiving PARP inhibitors (sham-treated vs olaprib-treated group; mean [SD] tumor volume of sham-treated vs olaprib-treated groups = 7377 [1451] m 3 vs 298 [312] m 3 , P = .001, n = 4). Conclusions Genomic alterations of DDR-associated genes including ATM, which regulates homologous recombination repair, were observed in almost half of NBs, suggesting that synthetic lethality could be induced by treatment with a PARP inhibitor. Indeed, DDR-defective NB cell lines were sensitive to PARP inhibitors. Thus, PARP inhibitors represent candidate NB therapeutics.

Diplotype analysis of NUDT15 variants and 6-mercaptopurine sensitivity in pediatric lymphoid neoplasms

Thiopurines are key to the treatment of acute lymphoblastic leukemia (ALL) and lymphoblastic lymphoma (LBL) in children and adults, and 6-mercaptopurine (6-MP) is commonly used in consolidation and maintenance therapy [1–3]. Leukopenia is a dose-limiting toxicity of 6-MP partly explained by hypomorphic variants of TPMT [2–5] and clinical importance of TMPT genotyping is well established. Recently, NUDT15 was identified as a novel thiopurine regulator conferring 6-MP sensitivity most prominently in Asians and Hispanics [4–9]. Patients with bi-allelic NUDT15 variants are extremely sensitive to 6-MP, and only 5–10% of the standard dose is sufficient to maintain the target leukocyte count [4, 8]. Thus far, a total of seven variants in NUDT15 with low diphosphatase activity resulting in excess myelosuppression by 6-MP have been identified [4, 10, 11] and haplotypes with different combinations of variants are known to exist (Fig. 1a). Given the clinical importance of NUDT15 genotyping for individualized dosing of 6-MP [6, 12], the diplotype should be precisely determined, especially for those with heterozygous genotype at multiple variants. For example, a case with heterozygous c.36_37insGGAGTC and c.415C>T, which is the most frequent combination [4], should be determined as compound heterozygosity (*3/*6) or mono-allelic variants (*1/*2), because the two diplotypes have significantly different impacts on total NUDT15 activity [4]. However, a convenient method to determine the diplotype of NUDT15 has not been established, and as shown in the previous study, the diplotype is currently inferred using a public catalog of human variants [4]. Herein we demonstrated a method for identifying the diplotype using droplet digital PCR (ddPCR), and confirmed the results by wildtype specific PCR by a restriction enzyme. In total 138 Japanese children with ALL or LBL were enrolled (Supplementary Table 1). Patients were treated at the National Center for Child Health and Development, the University of Tokyo Hospital, the Hirosaki University Hospital or the Saitama Children’s Medical Center. For maintenance therapy, the 6-MP and MTX dosages were adjusted to maintain a leukocyte count of 1500–3000 /μl by at least monthly blood tests. In this study, a tolerable 6-MP and MTX dose was defined as the average of the doses per day (per week for MTX) administered during the first These authors contributed equally: Shinichi Tsujimoto, Tomoo Osumi.