The NUDIX hydrolase NUDT5 regulates thiopurine metabolism and cytotoxicity

Abstract

Thiopurines 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.

Keywords: Genetics; Oncology; Pharmacogenetics; Therapeutics.

Figure 1. NUDIX-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 (TG_Day7) compared with control (No treat_Day7). (C) Volcano plot showing enrichment or depletion of the NUDIX genes in CRISPR/Cas9-transduced cells after 7 days of culturing (No treat_Day7) compared with day 0 of culturing (i.e., immediately after lentiviral transduction [No treat_Day0]), 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.

Figure 2. NUDT5 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 NUDT5^KO and parental cell lines. (C and D) NUDT5^KO Nalm6 cell sensitivity to TG and MP, respectively, after 72 hours of treatment. (E and F) NUDT5^KO 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.

Figure 3. NUDT5 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 NUDT5^KO 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.

Figure 4. Targeted 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 NUDT5^KO cells. (D) Levels of the purine intermediates hypoxanthine, guanosine, AICAR, and IMP in Nalm6 parental and NUDT5^KO 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.

Figure 5. Stable 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 NUDT5^KO 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 NUDT5^KO 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.

Figure 6. Characterization 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.