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Research ArticleGeneticsOncology
Open Access | 
10.1172/JCI190443
1Department of Pharmacy and Pharmaceutical Sciences and
2Department of Pathology, Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
3Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA.
4Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
5Institute for Cancer Outcomes and Survivorship and Division of Pediatric Hematology-Oncology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
6Departments of Clinical Pharmacology, and of Biochemistry and Pharmacy and
7Cluster of Excellence iFIT (EXC 2180) “Image-guided and Functionally Instructed Tumor Therapies,” University Tuebingen, Tuebingen, Germany.
8Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
Address correspondence to: Min Ni or Jun J. Yang, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS313, Memphis, Tennessee, 38105, USA. Phone: 1.901.595.7325; Email: min.ni@stjude.org (MN). Phone: 1.901.595.2517; Email: jun.yang@stjude.org (JJY).
Authorship note: MM and RN are co–first authors.
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1Department of Pharmacy and Pharmaceutical Sciences and
2Department of Pathology, Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
3Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA.
4Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
5Institute for Cancer Outcomes and Survivorship and Division of Pediatric Hematology-Oncology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
6Departments of Clinical Pharmacology, and of Biochemistry and Pharmacy and
7Cluster of Excellence iFIT (EXC 2180) “Image-guided and Functionally Instructed Tumor Therapies,” University Tuebingen, Tuebingen, Germany.
8Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
Address correspondence to: Min Ni or Jun J. Yang, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS313, Memphis, Tennessee, 38105, USA. Phone: 1.901.595.7325; Email: min.ni@stjude.org (MN). Phone: 1.901.595.2517; Email: jun.yang@stjude.org (JJY).
Authorship note: MM and RN are co–first authors.
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1Department of Pharmacy and Pharmaceutical Sciences and
2Department of Pathology, Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
3Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA.
4Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
5Institute for Cancer Outcomes and Survivorship and Division of Pediatric Hematology-Oncology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
6Departments of Clinical Pharmacology, and of Biochemistry and Pharmacy and
7Cluster of Excellence iFIT (EXC 2180) “Image-guided and Functionally Instructed Tumor Therapies,” University Tuebingen, Tuebingen, Germany.
8Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
Address correspondence to: Min Ni or Jun J. Yang, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS313, Memphis, Tennessee, 38105, USA. Phone: 1.901.595.7325; Email: min.ni@stjude.org (MN). Phone: 1.901.595.2517; Email: jun.yang@stjude.org (JJY).
Authorship note: MM and RN are co–first authors.
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1Department of Pharmacy and Pharmaceutical Sciences and
2Department of Pathology, Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
3Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA.
4Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
5Institute for Cancer Outcomes and Survivorship and Division of Pediatric Hematology-Oncology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
6Departments of Clinical Pharmacology, and of Biochemistry and Pharmacy and
7Cluster of Excellence iFIT (EXC 2180) “Image-guided and Functionally Instructed Tumor Therapies,” University Tuebingen, Tuebingen, Germany.
8Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
Address correspondence to: Min Ni or Jun J. Yang, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS313, Memphis, Tennessee, 38105, USA. Phone: 1.901.595.7325; Email: min.ni@stjude.org (MN). Phone: 1.901.595.2517; Email: jun.yang@stjude.org (JJY).
Authorship note: MM and RN are co–first authors.
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1Department of Pharmacy and Pharmaceutical Sciences and
2Department of Pathology, Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
3Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA.
4Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
5Institute for Cancer Outcomes and Survivorship and Division of Pediatric Hematology-Oncology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
6Departments of Clinical Pharmacology, and of Biochemistry and Pharmacy and
7Cluster of Excellence iFIT (EXC 2180) “Image-guided and Functionally Instructed Tumor Therapies,” University Tuebingen, Tuebingen, Germany.
8Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
Address correspondence to: Min Ni or Jun J. Yang, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS313, Memphis, Tennessee, 38105, USA. Phone: 1.901.595.7325; Email: min.ni@stjude.org (MN). Phone: 1.901.595.2517; Email: jun.yang@stjude.org (JJY).
Authorship note: MM and RN are co–first authors.
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1Department of Pharmacy and Pharmaceutical Sciences and
2Department of Pathology, Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
3Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA.
4Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
5Institute for Cancer Outcomes and Survivorship and Division of Pediatric Hematology-Oncology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
6Departments of Clinical Pharmacology, and of Biochemistry and Pharmacy and
7Cluster of Excellence iFIT (EXC 2180) “Image-guided and Functionally Instructed Tumor Therapies,” University Tuebingen, Tuebingen, Germany.
8Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
Address correspondence to: Min Ni or Jun J. Yang, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS313, Memphis, Tennessee, 38105, USA. Phone: 1.901.595.7325; Email: min.ni@stjude.org (MN). Phone: 1.901.595.2517; Email: jun.yang@stjude.org (JJY).
Authorship note: MM and RN are co–first authors.
Find articles by Li, J. in: PubMed | Google Scholar
1Department of Pharmacy and Pharmaceutical Sciences and
2Department of Pathology, Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
3Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA.
4Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
5Institute for Cancer Outcomes and Survivorship and Division of Pediatric Hematology-Oncology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
6Departments of Clinical Pharmacology, and of Biochemistry and Pharmacy and
7Cluster of Excellence iFIT (EXC 2180) “Image-guided and Functionally Instructed Tumor Therapies,” University Tuebingen, Tuebingen, Germany.
8Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
Address correspondence to: Min Ni or Jun J. Yang, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS313, Memphis, Tennessee, 38105, USA. Phone: 1.901.595.7325; Email: min.ni@stjude.org (MN). Phone: 1.901.595.2517; Email: jun.yang@stjude.org (JJY).
Authorship note: MM and RN are co–first authors.
Find articles by Hofmann, U. in: PubMed | Google Scholar
1Department of Pharmacy and Pharmaceutical Sciences and
2Department of Pathology, Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
3Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA.
4Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
5Institute for Cancer Outcomes and Survivorship and Division of Pediatric Hematology-Oncology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
6Departments of Clinical Pharmacology, and of Biochemistry and Pharmacy and
7Cluster of Excellence iFIT (EXC 2180) “Image-guided and Functionally Instructed Tumor Therapies,” University Tuebingen, Tuebingen, Germany.
8Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
Address correspondence to: Min Ni or Jun J. Yang, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS313, Memphis, Tennessee, 38105, USA. Phone: 1.901.595.7325; Email: min.ni@stjude.org (MN). Phone: 1.901.595.2517; Email: jun.yang@stjude.org (JJY).
Authorship note: MM and RN are co–first authors.
Find articles by Savic, D. in: PubMed | Google Scholar
1Department of Pharmacy and Pharmaceutical Sciences and
2Department of Pathology, Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
3Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA.
4Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
5Institute for Cancer Outcomes and Survivorship and Division of Pediatric Hematology-Oncology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
6Departments of Clinical Pharmacology, and of Biochemistry and Pharmacy and
7Cluster of Excellence iFIT (EXC 2180) “Image-guided and Functionally Instructed Tumor Therapies,” University Tuebingen, Tuebingen, Germany.
8Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
Address correspondence to: Min Ni or Jun J. Yang, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS313, Memphis, Tennessee, 38105, USA. Phone: 1.901.595.7325; Email: min.ni@stjude.org (MN). Phone: 1.901.595.2517; Email: jun.yang@stjude.org (JJY).
Authorship note: MM and RN are co–first authors.
Find articles by Bhatia, S. in: PubMed | Google Scholar
1Department of Pharmacy and Pharmaceutical Sciences and
2Department of Pathology, Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
3Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA.
4Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
5Institute for Cancer Outcomes and Survivorship and Division of Pediatric Hematology-Oncology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
6Departments of Clinical Pharmacology, and of Biochemistry and Pharmacy and
7Cluster of Excellence iFIT (EXC 2180) “Image-guided and Functionally Instructed Tumor Therapies,” University Tuebingen, Tuebingen, Germany.
8Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
Address correspondence to: Min Ni or Jun J. Yang, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS313, Memphis, Tennessee, 38105, USA. Phone: 1.901.595.7325; Email: min.ni@stjude.org (MN). Phone: 1.901.595.2517; Email: jun.yang@stjude.org (JJY).
Authorship note: MM and RN are co–first authors.
Find articles by Schwab, M. in: PubMed | Google Scholar
1Department of Pharmacy and Pharmaceutical Sciences and
2Department of Pathology, Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
3Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA.
4Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
5Institute for Cancer Outcomes and Survivorship and Division of Pediatric Hematology-Oncology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
6Departments of Clinical Pharmacology, and of Biochemistry and Pharmacy and
7Cluster of Excellence iFIT (EXC 2180) “Image-guided and Functionally Instructed Tumor Therapies,” University Tuebingen, Tuebingen, Germany.
8Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
Address correspondence to: Min Ni or Jun J. Yang, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS313, Memphis, Tennessee, 38105, USA. Phone: 1.901.595.7325; Email: min.ni@stjude.org (MN). Phone: 1.901.595.2517; Email: jun.yang@stjude.org (JJY).
Authorship note: MM and RN are co–first authors.
Find articles by Ni, M. in: PubMed | Google Scholar
1Department of Pharmacy and Pharmaceutical Sciences and
2Department of Pathology, Center of Excellence for Leukemia Studies, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
3Karmanos Cancer Institute, Wayne State University School of Medicine, Detroit, Michigan, USA.
4Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany.
5Institute for Cancer Outcomes and Survivorship and Division of Pediatric Hematology-Oncology, University of Alabama at Birmingham, Birmingham, Alabama, USA.
6Departments of Clinical Pharmacology, and of Biochemistry and Pharmacy and
7Cluster of Excellence iFIT (EXC 2180) “Image-guided and Functionally Instructed Tumor Therapies,” University Tuebingen, Tuebingen, Germany.
8Department of Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee, USA.
Address correspondence to: Min Ni or Jun J. Yang, St. Jude Children’s Research Hospital, 262 Danny Thomas Place, MS313, Memphis, Tennessee, 38105, USA. Phone: 1.901.595.7325; Email: min.ni@stjude.org (MN). Phone: 1.901.595.2517; Email: jun.yang@stjude.org (JJY).
Authorship note: MM and RN are co–first authors.
Find articles by Yang, J. in: PubMed | Google Scholar
Authorship note: MM and RN are co–first authors.
Published July 15, 2025 - More info
Published in Volume 135, Issue 14 on July 15, 2025
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Abstract
Purine nucleotides are critical for nucleic acid synthesis, signaling, and cellular metabolism. Thiopurines (TPs), including 6-mercaptopurine and 6-thioguanine, are cornerstone agents for the treatment of acute lymphoblastic leukemia (ALL). TP efficacy and cytotoxicity depend on the metabolism and intracellular activation of TPs, a process influenced by pharmacogenes such as thiopurine-S methyltransferase (TPMT) and NUDIX (nucleoside diphosphates linked to moiety-X) hydrolase 15 (NUDT15). In this issue of the JCI, Maillard et al. identified NUDT5 as a determinant of TP pharmacology. They demonstrated that loss of NUDT5 conferred TP resistance by impairing drug activation and DNA damage responses. Metabolomics studies by Maillard and others revealed that NUDT5 may regulate the balance between the de novo purine synthesis and salvage pathways. Clinically, NUDT5 expression variants were associated with altered TP tolerance. These findings position NUDT5 as a key modulator of nucleotide metabolism and TP efficacy, with potential implications for pharmacogenomics-guided therapy optimization in ALL.
Authors
× AbstractThiopurines are anticancer agents used for the treatment of leukemia and autoimmune diseases. These purine analogs are characterized by a narrow therapeutic index because of the risk of myelosuppression. With the discovery of NUDIX hydrolase 15 (NUDT15) as a major modulator of thiopurine metabolism and toxicity, we sought to comprehensively examine all members of the NUDIX hydrolase family for their effect on the pharmacologic effects of thiopurine. By performing a NUDIX-targeted CRISPR/Cas9 screen in leukemia cells, we identified NUDT5, whose depletion led to drastic thiopurine resistance. NUDT5 deficiency resulted in a nearly complete depletion of active metabolites of thiopurine and the loss of thioguanine incorporation into DNA. Mechanistically, NUDT5 deletion resulted in substantial alteration in purine nucleotide biosynthesis, as determined by steady-state metabolomics profiling. Stable isotope tracing demonstrated that the loss of NUDT5 was linked to a marked suppression of the purine salvage pathway but with minimal effects on purine de novo synthesis. Finally, we comprehensively identified germline genetic variants in NUDT5 associated with thiopurine-induced myelosuppression in 582 children with acute lymphoblastic leukemia. Collectively, these results pointed to NUDT5 as a key regulator of the thiopurine response primarily through its effects on purine homeostasis, highlighting its potential to inform individualized thiopurine therapy.
IntroductionThiopurines, including 6-thioguanine (TG), 6-mercaptopurine (MP), and its prodrug azathioprine, are widely used therapeutic agents for treating cancers such as acute lymphoblastic leukemia (ALL), as well as autoimmune diseases like inflammatory bowel disease (IBD) (1, 2). However, these synthetic guanine analogs have a narrow therapeutic index due to severe hematopoietic toxicity that can lead to treatment discontinuation and life-threatening infections (3, 4).
Thiopurines exert their antileukemic activity only after being extensively metabolized into thioguanine nucleotides (TGNs). MP and TG are first converted by hypoxanthine-guanine phosphoribosyl transferase (HPRT) into thioinosine monophosphate (TIMP) and thioguanosine monophosphate (TGMP), respectively, and subsequently into active cytotoxic thioguanosine triphosphate (TGTP). The incorporation of TGTP into DNA (as DNA-TG) triggers post-replication mismatch repair (MMR), DNA strand breaks, and, ultimately, apoptosis (5–7). Thiopurine methyltransferase (TPMT) and NUDIX hydrolase 15 (NUDT15) are 2 major metabolizing enzymes of thiopurines, both acting to prevent the excessive build-up of TGNs. More important, germline loss-of-function variants in these 2 genes have been associated with thiopurine toxicity (8–18). This led to the recommendations of a TPMT/NUDT15 genotype–guided dose adjustment by the Clinical Pharmacogenetics Implementation Consortium (CPIC) (19), which have since been added to the drug labels issued by the US FDA, the European Medicines Agency, and other regulatory agencies globally.
NUDT15 belongs to a family of 22 hydrolases that dephosphorylate a wide variety of naturally occurring nucleotides as well as synthetic analogs that are used in cancer or infectious disease therapies (20, 21). With the exception of NUDT15, the role of these enzymes in thiopurine disposition has not been systematically examined. Yet, the promiscuity in their substrate recognition suggests that other NUDIX enzymes may also be involved in regulating thiopurine metabolism and/or response.
In this study, we performed a NUDIX-targeted CRISPR/Cas9 screen in human B-ALL cells treated with thiopurine and found that NUDIX hydrolase 5–deficient cells were resistant to thiopurine. We performed extensive metabolomics profiling followed by stable isotope tracing of the purine synthesis pathway to identify the molecular mechanisms supporting this phenotype. Finally, we systematically evaluated the association of germline genetic variants in NUDT5 with thiopurine-related myelosuppression in 582 children with ALL.
ResultsNUDIX-targeted CRISPR/Cas9 screen for modulators of thiopurine cytotoxicity. To identify which of the 22 NUDIX hydrolases regulate the cytotoxic effects of thiopurines, we performed a NUDIX-targeted CRISPR/Cas9 screen in 2 human B-ALL cell lines, Nalm6 (DUX4-IGH rearranged B-ALL) and 697 (TCF3-PBX1 fusion B-ALL) (Figure 1A). A library of 46 sgRNAs was assembled with 2 sgRNAs per gene, except NUDT4, for which 2 pairs of flanking sgRNAs were designed because of the small exon size. Following a 7-day exposure to TG, sgRNAs targeting NUDT15 were significantly depleted, confirming the loss of this gene linked to thiopurine sensitivity (fold-change [FC] vs. no treatment control: 0.41 and 0.52, P = 0.006 and 0.011, in Nalm6 and 697 cells, respectively) (Figure 1B). Conversely, NUDT5 sgRNAs were significantly enriched in B-ALL cells that survived thiopurine treatment (FC = 18.4 and 3.2, respectively, P = 0.002), indicating that NUDT5 deficiency drives drug resistance. The depletion of NUDT21 in the control condition (without thiopurine) suggested it is essential for ALL survival (FC = 0.35 and 0.25, P = 0.006 and 0.002, respectively) (22) (Figure 1C). Using a FC of 2 or less or greater than 2 and a P value of less than 0.05 as the threshold of statistical significance, we identified no other NUDIX genes with significant effects on the thiopurine response.
Figure 1NUDIX-targeted CRISPR/Cas9 screen for modulators of thiopurine cytotoxicity. (A) Workflow for NUDIX-targeted CRISPR/Cas9 screen in B-ALL cell lines. Illustration was created with BioRender.com. (B) Volcano plot showing the enrichment or depletion of the sgRNAs targeting the respective NUDIX genes in CRISPR/Cas9-transduced cells after 7 days of TG (TGDay7) compared with control (No treatDay7). (C) Volcano plot showing enrichment or depletion of the NUDIX genes in CRISPR/Cas9-transduced cells after 7 days of culturing (No treatDay7) compared with day 0 of culturing (i.e., immediately after lentiviral transduction [No treatDay0]), without drug exposure. The x and y axes represent the FC (in logarithmic scale) and the nominal P value of the enrichment or depletion, respectively. The vertical dotted line indicates a log2(FC) of –1 or 1, and the horizontal dotted line represents a P value of 0.05.
NUDT5 deletion results in marked thiopurine drug resistance. To characterize the molecular mechanism by which NUDT5 modulates the thiopurine response, we first generated NUDT5KO B-ALL cell lines using one of the sgRNAs from the original CRISPR library and confirmed protein depletion by Western blotting (Figure 2A). In the absence of thiopurine drugs, NUDT5 deletion did not alter ALL cell proliferation (Figure 2B). After exposing the cells to increasing concentrations of TG or MP, NUDT5-KO (NUDT5KO) Nalm6 cells showed minimal cell death, whereas both drugs were highly cytotoxic in parental cells in a dose-dependent manner (Figure 2, C and D). These results were replicated in the 697 cell line (Figure 2, E and F). Reexpression of NUDT5 in the NUDT5KO cells largely restored drug sensitivity (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI190443DS1). As expected, the MMR-dependent DNA damage signaling pathway was activated in parental cells exposed to MP, e.g., histone H2AX phosphorylation and checkpoint kinase Chk1/Chk2 activation (23). However, no change was observed when NUDT5 was knocked out, similar to the parental cells without MP treatment (Supplemental Figure 2).
Figure 2NUDT5 is required for thiopurine-induced apoptosis. (A) Western blots confirming NUDT5 KO in the Nalm6 and 697 B-ALL cell lines. (B) Proliferation assay showing no difference in cell growth between NUDT5KO and parental cell lines. (C and D) NUDT5KO Nalm6 cell sensitivity to TG and MP, respectively, after 72 hours of treatment. (E and F) NUDT5KO 697 cell sensitivity to TG and MP, respectively, after 72 hours of treatment. Data are presented as the mean ± SD. n = 3 replicates. **P < 0.01, by 2-tailed, unpaired t test for comparisons between groups. LC50, lethal concentration 50.
KO of NUDT5 compromises thiopurine biotransformation. To determine whether thiopurine metabolism was altered in NUDT5KO cells, we measured cytosolic concentrations of 12 thiopurine metabolites (namely, thioguanine mono-, di-, and triphosphate [TGMP, TGDP, and TGTP, respectively], thioinosine mono-, di-, and triphosphate [TIMP, TIDP, and TITP, respectively], and their respective methylated forms [meTGMP, meTGDP, meTGTP, meTIMP, meTIDP, and meTITP]), plus the degree of thioguanine incorporation into DNA (DNA-TG) (Figure 3A). The first product of MP biotransformation, TIMP, was significantly decreased in both NUDT5KO cell lines (Nalm6: FC = 0.31, P < 0.001, 697: FC = 0.005, P < 0.001), as was its methylated species, meTIMP (FC = 0.23 and 0.017, P < 0.0001) (Figure 3, B and C). Similarly, the final product of thiopurine activation, TGTP, was significantly reduced in both NUDT5KO cell lines (Nalm6: FC = 0.35, P = 0.0016, 697: not detected in NUDT5KO, P = 0.0002). Last, DNA-TG was almost completely absent in cells without NUDT5 (Nalm6: FC = 0.009, P < 0.001, 697: not detected in NUDT5KO, P < 0.0001). This depletion in DNA-TG was also observed when these cells were treated with TG, which utilizes a similar but not identical biotransformation pathway (Supplemental Figure 3). Together, these data indicate that NUDT5 is essential for the activation of thiopurines to exert their cytotoxicity.
Figure 3NUDT5 deletion impairs intracellular metabolism of thiopurines. (A) Thiopurine metabolism pathway. Illustration was created with BioRender.com. (B and C) Cytosolic metabolites were measured in parental and NUDT5KO Nalm6 (B) and 697 (C) cell lines after treatment with 10 μM MP for 24 hours. Nuclear DNA-TG was measured after treatment with 5 μM MP for 24 hours. Data are presented as the mean ± SD. n = 3 replicates. **P < 0.01. ***P < 0.001, and ****P < 0.0001, by 2-tailed, unpaired t test.
Depletion of NUDT5 inhibits the purine salvage pathway. As a phosphatase, NUDT5 has been linked to the hydrolysis of adenosine diphosphate ribose (ADP-R) to generate ribose-5-phosphate (R-5-P) (Figure 4A), the precursor of phosphoribosyl pyrophosphate (PRPP) that is used for nucleotide biosynthesis, including both de novo and salvage purine synthesis (Figure 4B) (24). Building upon this, we first subjected both parental and NUDT5KO cell lines to a broad metabolomics analysis examining 166 cellular metabolites involved in nucleotide, amino acid, and carbohydrate biosynthesis and degradation (Supplemental Table 1). The most significant change was seen with hypoxanthine and guanosine, 400-fold and 16-fold higher in NUDT5KO compared with parental cells, respectively (P = 0.0004 and 0.003, Figure 4, C and D). Of the 23 purine-related metabolites detected, we also observed a significant increase of 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and inosine monophosphate (IMP) in NUDT5KO cells (2-fold, P = 0.0007, and 1.6-fold, P = 0.02, respectively). These changes pointed to the profound effect of NUDT5 expression on purine metabolism.
Figure 4Targeted metabolomics profiling identifies the effects of NUDT5 on purine nucleotide homeostasis. (A) NUDT5 hydrolyzes ADP-R into AMP and R-5-P. (B) Illustration of de novo and salvage pathways for purine synthesis. (C) Volcano plot generated after data transformation and normalization of the concentrations of metabolites measured in Nalm6 parental and NUDT5KO cells. (D) Levels of the purine intermediates hypoxanthine, guanosine, AICAR, and IMP in Nalm6 parental and NUDT5KO cells. The concentrations (conc.) of the metabolites were normalized by the protein concentration in the sample. Data are represented as the mean ± SD. n = 3 replicates. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed, unpaired t test. Illustrations in A and B were created with BioRender.com.
To more precisely determine the role of NUDT5 in purine metabolism, we performed a series of isotope-tracing experiments focusing on metabolites related to either de novo purine synthesis (DNPS) or purine salvage pathways. Using nitrogen-labeled hypoxanthine ([15N4]hypoxanthine), we sought to track the 15N-labeled purine ring into IMP, guanosine monophosphate (GMP), and adenosine monophosphate (AMP) (m+4 labeling) through the salvage pathway (Figure 5A). As shown in Figure 5B, the loss of NUDT5 markedly reduced m+4 labeling of all purine nucleotides (all P < 0.05), consistent with downregulation of the salvage pathway. Exposure to MP had a minimal effect on these hypoxanthine-derived purine nucleotides in either WT (P = 0.14, 0.26, and 0.20, for IMP, GMP, and AMP, respectively) or NUDT5KO (P = 0.11, 0.02, and 0.04, for IMP, GMP and AMP, respectively) cells. These results suggested that the first step of purine salvage, i.e., HPRT-catalyzed conversion of hypoxanthine to IMP, was impaired in NUDT5KO cells. This was likely due to the loss of HPRT catalytic activity (or enzymatic inhibition), whereas HPRT1 expression was unaffected in the absence of NUDT5 (Supplemental Figure 4).
Figure 5Stable isotope tracing of purine synthesis pathways highlights the contribution of NUDT5 to purine metabolism. (A) Schematic illustrating 15N labeling of purine nucleotides from [15N4]hypoxanthine during the purine salvage pathway. (B) Fractional enrichment of m+4 IMP, AMP, and GMP from [15N4]hypoxanthine in parental and NUDT5KO Nalm6 cells during the purine salvage pathway, with and without MP. (C) Schematic illustrating 15N labeling of purine nucleotides from [amide-15N]glutamine during the de novo purine synthesis pathway. (D) Fractional enrichment of m+2 IMP and AMP, and m+3 GMP from [amide-15N]glutamine in parental and NUDT5KO Nalm6 cells during the purine de novo synthesis, with and without MP. Data are presented as the mean ± SD. n = 2 replicates. *P < 0.05, **P < 0.01, and ***P < 0.001, by 2-tailed, unpaired t test. Panels A and C were created with BioRender.com.
In parallel, we examined DNPS using [amide-15N]glutamine, which is used by phosphoribosyl pyrophosphate amidotransferase (PPAT) to convert PRPP to phosphoribosylamine (PRA), the first precursor of purine nucleotides during DNPS (Figure 5C). Labeled glutamine provides two 15N atoms for the de novo synthesis of IMP, which is further converted to AMP (m+2 labeling) or to GMP using a second molecule of glutamine (m+3 labeling). As shown in Figure 5D, there was no change in m+2 labeling of IMP or AMP in NUDT5KO cells relative to parental control cells, although we observed a modest decrease in m+3 labeling of GMP (P = 0.006), suggesting a minimal effect of NUDT5 on DNPS at baseline. In the presence of MP, we found that DNPS was greatly inhibited in parental cells, as reflected by the reduction of IMP, AMP, and GMP, whereas NUDT5KO cells showed no MP-induced changes in DNPS.
Therefore, we reason that NUDT5 deficiency blunted HPRT activity, downregulating purine salvage and hampering the conversion of thiopurine to cytotoxic metabolites. By contrast, NUDT5 may have a minimal role in DNPS under physiological conditions but is required for DNPS inhibition by thiopurines.
Association of NUDT5 germline variants with hematopoietic toxicity in patients receiving thiopurine therapy. To evaluate the clinical relevance of the NUDT5-mediated thiopurine response, we comprehensively characterized genetic variations of NUDT5 in 582 children with ALL enrolled on the Children’s Oncology Group AALL03N1 trial, for whom systematic data on MP-related myelosuppression were collected (13). Patients with known pharmacogenetic variants in TPMT and NUDT15 were excluded to avoid confounding effects. A total of 2,108 variants were identified within NUDT5 exons and 100 kb upstream of the gene (Figure 6A). Among 416 common variants (minor allele frequency [MAF] >0.03), the only coding variant in NUDT5 was a synonymous variant, rs6686 (c.609A>G, p.A203A), in exon 10, which was not associated with MP dose intensity (DI) (nominal P = 0.472).
Figure 6Characterization of NUDT5 germline variants associated with thiopurine-induced myelosuppression in children with ALL. (A) NUDT5 sequencing and genotyping data were retrieved for 582 children with ALL, who were enrolled in the Children’s Oncology Group AALL03N1 clinical trial. All patients were TPMT and NUDT15 WT. Common variants (MAF >0.03) were considered for a multiple regression association analysis with MP sensitivity. HWE, Hardy-Weinberg Equilibrium. (B) The intergenic variant rs55713253 C>T on chromosome 10 was associated with a significant decrease in MP DI during maintenance therapy. Shown is the nominal P value of the multiple regression model, assuming an additive genetic model. (C) View of the genomic environment of rs55713253 C>T from the UCSC Genome Browser (https://genome.ucsc.edu/). CLP, common lymphoid progenitor; CMP, common myeloid progenitor; DHS, DNase hypersensitivity site; GMP, granulocyte-monocyte progenitor; HSC, hematopoietic stem cell; LMPP, lymphoid-primed multipotent progenitor cell; MEP, megakaryocyte-erythrocyte progenitor; MMP, multipotent progenitor. (D) Luciferase reporter gene assay in Nalm6 cells expressing the NUDT5 rs55713253-mutant T allele showed increased expression of the reporter gene. Ref, reference allele; Alt, alternative allele. n = 3 replicates. Data are presented as the mean ± SD. **P < 0.01, by 2-tailed, unpaired t test.
Because NUDT5 expression was linked to the thiopurine response, we hypothesized that cis-regulatory variants at this locus (i.e., expression quantitative trait loci, [eQTL], of NUDT5) are associated with MP dose intensity (DI, in %), a proxy marker for drug-induced myelosuppression. Among the 415 common noncoding variants, 32 were associated with MP DI during the maintenance therapy according to the multivariable regression model adjusting for genetic ancestry (P < 0.05), and 4 of them were related to NUDT5 transcription in blood cells according to the public eQTLGen gene expression database (P < 0.0001) (Supplemental Table 2).
Only the intergenic variant rs55713253 (C>T, Chr10:12,305,358, MAF = 0.06 in the cohort) remained significant in a permutation-based analysis to account for multiple testing (nominal P = 0.0019 and adjusted P = 0.036). Patients with the WT genotype (CC, n = 514) tolerated a median DI of 89% (IQR, 72%–97.2%), which was higher than for patients heterozygous for rs55713253 (CT, n = 64, 79.2%, [63.1–92.7]), and the DI was the lowest in homozygous patients (TT, n = 4, DI = 52.7%, [37.4–90.0], Figure 6B). Consistent with our observation that NUDT5 potentiated thiopurine cytotoxicity, the T allele at this locus was also associated with elevated NUDT5 expression in blood (P = 5.0 × 10–19, z score = 9.0, per eQTLGen) (25). We queried the RegulomeDB public database and found that rs55713253 was likely to affect NUDT5 transcription in hematopoietic cells (probability score of 0.97 based on chromatin accessibility and transcription factor binding data); it occurs 67 kb upstream of the NUDT5 transcription start site, in a region enriched in histone 3 methylation marks (H3K4Me1 and H3K4Me3) (Figure 6C). This variant overlaps with an YY1 ChIP-Seq peak in GM12878 lymphoblastoid cells according to the Encyclopedia of DNA Elements (ENCODE) database. We experimentally tested the effect of rs55713253 on enhancer activity with a reporter gene assay and found that the T allele significantly increased luciferase expression, confirming its potential to influence NUDT5 expression (FC = 1.29, P = 0.01, Figure 6D).
DiscussionIn this study, we comprehensively explored the role of NUDIX hydrolases on thiopurine metabolism, efficacy, and toxicity. Our NUDIX-targeted CRISPR/Cas9 screen confirmed NUDT15 as a negative regulator of cytotoxicity of this class of drugs, but, surprisingly, we discovered that NUDT5 was required for thiopurine-induced cell death. In fact, several prior studies have reported similar findings, so much so that NUDT5 was the top thiopurine resistance gene in a genome-wide fashion (26), although the underlying biology is not understood. Physiologically, NUDT5 plays a critical role in metabolic homeostasis by hydrolyzing cytosolic ADP-R into AMP and R-5-P (24, 27–29). Because R-5-P is the precursor of PRPP, which is required for the conversion of MP to TIMP or TG to TGMP (24, 28), we initially hypothesized that NUDT5 deletion causes the loss of PRPP and thus impairs thiopurine biotransformation (Figure 4A). A few lines of evidence from us and others seem to refute this hypothesis: (a) leukemia cells generally favor the pentose phosphate pathway to produce R-5-P from glucose (30), and NUDT5-mediated hydrolysis of ADP-R is probably a minor source for this metabolite; (b) NUDT5 deletion specifically resulted in drastic reduction of purine salvage without affecting DNPS (Figure 5), even though both pathways depend on PRPP. Instead, our data suggest that the loss of NUDT5 impaired HPRT function (directly or indirectly), which limited the cell’s ability to convert thiopurine prodrug into active metabolites (TIMP, TGTP, and DNA-TG) and, in turn, reduced drug cytotoxicity. However, the exact molecular process by which NUDT5 modulates HPRT activity and purine salvage in this context remains unclear.
It should not go unnoticed that NUDT5 deletion also led to nearly complete depletion of meTIMP, the thiopurine metabolite responsible for DNPS inhibition (31, 32). These data raise the question of whether thiopurines exert their cytotoxic effects by being incorporated into nucleic acids and causing DNA mismatch (historically considered the major toxic pathway), or by inhibiting DNPS, previously deemed to be a minor pathway (33). The fact that NUDT5 KO also led to resistance to TG (which does not affect DNPS) points to DNA damage as the main cause of thiopurine-induced cell death in our model systems. Nonetheless, NUDT5 clearly contributed to thiopurine activation, regardless of the exact pathways leading to cytotoxicity. Thiopurine cytotoxicity and the rate of DNPS both vary substantially by ALL subtype (34, 35), but this is not likely to be related to NUDT5 because its expression is relatively consistent across ALL subtypes (St. Jude Cloud, ALL expression dataset of 925 cases: https://pecan.stjude.cloud/expression/gene-expression), However, we cannot completely rule out the subtype-dependent effects of NUDT5 on thiopurine metabolism.
Our comprehensive analysis of NUDT5 genetics in children with ALL suggests that increased expression of NUDT5 may be linked to reduced tolerance to thiopurines. In this context, higher enzymatic activity would be associated with increased thiopurine activation, leading to elevated TGNs and a higher risk of hematopoietic toxicity. For example, 3 of the 4 patients homozygous at rs55713253 (TT genotype) required a greater than 50% dose reduction, with a median DI lower than that in patients with the CT genotype, although this difference did not reach statistical significance plausibly because of our limited sample size. Therefore, we reason that NUDT5 variants may have a sizable effect on thiopurine tolerance but that the effects seem more variable than what we have seen with NUDT15 and TPMT variants. Assuming an additive allelic effect, TPMT variants (rs1142345, rs1800462, rs1800460) were estimated to explain 4.9% of the interpatient variability in DI in our study cohort, whereas NUDT15 rs116855232 would explain 6.1% of it. By comparison, the NUDT5 variant (rs55713253) explains an additional 1.8% variability in this phenotype. Larger cohorts will be needed in the future to define the precise clinical relevance of the NUDT5 genotype in thiopurine dosing. Finally, our findings also suggest that somatic loss-of-function mutations in NUDT5 could lead to thiopurine resistance, potentially reducing the efficacy of ALL therapy. However, when we queried whole-genome or whole-exome sequencing data on 2,754 ALL cases, we found no NUDT5 mutations in leukemia geno
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