Personalization of thiopurine therapy: Current recommendations and future perspectives.

Despite great therapeutic advances in the field of biologics, small synthetic molecules such as thiopurines, including azathioprine, mercaptopurine, and thioguanine, remain an important therapeutic pillar in the treatment of inflammatory bowel disease, other autoimmune disorders, and cancer. This review presents the latest guidelines for thiopurine administration, highlighting the importance of individualized therapy guided by pharmacogenomics. It emphasizes dose adjustment based on nudix hydrolase 15 (NUDT15) and thiopurine S-methyltransferase (TPMT) genotype, along side thiopurine S-methyltransferase activity and thiopurine metabolic profile. In addition, the article takes a critical look at emerging research in the field of thiopurine pharmaco genomics featuring novel genetic markers and technological developments in genetic testing. Finally, the potential of integrated approaches that combine genetic, meta bolic, and clinical factors to further individualize thiopurine therapy is highlighted.

Association of TPMT and NUDT15 gene polymorphisms with azathioprine-induced leukopenia: A case-control study in Eastern India.

BACKGROUND: Azathioprine (AZA) is a widely used immunosuppressant drug. Leukopenia is a serious adverse effect of the drug which often necessitates dose reduction or drug withdrawal. Predictors of leukopenia include genetic and nongenetic factors. Genetic polymorphism of AZA-metabolizing enzyme, thiopurine S-methyltransferase (TPMT) is well established. There is inconclusive evidence about the role of Nudix hydrolase (NUDT15) gene polymorphism. This case-control study assessed the association of genetic polymorphisms of NUDT15 and TPMT with leukopenia induced by AZA. MATERIALS AND METHODS: Cases were patients on AZA who developed leukopenia (white blood cell count <4000/µl) within 1 year of treatment initiation that necessitated dose reduction or drug withdrawal. Age and gender-matched patients without leukopenia within 1 year of treatment with AZA served as controls. TPMT (3 loci: c238G to C, c460G to A, c719A to G) and NUDT15 (c 415C to T, rs116855232) genotyping were done using TPMT strip assay and polymerase chain reaction-restriction fragment length polymorphism, respectively. Genotype frequencies were noted, and the odds ratio was calculated to determine the association between genotypes and leukopenia. RESULTS: Twenty-nine subjects (15 cases and 14 controls) were enrolled. Statistically significant differences were not observed in the TPMT genotype (*1/*1 and *1/*3C) (P = 0.23) between cases and controls. NUDT15 genotypes (*1/*1 and *1/*3) (P = 0.65) also showed no statistically significant difference between cases and controls. CONCLUSION: The above genotypes do not appear to be associated with AZA-induced leukopenia in an eastern Indian population.

USP9X regulates the proliferation, survival, migration and invasion of gastric cancer cells by stabilizing MTH1.

BACKGROUND: MutT homolog 1 (MTH1) sanitizes oxidized dNTP pools to promote the survival of cancer cells and its expression is frequently upregulated in cancers. Polyubiquitination stabilizes MTH1 to facilitate the proliferation of melanoma cells, suggesting the ubiquitin system controls the stability and function of MTH1. However, whether ubiquitination regulates MTH1 in gastric cancers has not been well defined. This study aims to investigate the interaction between MTH1 and a deubiquitinase, USP9X, in regulating the proliferation, survival, migration, and invasion of gastric cancer cells. METHODS: The interaction between USP9X and MTH1 was evaluated by co-immunoprecipitation (co-IP) in HGC-27 gastric cancer cells. siRNAs were used to interfere with USP9X expression in gastric cancer cell lines HGC-27 and MKN-45. MTT assays were carried out to examine the proliferation, propidium iodide (PI) and 7-AAD staining assays were performed to assess the cell cycle, Annexin V/PI staining assays were conducted to examine the apoptosis, and transwell assays were used to determine the migration and invasion of control, USP9X-deficient, and USP9X-deficient plus MTH1-overexpressing HGC-27 and MKN-45 gastric cancer cells. RESULTS: Co-IP data show that USP9X interacts with and deubiquitinates MTH1. Overexpression of USP9X elevates MTH1 protein level by downregulating its ubiquitination, while knockdown of USP9X has the opposite effect on MTH1. USP9X deficiency in HGC-27 and MKN-45 cells causes decreased proliferation, cell cycle arrest, extra apoptosis, and defective migration and invasion, which could be rescued by excessive MTH1. CONCLUSION: USP9X interacts with and stabilizes MTH1 to promote the proliferation, survival, migration and invasion of gastric cancer cells.

Innovating Thiopurine Therapeutic Drug Monitoring: A Systematic Review and Meta-Analysis on DNA-Thioguanine Nucleotides (DNA-TG) as an Inclusive Biomarker in Thiopurine Therapy.

BACKGROUND AND OBJECTIVE: Thioguanine (TG), azathioprine (AZA), and mercaptopurine (MP) are thiopurine prodrugs commonly used to treat diseases, such as leukemia and inflammatory bowel disease (IBD). 6-thioguanine nucleotides (6-TGNs) have been commonly used for monitoring treatment. High levels of 6-TGNs in red blood cells (RBCs) have been associated with leukopenia, the cutoff levels that predict this side effect remain uncertain. Thiopurines are metabolized and incorporated into leukocyte DNA. Measuring levels of DNA-incorporated thioguanine (DNA-TG) may be a more suitable method for predicting clinical response and toxicities such as leukopenia. Unfortunately, most methodologies to assay 6-TGNs are unable to identify the impact of NUDT15 variants, effecting mostly ethnic populations (e.g., Chinese, Indian, Malay, Japanese, and Hispanics). DNA-TG tackles this problem by directly measuring thioguanine in the DNA, which can be influenced by both TPMT and NUDT15 variants. While RBC 6-TGN concentrations have traditionally been used to optimize thiopurine therapy due to their ease and affordability of measurement, recent developments in liquid chromatography-tandem mass spectrometry (LC-MS/MS) techniques have made measuring DNA-TG concentrations in lymphocytes accurate, reproducible, and affordable. The objective of this systematic review was to assess the current evidence of DNA-TG levels as marker for thiopurine therapy, especially with regards to NUDT15 variants. METHODS: A systematic review and meta-analysis were performed on the current evidence for DNA-TG as a marker for monitoring thiopurine therapy, including methods for measurement and the illustrative relationship between DNA-TG and various gene variants (such as TPMT, NUDT15, ITPA, NT5C2, and MRP4). PubMed and Embase were systematically searched up to April 2024 for published studies, using the keyword "DNA-TG" with MeSH terms and synonyms. The electronic search strategy was augmented by a manual examination of references cited in articles, recent reviews, editorials, and meta-analyses. A meta-analysis was performed using R studio 4.1.3. to investigate the difference between the coefficients (Fisher's z-transformed correlation coefficient) of DNA-TG and 6-TGNs levels. A meta-analysis was performed using RevMan version 5.4 to investigate the difference in DNA-TG levels between patients with or without leukopenia using randomized effect size model. The risk of bias was assessed using the Newcastle-Ottowa quality assessment scale. RESULTS: In this systematic review, 21 studies were included that measured DNA-TG levels in white blood cells for either patients with ALL (n = 16) or IBD (n = 5). In our meta-analysis, the overall mean difference between patients with leukopenia (ALL + IBD) versus no leukopenia was 134.15 fmol TG/µg DNA [95% confidence interval (CI) (83.78-184.35), P < 0.00001; heterogeneity chi squared of 5.62, I2 of 47%]. There was a significant difference in DNA-TG levels for patients with IBD with and without leukopenia [161.76 fmol TG/µg DNA; 95% CI (126.23-197.29), P < 0.00001; heterogeneity chi squared of 0.20, I2 of 0%]. No significant difference was found in DNA-TG level between patients with ALL with or without leukopenia (57.71 fmol TG/µg DNA [95% CI (- 22.93 to 138.35), P < 0.80]). DNA-TG monitoring was found to be a promising method for predicting relapse rates in patients with ALL, and DNA-TG levels are likely a better predictor for leukopenia in patients with IBD than RBC 6-TGNs levels. DNA-TG levels have been shown to correlate with various gene variants (TPMT, NUDT15, ITPA, and MRP4) in various studies, points to its potential as a more informative marker for guiding thiopurine therapy across diverse genetic backgrounds. CONCLUSIONS: This systematic review strongly supports the further investigation of DNA-TG as a marker for monitoring thiopurine therapy. Its correlation with treatment outcomes, such as relapse-free survival in ALL and the risk of leukopenia in IBD, underscores its role in enhancing personalized treatment approaches. DNA-TG effectively identifies NUDT15 variants and predicts late leukopenia in patients with IBD, regardless of their NUDT15 variant status. The recommended threshold for late leukopenia prediction in patients with IBD with DNA-TG is suggested to be between 320 and 340 fmol/µg DNA. More clinical research on DNA-TG implementation is mandatory to improve patient care and to improve inclusivity in thiopurine treatment.

Influence of TPMT and NUDT15 Genetic Polymorphisms on Mercaptopurine Pharmacokinetics in Healthy Volunteers.

Aims: This study aimed to investigate the impact of genetic polymorphisms of thiopurine methyltransferase (TPMT) and NUDT15 on pharmacokinetics profile of mercaptopurine in healthy adults in China. Methods: Blood samples were obtained from 45 healthy adult volunteers who were administered azathioprine. Genomic DNA was extracted and sequenced for TPMT and NUDT15. The plasma concentrations of 6-mercaptopurine (6-MP) were determined by ultra-performance liquid chromatography-tandem mass spectrometry. Finally, pharmacokinetic parameters were calculated based on the time-concentration curve. Results: Among the 45 healthy adult volunteers enrolled in the study, two TPMT allelic variants and three NUDT15 allelic variants were detected. In total, six genotypes were identified, including TPMT*1/*1&NUDT15*1/*1, TPMT*1/*1&NUDT15*1/*2, TPMT*1/*1&NUDT15*1/*9, TPMT*1/*1&NUDT15*2/*5, TPMT*1/*6&NUDT15*1/*2, and TPMT*1/*3&NUDT15*1/*2. The results indicated that Area Under Curve (AUC) of 6-MP in volunteers with TPMT*1/*3&NUDT15*1/*2 and TPMT*1/*6&NUDT15*1/*2 were 1.57-1.62-fold higher than in individuals carrying the wild type (TPMT*1/*1&NUDT15*1/*1). Compared with wild type, the half-life (T1/2) of TPMT*1/*6&NUDT15*1/*2 was extended by 1.98 times, whereas T1/2 of TPMT*1/*3&NUDT15*1/*2 decreased by 67%. The maximum concentration (Cmax) of TPMT*1/*3&NUDT15*1/*2 increased significantly by 2.15-fold, whereas the corresponding clearance (CL/F) decreased significantly by 58.75%. Conclusion: The findings of this study corroborate the notion that various genotypes of TPMT and NUDT15 can impact the pharmacokinetics of mercaptopurine, potentially offering foundational insights for personalized mercaptopurine therapy.

NUDT15 Polymorphism and Its Association With Mercaptopurine Hematotoxicity in Acute Lymphoblastic Leukemia in Indonesian Children.

BACKGROUND/AIM: Hematotoxicity is a life-threatening condition that has become the major cause of drug discontinuation in patients with acute lymphoblastic leukemia (ALL). The nudix hydrolase 15 (NUDT15) gene polymorphism (c.415C>T) is reported to have an association with the hematotoxicity of 6-mercaptopurine (6-MP) as maintenance therapy in patients with ALL. However, the prevalence of this genetic polymorphism in the Indonesian population is unknown. This study aimed to assess the frequency of NUDT15 polymorphism among Indonesian pediatric patients with ALL and its association with the hematotoxicity of 6-MP. PATIENTS AND METHODS: A total of 101 stored DNA samples from pediatric patients with ALL receiving 6-MP treatment were used for genetic testing. Direct sequencing was conducted to determine the NUDT15 c.415C>T genotype. Chi-square or Fisher's exact test were employed to examine the association between the NUDT15 c.415C>T genotype and hematotoxicity. RESULTS: All (100%) of the DNA samples from patients with ALL treated with 6-MP exhibited a homozygous variant of the NUDT15 c.415C>T genotype, 70.3% of which showed hematotoxicity to some extent. We found no significant differences in NUDT15 gene polymorphism among patients with ALL with different states of hematotoxicity. CONCLUSION: The observed high frequency of NUDT15 c.415C>T in our study population might explain the elevated prevalence of 6-MP-associated hematotoxicity in pediatric patients with ALL within the Indonesian population. Our study provides new insight regarding the NUDT15 gene polymorphism and its relation to hematotoxicity. Further studies are required to determine the necessity of adjusting the initial dose of 6-MP for Indonesian pediatric patients with ALL.

Predictive role of ITPA genetic variants in thiopurine-related myelotoxicity in Crohn's disease patients.

Thiopurines, an effective therapy for Crohn's disease (CD), often lead to adverse events (AEs). Gene polymorphisms affecting thiopurine metabolism may predict AEs. This retrospective study in CD patients (n = 114) with TPMT activity > 5 Units/Red Blood Cells analyzed TPMT (c.238 G > C, c.460 G > A, c.719 A > G), ITPA (c.94 C > A, IVS2 + 21 A > C), and NUDT15 (c.415 C > T) polymorphisms. All patients received azathioprine (median dose 2.2 mg/kg) with 41.2% experiencing AEs, mainly myelotoxicity (28.1%). No NUDT15 polymorphisms were found, 7% had TPMT, and 31.6% had ITPA polymorphisms. AEs led to therapy modifications in 41.2% of patients. Multivariate analysis identified advanced age (OR 1.046, p = 0.007) and ITPA IVS2 + 21 A > C (OR 3.622, p = 0.015) as independent predictors of AEs. IVS2 + 21 A > C was also associated with myelotoxicity (OR 2.863, p = 0.021). These findings suggest that ITPA IVS2 + 21 A > C polymorphism and advanced age predict AEs during thiopurine therapy for CD with intermediate-normal TPMT activity.

Activities and genetic interactions of fission yeast Aps1, a Nudix-type inositol pyrophosphatase and inorganic polyphosphatase.

Inositol pyrophosphate 1,5-IP8 regulates expression of a fission yeast phosphate homeostasis regulon, comprising phosphate acquisition genes pho1, pho84, and tgp1, via its action as an agonist of precocious termination of transcription of the upstream lncRNAs that repress PHO mRNA synthesis. 1,5-IP8 levels are dictated by a balance between the Asp1 N-terminal kinase domain that converts 5-IP7 to 1,5-IP8 and three inositol pyrophosphatases-the Asp1 C-terminal domain (a histidine acid phosphatase), Siw14 (a cysteinyl-phosphatase), and Aps1 (a Nudix enzyme). In this study, we report the biochemical and genetic characterization of Aps1 and an analysis of the effects of Asp1, Siw14, and Aps1 mutations on cellular inositol pyrophosphate levels. We find that Aps1's substrate repertoire embraces inorganic polyphosphates, 5-IP7, 1-IP7, and 1,5-IP8. Aps1 displays a ~twofold preference for hydrolysis of 1-IP7 versus 5-IP7 and aps1∆ cells have twofold higher levels of 1-IP7 vis-à-vis wild-type cells. While neither Aps1 nor Siw14 is essential for growth, an aps1∆ siw14∆ double mutation is lethal on YES medium. This lethality is a manifestation of IP8 toxicosis, whereby excessive 1,5-IP8 drives derepression of tgp1, leading to Tgp1-mediated uptake of glycerophosphocholine. We were able to recover an aps1∆ siw14∆ mutant on ePMGT medium lacking glycerophosphocholine and to suppress the severe growth defect of aps1∆ siw14∆ on YES by deleting tgp1. However, the severe growth defect of an aps1∆ asp1-H397A strain could not be alleviated by deleting tgp1, suggesting that 1,5-IP8 levels in this double-pyrophosphatase mutant exceed a threshold beyond which overzealous termination affects other genes, which results in cytotoxicity. IMPORTANCE: Repression of the fission yeast PHO genes tgp1, pho1, and pho84 by lncRNA-mediated interference is sensitive to changes in the metabolism of 1,5-IP8, a signaling molecule that acts as an agonist of precocious lncRNA termination. 1,5-IP8 is formed by phosphorylation of 5-IP7 and catabolized by inositol pyrophosphatases from three distinct enzyme families: Asp1 (a histidine acid phosphatase), Siw14 (a cysteinyl phosphatase), and Aps1 (a Nudix hydrolase). This study entails a biochemical characterization of Aps1 and an analysis of how Asp1, Siw14, and Aps1 mutations impact growth and inositol pyrophosphate pools in vivo. Aps1 catalyzes hydrolysis of inorganic polyphosphates, 5-IP7, 1-IP7, and 1,5-IP8 in vitro, with a ~twofold preference for 1-IP7 over 5-IP7. aps1∆ cells have twofold higher levels of 1-IP7 than wild-type cells. An aps1∆ siw14∆ double mutation is lethal because excessive 1,5-IP8 triggers derepression of tgp1, leading to toxic uptake of glycerophosphocholine.

Frequency of pharmacogenomic variants affecting efficacy and safety of anti-cancer drugs in a south Asian population from Sri Lanka.

BACKGROUND: Therapy with anti-cancer drugs remain the cornerstone of treating cancer. The effectiveness and safety of anti-cancer drugs vary significantly among individuals due to genetic factors influencing the drug response and metabolism. Data on the pharmacogenomic variations in Sri Lankans related to anti-cancer therapy is sparse. As current treatment guidelines in Sri Lanka often do not consider local pharmacogenomic variants, this study aimed to explore the diversity of pharmacogenomic variants in the Sri Lankan population to pave the way for personalized treatment approaches and improve patient outcomes. METHODS: Pharmacogenomic data regarding variant-drug pairs of genes CYP2D6, DPYD, NUDT15, EPAS1, and XRCC1 with clinical annotations labelled as evidence levels 1A-2B were obtained from the Pharmacogenomics Knowledgebase database. Their frequencies in Sri Lankans were obtained from an anonymized database that was derived from 541 Sri Lankans who underwent exome sequencing at the Human Genetics Unit, Faculty of Medicine, University of Colombo. Variations in DPYD, NUDT15, and EPAS1 genes are related to increased toxicity to fluoropyrimidines, mercaptopurines, and sorafenib respectively. Variations in CYP2D6 and XRCC1 genes are related to changes in efficacy of tamoxifen and platinum compounds, respectively. Minor allele frequencies of these variants were calculated and compared with other populations. RESULTS: MAFs of rs1065852 c.100 C > T (CYP2D6), rs3918290 c.1905 + 1G > A (DPYD), rs56038477 c.1236G > A (DPYD), rs7557402 c.1035-7 C > G (EPAS1), rs116855232 c.415 C > T (NUDT15*3), and rs25487 c.1196 A > G (XRCC1) were: 12.9% [95%CI:10.9-14.9], 1.5% [95%CI:0.8-2.2], 1.2% [95%CI:0.5-1.8], 37.7% [95%CI:34.8-40.6], 8.3% [95%CI:6.7-10.0], and 64.0% [95%CI:61.1-66.8], respectively. Frequencies of rs1065852 c.100 C > T (CYP2D6), rs7557402 c.1035-7 C > G (EPAS1), and rs25487 (XRCC1) were significantly lower in Sri Lankans, while frequencies of rs116855232 c.415 C > T (NUDT15*3) and rs56038477 c.1236G > A (DPYD) were significantly higher in Sri Lankans when compared to some Western and Asian populations. CONCLUSION: Sri Lankans are likely to show lower toxicity risk with sorafenib (rs7557402 c.84,131 C > G) and, higher toxicity risk with fluoropyrimidines (rs56038477 c.1236G > A) and mercaptopurine (rs116855232 c.415 C > T), and reduced effectiveness with tamoxifen (rs1065852 c.100 C > T) and platinum compounds (rs25487). These findings highlight the potential contribution of these genetic variations to the individual variability in anti-cancer dosage requirements among Sri Lankans.

Combined or High Dose Immunosuppression in Inflammatory Bowel Disease patients who are NUDT15 Homozygous Harbors Risk of Developing Invasive Fungal Infections: A Case Series.

NUDT15 homozygous mutations predispose patients to severe leucopenia, which invites risk of disseminated fungal infections when high doses or a combination of immunosuppressives are administered in this patient population.

Real-world NUDT15 genotyping and thiopurine treatment optimization in inflammatory bowel disease: a multicenter study.

BACKGROUND: This study evaluated the effectiveness of NUDT15 codon 139 genotyping in optimizing thiopurine treatment for inflammatory bowel disease (IBD) in Japan, using real-world data, and aimed to establish genotype-based treatment strategies. METHODS: A retrospective analysis of 4628 IBD patients who underwent NUDT15 codon 139 genotyping was conducted. This study assessed the purpose of the genotyping test and subsequent prescriptions following the obtained results. Outcomes were compared between the Genotyping group (thiopurine with genotyping test) and Non-genotyping group (thiopurine without genotyping test). Risk factors for adverse events (AEs) were analyzed by genotype and prior genotyping status. RESULTS: Genotyping test for medical purposes showed no significant difference in thiopurine induction rates between Arg/Arg and Arg/Cys genotypes, but nine Arg/Cys patients opted out of thiopurine treatment. In the Genotyping group, Arg/Arg patients received higher initial doses than the Non-genotyping group, while Arg/Cys patients received lower ones (median 25 mg/day). Fewer AEs occurred in the Genotyping group because of their lower incidence in Arg/Cys cases. Starting with < 25 mg/day of AZA reduced AEs in Arg/Cys patients, while Arg/Arg patients had better retention rates when maintaining ≥ 75 mg AZA. Nausea and liver injury correlated with thiopurine formulation but not dosage. pH-dependent mesalamine reduced leukopenia risk in mesalamine users. CONCLUSIONS: NUDT15 codon 139 genotyping effectively reduces thiopurine-induced AEs and improves treatment retention rates in IBD patients after genotype-based dose adjustments. This study provides data-driven treatment strategies based on genotype and identifies risk factors for specific AEs, contributing to a refined thiopurine treatment approach.

Novel variant in Nudix hydrolase 15 gene influences 6-mercaptopurine toxicity in childhood acute lymphoblastic leukemia patients.

Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer. 6-Mercaptopurine (6-MP) is a key component of ALL treatment. Its use, however, is also associated with adverse drug reactions, particularly myelosuppression. Thiopurine S-methyltransferase (TPMT) and, more recently, Nudix hydrolase 15 (NUDT15) deficiency, due to no-function variants in their respective genes, are well known for their role in the development of this toxicity. Two novel genetic variants, rs12199316 in TPMT and rs73189762 in the NUDT15 gene, were recently identified by targeted sequencing. The latter is particularly interesting because of its potential association with 6-MP intolerance. Here, we assessed the relationship of this variant with the risk of myelosuppression and 6-MP dose intensity in 275 patients treated with Dana Farber Cancer Institute ALL protocols at the Sainte Justine University Health Center. Carriers of the NUDT15 rs73189762 variant allele were at a higher risk of myelosuppression, as shown by absolute phagocyte count reduction during consolidation II and maintenance phases of therapy. Reduction in 6-MP dose intensity was observed in patients with both rs73189762 and known no-function variants in the NUDT15 and TPMT genes. This finding supports the initial observation and suggests that 6-MP dose reduction might be beneficial for individuals with this genotype combination.

Therapeutic targeting nudix hydrolase 1 creates a MYC-driven metabolic vulnerability.

Tumor cells must rewire nucleotide synthesis to satisfy the demands of unbridled proliferation. Meanwhile, they exhibit augmented reactive oxygen species (ROS) production which paradoxically damages DNA and free deoxy-ribonucleoside triphosphates (dNTPs). How these metabolic processes are integrated to fuel tumorigenesis remains to be investigated. MYC family oncoproteins coordinate nucleotide synthesis and ROS generation to drive the development of numerous cancers. We herein perform a Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-based functional screen targeting metabolic genes and identified nudix hydrolase 1 (NUDT1) as a MYC-driven dependency. Mechanistically, MYC orchestrates the balance of two metabolic pathways that act in parallel, the NADPH oxidase 4 (NOX4)-ROS pathway and the Polo like kinase 1 (PLK1)-NUDT1 nucleotide-sanitizing pathway. We describe LC-1-40 as a potent, on-target degrader that depletes NUDT1 in vivo. Administration of LC-1-40 elicits excessive nucleotide oxidation, cytotoxicity and therapeutic responses in patient-derived xenografts. Thus, pharmacological targeting of NUDT1 represents an actionable MYC-driven metabolic liability.

Nudix hydrolase 23 post-translationally regulates carotenoid biosynthesis in plants.

Carotenoids are essential for photosynthesis and photoprotection. Plants must evolve multifaceted regulatory mechanisms to control carotenoid biosynthesis. However, the regulatory mechanisms and the regulators conserved among plant species remain elusive. Phytoene synthase (PSY) catalyzes the highly regulated step of carotenogenesis and geranylgeranyl diphosphate synthase (GGPPS) acts as a hub to interact with GGPP-utilizing enzymes for the synthesis of specific downstream isoprenoids. Here, we report a function of Nudix hydrolase 23 (NUDX23), a Nudix domain-containing protein, in post-translational regulation of PSY and GGPPS for carotenoid biosynthesis. NUDX23 expresses highly in Arabidopsis (Arabidopsis thaliana) leaves. Overexpression of NUDX23 significantly increases PSY and GGPPS protein levels and carotenoid production, whereas knockout of NUDX23 dramatically reduces their abundances and carotenoid accumulation in Arabidopsis. NUDX23 regulates carotenoid biosynthesis via direct interactions with PSY and GGPPS in chloroplasts, which enhances PSY and GGPPS protein stability in a large PSY-GGPPS enzyme complex. NUDX23 was found to co-migrate with PSY and GGPPS proteins and to be required for the enzyme complex assembly. Our findings uncover a regulatory mechanism underlying carotenoid biosynthesis in plants and offer promising genetic tools for developing carotenoid-enriched food crops.

Nudix hydrolase WvNUDX24 is involved in borneol biosynthesis in Wurfbainia villosa.

Borneol, camphor, and bornyl acetate are highly promising monoterpenoids widely used in medicine, flavor, food, and chemical applications. Bornyl diphosphate (BPP) serves as a common precursor for the biosynthesis of these monoterpenoids. Although bornyl diphosphate synthase (BPPS) that catalyzes the cyclization of geranyl diphosphate (GPP) to BPP has been identified in multiple plants, the enzyme responsible for the hydrolysis of BPP to produce borneol has not been reported. Here, we conducted in vitro and in vivo functional characterization to identify the Nudix hydrolase WvNUDX24 from W. villosa, which specifically catalyzes the hydrolysis of BPP to generate bornyl phosphate (BP), and then BP forms borneol under the action of phosphatase. Subcellular localization experiments indicated that the hydrolysis of BPP likely occurs in the cytoplasm. Furthermore, site-directed mutagenesis experiments revealed that four critical residues (R84, S96, P98, and G99) for the hydrolysis activity of WvNUDX24. Additionally, the functional identification of phosphatidic acid phosphatase (PAP) demonstrated that WvPAP5 and WvPAP10 were able to hydrolyze geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate (FPP) to generate geranylgeranyl phosphate (GGP) and farnesyl phosphate (FP), respectively, but could not hydrolyze BPP, GPP, and neryl diphosphate (NPP) to produce corresponding monophosphate products. These findings highlight the essential role of WvNUDX24 in the first step of BPP hydrolysis to produce borneol and provide genetic elements for the production of BPP-related terpenoids through plant metabolic engineering and synthetic biology.

The first case of intellectual disability caused by novel compound heterozygosity for NUDT2 variants.

NUDT2 is an enzyme important for maintaining the intracellular level of the diadenosine tetraphosphate (Ap4A). Bi-allelic loss of function variants in NUDT2 has recently been reported as a rare cause of intellectual disability (ID). Herein, we describe a Chinese girl with ID, attention deficit hyperactivity disorder (ADHD), and motor delays with abnormal walking posture and difficulty climbing stairs, who bears compound heterozygous variants c.34 C > T (p.R12*) and c.194T > G (p.I65R) in NUDT2. Homozygous variants c.34 C > T (p.R12*) or c.186del (p.A63Qfs*3) in NUDT2 were previously reported to cause ID. This is the first patient with ID due to compound heterozygous variants in NUDT2 and p.I65R is a novel missense variant. This study enriched the genotype and phenotype of NUDT2-related ID and supported the critical developmental involvement of NUDT2.

Measurement of the intracellular active metabolites of thiopurine drugs to evaluate the enzymatic activity of nudix hydrolase 15 in human blood samples.

Thiopurine is metabolized to 6-thio-(deoxy) guanosine triphosphate (6-thio-(d) GTP), which is then incorporated into DNA or RNA and causes cytotoxicity. Nudix hydrolase 15 (NUDT15) reduces the cytotoxic effects of thiopurine by converting 6-thio-(d) GTP to 6-thio-(d) guanosine monophosphate (6-thio-(d) GMP). NUDT15 polymorphisms like the Arg139Cys variant are strongly linked to thiopurine-induced severe leukocytopenia and alopecia. Therefore, measurement of NUDT15 enzymatic activity in individual patients can help predict thiopurine tolerability and adjust the dosage. We aimed to develop a quantitative assay for NUDT15 enzymatic activity in human blood samples. Blood samples were collected from donors whose NUDT15 genetic status was determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the 6-thio-GTP metabolic activity in cell extracts. Because 6-thio-guanosine diphosphate (6-thio-GDP) and 6-thio-GMP were generated upon incubation of 6-thio-GTP with human blood cell extracts, the method detecting 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP was validated. All three metabolites were linearly detected, and the lower limit of quantification (LLOQ) of 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 5 µM, 1 µM, and 2 µM, respectively. Matrix effects of human blood cell extracts to detect 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 99.0 %, 100.5 %, and 101.4 %, respectively, relative to the signals in the absence of blood cell extracts. The accuracy and precision of the method and the stability of the samples were also assessed. Using this established method, the genotype-dependent differences in NUDT15 activities were successfully determined using cell extracts derived from human blood cells with NUDT15 wild-type (WT) or Arg139Cys variant and 6-thio-GTP (100 µM) as a substrate (18.1, 14.9, and 6.43 µM/h/106 cells for WT, Arg139Cys heterozygous, and homozygous variant, respectively). We developed a method for quantifying intracellular NUDT15 activity in peripheral blood mononuclear cells (PBMCs), which we defined as the conversion of 6-thio-GTP to 6-thio-GMP. Although PBMCs preparation takes some time, its reproducibility in experiments makes it a promising candidate for clinical application. This method can tell the difference between WT and Arg139Cys homozygous blood samples. Even in patients with WT NUDT15, WT samples showed variations in NUDT15 activity, which may correlate with variations in thiopurine dosage.

Additive effects of TPMT and NUDT15 on thiopurine toxicity in children with acute lymphoblastic leukemia across multiethnic populations.

BACKGROUND: Thiopurines such as mercaptopurine (MP) are widely used to treat acute lymphoblastic leukemia (ALL). Thiopurine-S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15) inactivate thiopurines, and no-function variants are associated with drug-induced myelosuppression. Dose adjustment of MP is strongly recommended in patients with intermediate or complete loss of activity of TPMT and NUDT15. However, the extent of dosage reduction recommended for patients with intermediate activity in both enzymes is currently not clear. METHODS: MP dosages during maintenance were collected from 1768 patients with ALL in Singapore, Guatemala, India, and North America. Patients were genotyped for TPMT and NUDT15, and actionable variants defined by the Clinical Pharmacogenetics Implementation Consortium were used to classify patients as TPMT and NUDT15 normal metabolizers (TPMT/NUDT15 NM), TPMT or NUDT15 intermediate metabolizers (TPMT IM or NUDT15 IM), or TPMT and NUDT15 compound intermediate metabolizers (TPMT/NUDT15 IM/IM). In parallel, we evaluated MP toxicity, metabolism, and dose adjustment using a Tpmt/Nudt15 combined heterozygous mouse model (Tpmt+/-/Nudt15+/-). RESULTS: Twenty-two patients (1.2%) were TPMT/NUDT15 IM/IM in the cohort, with the majority self-reported as Hispanics (68.2%, 15/22). TPMT/NUDT15 IM/IM patients tolerated a median daily MP dose of 25.7 mg/m2 (interquartile range = 19.0-31.1 mg/m2), significantly lower than TPMT IM and NUDT15 IM dosage (P < .001). Similarly, Tpmt+/-/Nudt15+/- mice displayed excessive hematopoietic toxicity and accumulated more metabolite (DNA-TG) than wild-type or single heterozygous mice, which was effectively mitigated by a genotype-guided dose titration of MP. CONCLUSION: We recommend more substantial dose reductions to individualize MP therapy and mitigate toxicity in TPMT/NUDT15 IM/IM patients.

Diadenosine Tetraphosphate (Ap4 A) Serves as a 5' RNA Cap in Mammalian Cells.

The recent expansion of the field of RNA chemical modifications has changed our understanding of post-transcriptional gene regulation. Apart from internal nucleobase modifications, 7-methylguanosine was long thought to be the only eukaryotic RNA cap. However, the discovery of non-canonical RNA caps in eukaryotes revealed a new niche of previously undetected RNA chemical modifications. We are the first to report the existence of a new non-canonical RNA cap, diadenosine tetraphosphate (Ap4 A), in human and rat cell lines. Ap4 A is the most abundant dinucleoside polyphosphate in eukaryotic cells and can be incorporated into RNA by RNA polymerases as a non-canonical initiating nucleotide (NCIN). Using liquid chromatography-mass spectrometry (LC-MS), we show that the amount of capped Ap4 A-RNA is independent of the cellular concentration of Ap4 A. A decapping enzyme screen identifies two enzymes cleaving Ap4 A-RNA,NUDT2 and DXO, both of which also cleave other substrate RNAs in vitro. We further assess the translatability and immunogenicity of Ap4 A-RNA and show that although it is not translated, Ap4 A-RNA is recognized as self by the cell and does not elicit an immune response, making it a natural component of the transcriptome. Our findings open a previously unexplored area of eukaryotic RNA regulation.

Biallelic NUDT2 variants defective in mRNA decapping cause a neurodevelopmental disease.

Dysfunctional RNA processing caused by genetic defects in RNA processing enzymes has a profound impact on the nervous system, resulting in neurodevelopmental conditions. We characterized a recessive neurological disorder in 18 children and young adults from 10 independent families typified by intellectual disability, motor developmental delay and gait disturbance. In some patients peripheral neuropathy, corpus callosum abnormalities and progressive basal ganglia deposits were present. The disorder is associated with rare variants in NUDT2, a mRNA decapping and Ap4A hydrolysing enzyme, including novel missense and in-frame deletion variants. We show that these NUDT2 variants lead to a marked loss of enzymatic activity, strongly implicating loss of NUDT2 function as the cause of the disorder. NUDT2-deficient patient fibroblasts exhibit a markedly altered transcriptome, accompanied by changes in mRNA half-life and stability. Amongst the most up-regulated mRNAs in NUDT2-deficient cells, we identified host response and interferon-responsive genes. Importantly, add-back experiments using an Ap4A hydrolase defective in mRNA decapping highlighted loss of NUDT2 decapping as the activity implicated in altered mRNA homeostasis. Our results confirm that reduction or loss of NUDT2 hydrolase activity is associated with a neurological disease, highlighting the importance of a physiologically balanced mRNA processing machinery for neuronal development and homeostasis.