Increased DNA-incorporated thiopurine metabolite as a possible mechanism for leukocytopenia through cell apoptosis in inflammatory bowel disease patients with NUDT15 mutation.

BACKGROUND AND AIMS: Polymorphisms in the nucleotide diphosphate-linked moiety X-type motif 15 (NUDT15) gene are associated with thiopurine-induced leukopenia in patients with inflammatory bowel disease (IBD). NUDT15-associated subcellular thiopurine metabolism has not been investigated in primary lymphocytes. We hypothesized that NUDT15 mutation increases DNA-incorporated deoxythioguanosine (dTG) and induces apoptosis in lymphocytes. METHODS: DNA-incorporated dTG in peripheral blood mononuclear cells (PBMCs) and 6-thioguanine nucleotides (6-TGN) in red blood cells were measured in patients with IBD undergoing thiopurine treatment. The association of a single nucleotide polymorphism for NUDT15 (rs116855232) with dTGPBMC was examined. The pro-apoptotic effect of DNA-incorporated dTG was examined ex vivo in association with NUDT15 genotypes by co-culturing patient-derived peripheral CD4+ T lymphocytes with 6-thioguanine (6-TG). RESULTS: dTGPBMC was significantly higher in NUDT15 variants than in non-variants. dTGPBMC, but not 6-TGNRBC, negatively correlated with peripheral lymphocyte counts (r = - 0.31 and - 0.12, p = 0.012 and 0.173, respectively). DNA-incorporated dTG significantly accumulated to a greater extent in lymphocytes from NUDT15 variants when co-cultured with 6-TG ex vivo than in those from non-variants and was associated with decreased proliferation and increased apoptosis. CONCLUSION: Increased DNA-incorporated dTG may be responsible for thiopurine-induced leukocytopenia through cell apoptosis in IBD patients with NUDT15 mutation.

Three Faces of Mercaptopurine Cytotoxicity In Vitro: Methylation, Nucleotide Homeostasis, and Deoxythioguanosine in DNA.

Mercaptopurine (MP) is a cytotoxic thiopurine important for the treatment of cancer and autoimmune diseases. MP and other thiopurine drugs undergo extensive intracellular metabolism, but the mechanisms of action are poorly characterized. In particular, it is unknown how different metabolites contribute to cytotoxicity and incorporation of thiopurine bases into DNA. The aim of this study was to ask whether cytotoxicity results from the incorporation of thioguanosine nucleotides into DNA, an alternative thiopurine metabolite, or a combination of factors. Therefore, we measured the cytotoxicity, metabolism, and incorporation of thioguanosine into DNA in response to MP or MP metabolites. Thiopurine metabolites varied in cytotoxicity, with methyl-thioinosine-mono-phosphate and thioguanosine-tri-phosphate the most toxic, and the methyl-thioguanosine nucleotides the least. We show, using liquid chromatography-tandem mass spectrometry, how different metabolites may perturb biochemical pathways, particularly disrupting guanosine nucleotide homeostasis, that may contribute to the mechanism of action of thiopurines. Although there was no correlation between metabolite cytotoxicity and the levels of 6-methylthioinosine-mono-phosphate or thioguanosine incorporation into DNA as individual factors, a combined analysis suggested that these factors together had a major influence on cytotoxicity. This study emphasizes the importance of enzymes of nucleotide homeostasis, methylation, and demethylation in thiopurine effects. These results will facilitate the development of dynamic biochemical models of thiopurine biochemistry that will improve our understanding of mechanisms of action in relevant target tissues.

Metabolites of endophytic fungi from Australian native plants as potential anticancer agents.

Interest in endophytes as natural sources for new medicines was inspired by the discovery of paclitaxel-producing endophytic fungi. This study investigated the anti-cancer activity of extracts of endophytes isolated from two Australian plants, Eremophila longifolia (EL) and Eremophila maculata (EM). Endophytes were isolated from surface-sterilised leaf tissue, grown as pure cultures and identified by sequencing of Internal Transcribed Spacer (ITS) regions of the ribosomal DNA. To determine cytotoxicity, two leukaemic (MOLT-4, T-cell leukaemia; PreB-697, Pre-B leukaemia), a lung cancer cell line (A549) and a normal human fibroblast cell line were treated with endophyte extracts to assess cytotoxicity in relation to alternariol monomethyl ether (AME) and alternariol (AOH). Endophyte extracts that showed cell cytotoxicity were analysed by UV-HPLC to determine the metabolites. Pure AME and AOH, three extracts form Alternaria sp. (EM-6, EM-7 and EM-9) and one from Preussia minima (EL-14) were cytotoxic to the cancer cell lines. All cytotoxic endophytes contained AME and AOH, the most cytotoxic endophyte EM-6 also contained two unique peaks. These data indicate that these four endophyte extracts may have anti-cancer properties due to the presence of AME and AOH; however, the unique compounds found in the EM-6 extract may be exclusively cytotoxic and warrant further investigation.

Azathioprine with Allopurinol: Lower Deoxythioguanosine in DNA and Transcriptome Changes Indicate Mechanistic Differences to Azathioprine Alone.

BACKGROUND: Use of azathioprine (AZA) for inflammatory bowel disease is limited by side effects or poor efficacy. Combining low-dose azathioprine with allopurinol (LDAA) bypasses side effects, improves efficacy, and may be appropriate as first-line therapy. We test the hypothesis that standard-dose azathioprine (AZA) and LDAA treatments work by similar mechanisms, using incorporation of the metabolite deoxythioguanosine into patient DNA, white-blood cell counts, and transcriptome analysis as biological markers of drug effect. METHODS: DNA was extracted from peripheral whole-blood from patients with IBD treated with AZA or LDAA, and analyzed for DNA-incorporated deoxythioguanosine. Measurement of red-blood cell thiopurine metabolites was part of usual clinical practice, and pre- and on-treatment (12 wk) blood samples were used for transcriptome analysis. RESULTS: There were no differences in reduction of white-cell counts between the 2 treatment groups, but patients on LDAA had lower DNA-incorporated deoxythioguanosine than those on AZA; for both groups, incorporated deoxythioguanosine was lower in patients on thiopurines for 24 weeks or more (maintenance of remission) compared to patients treated for less than 24 weeks (achievement of remission). Patients on LDAA had higher levels of red-blood cell thioguanine nucleotides than those on AZA, but there was no correlation between these or their methylated metabolites, and incorporated deoxythioguanosine. Transcriptome analysis suggested down-regulation of immune responses consistent with effective immunosuppression in patients receiving LDAA, with evidence for different mechanisms of action between the 2 therapies. CONCLUSIONS: LDAA is biologically effective despite lower deoxythioguanosine incorporation into DNA, and has different mechanisms of action compared to standard-dose azathioprine.

Association of Liver Injury From Specific Drugs, or Groups of Drugs, With Polymorphisms in HLA and Other Genes in a Genome-Wide Association Study.

BACKGROUND & AIMS: We performed a genome-wide association study (GWAS) to identify genetic risk factors for drug-induced liver injury (DILI) from licensed drugs without previously reported genetic risk factors. METHODS: We performed a GWAS of 862 persons with DILI and 10,588 population-matched controls. The first set of cases was recruited before May 2009 in Europe (n = 137) and the United States (n = 274). The second set of cases were identified from May 2009 through May 2013 from international collaborative studies performed in Europe, the United States, and South America. For the GWAS, we included only cases with patients of European ancestry associated with a particular drug (but not flucloxacillin or amoxicillin-clavulanate). We used DNA samples from all subjects to analyze HLA genes and single nucleotide polymorphisms. After the discovery analysis was concluded, we validated our findings using data from 283 European patients with diagnosis of DILI associated with various drugs. RESULTS: We associated DILI with rs114577328 (a proxy for A*33:01 a HLA class I allele; odds ratio [OR], 2.7; 95% confidence interval [CI], 1.9-3.8; P = 2.4 × 10-8) and with rs72631567 on chromosome 2 (OR, 2.0; 95% CI, 1.6-2.5; P = 9.7 × 10-9). The association with A*33:01 was mediated by large effects for terbinafine-, fenofibrate-, and ticlopidine-related DILI. The variant on chromosome 2 was associated with DILI from a variety of drugs. Further phenotypic analysis indicated that the association between DILI and A*33:01 was significant genome wide for cholestatic and mixed DILI, but not for hepatocellular DILI; the polymorphism on chromosome 2 was associated with cholestatic and mixed DILI as well as hepatocellular DILI. We identified an association between rs28521457 (within the lipopolysaccharide-responsive vesicle trafficking, beach and anchor containing gene) and only hepatocellular DILI (OR, 2.1; 95% CI, 1.6-2.7; P = 4.8 × 10-9). We did not associate any specific drug classes with genetic polymorphisms, except for statin-associated DILI, which was associated with rs116561224 on chromosome 18 (OR, 5.4; 95% CI, 3.0-9.5; P = 7.1 × 10-9). We validated the association between A*33:01 terbinafine- and sertraline-induced DILI. We could not validate the association between DILI and rs72631567, rs28521457, or rs116561224. CONCLUSIONS: In a GWAS of persons of European descent with DILI, we associated HLA-A*33:01 with DILI due to terbinafine and possibly fenofibrate and ticlopidine. We identified polymorphisms that appear to be associated with DILI from statins, as well as 2 non-drug-specific risk factors.

Liquid chromatography-mass spectrometry for measuring deoxythioguanosine in DNA from thiopurine-treated patients.

Adverse reactions and non-response are common in patients treated with thiopurine drugs. Current monitoring of drug metabolite levels for guiding treatment are limited to analysis of thioguanine nucleotides (TGNs) in erythrocytes after chemical derivatisation. Erythrocytes are not the target tissue and TGN levels show poor correlations with clinical response. We have developed a sensitive assay to quantify deoxythioguanosine (dTG) without derivatisation in the DNA of nucleated blood cells. Using liquid chromatography and detection by tandem mass spectrometry, an intra- and inter-assay variability below 7.8% and 17.0% respectively were achieved. The assay had a detection limit of 0.0003125ng (1.1 femtomoles) dTG and was quantified in DNA samples relative to endogenous deoxyadenosine (dA) in a small group of 20 patients with inflammatory bowel disease, all of whom had been established on azathioprine (AZA) therapy for more than 25 weeks. These patients had dTG levels of 20-1360mol dTG/10(6)mol dA; three patients who had not started therapy had no detectable dTG. This method, comparable to previous methods in sensitivity, enables the direct detection of a cytotoxic thiopurine metabolite without derivatisation in an easily obtainable, stable sample and will facilitate a better understanding of the mechanisms of action of these inexpensive yet effective drugs.

HLA-DRB1*16: 01-DQB1*05: 02 is a novel genetic risk factor for flupirtine-induced liver injury.

OBJECTIVE: Flupirtine is a nonopioid analgesic with regulatory approval in a number of European countries. Because of the risk of serious liver injury, its use is now limited to short-term pain management. We aimed to identify genetic risk factors for flupirtine-related drug-induced liver injury (DILI) as these are unknown. MATERIALS AND METHODS: Six flupirtine-related DILI patients from Germany were included in a genome-wide association study (GWAS) involving a further 614 European cases of DILI because of other drugs and 10,588 population controls. DILI was diagnosed by causality assessment and expert review. Human leucocyte antigen (HLA) and single nucleotide polymorphism genotypes were imputed from the GWAS data, with direct HLA typing performed on selected cases to validate HLA predictions. Four replication cases that were unavailable for the GWAS were genotyped by direct HLA typing, yielding an overall total of 10 flupirtine DILI cases. RESULTS: In the six flupirtine DILI cases included in the GWAS, we found a significant enrichment of the DRB1*16:01-DQB1*05:02 haplotype compared with the controls (minor allele frequency cases 0.25 and minor allele frequency controls 0.013; P=1.4 × 10(-5)). We estimated an odds ratio for haplotype carriers of 18.7 (95% confidence interval 2.5-140.5, P=0.002) using population-specific HLA control data. The result was replicated in four additional cases, also with a haplotype frequency of 0.25. In the combined cohort (six GWAS plus four replication cases), the haplotype was also significant (odds ratio 18.7, 95% confidence interval 4.31-81.42, P=6.7 × 10(-5)). CONCLUSION: We identified a novel HLA class II association for DILI, confirming the important contribution of HLA genotype towards the risk of DILI generally.

Nomenclature for alleles of the thiopurine methyltransferase gene.

The drug-metabolizing enzyme thiopurine methyltransferase (TPMT) has become one of the best examples of pharmacogenomics to be translated into routine clinical practice. TPMT metabolizes the thiopurines 6-mercaptopurine, 6-thioguanine, and azathioprine, drugs that are widely used for treatment of acute leukemias, inflammatory bowel diseases, and other disorders of immune regulation. Since the discovery of genetic polymorphisms in the TPMT gene, many sequence variants that cause a decreased enzyme activity have been identified and characterized. Increasingly, to optimize dose, pretreatment determination of TPMT status before commencing thiopurine therapy is now routine in many countries. Novel TPMT sequence variants are currently numbered sequentially using PubMed as a source of information; however, this has caused some problems as exemplified by two instances in which authors' articles appeared on PubMed at the same time, resulting in the same allele numbers given to different polymorphisms. Hence, there is an urgent need to establish an order and consensus to the numbering of known and novel TPMT sequence variants. To address this problem, a TPMT nomenclature committee was formed in 2010, to define the nomenclature and numbering of novel variants for the TPMT gene. A website (http://www.imh.liu.se/tpmtalleles) serves as a platform for this work. Researchers are encouraged to submit novel TPMT alleles to the committee for designation and reservation of unique allele numbers. The committee has decided to renumber two alleles: nucleotide position 106 (G>A) from TPMT*24 to TPMT*30 and position 611 (T>C, rs79901429) from TPMT*28 to TPMT*31. Nomenclature for all other known alleles remains unchanged.

Poly(ADP-ribose) polymerase-1 (PARP-1) pharmacogenetics, activity and expression analysis in cancer patients and healthy volunteers.

There is a wide inter-individual variation in PARP-1 {PAR [poly(ADP-ribose)] polymerase 1} activity, which may have implications for health. We investigated if the variation: (i) is due to polymorphisms in the PARP-1 gene or PARP-1 protein expression; and (ii) affects patients' response to anticancer treatment. We studied 56 HV (healthy volunteers) and 118 CP (cancer patients) with supporting in vivo experiments. PARP activity ranged between 10 and 2600 pmol of PAR/106 cells and expression between 0.02-1.55 ng of PARP-1/µg of protein. PARP-1 expression correlated with activity in HV (R2=0.19, P=0.003) and CP (R2=0.06, P=0.01). A short CA repeat in the promoter was significantly associated with increased cancer risk [OR (odds ratio), 5.22; 95% CI (confidence interval), 1.79-15.24]. PARP activity was higher in men than women (P=0.04) in the HV. Male mice also had higher PARP activity than females or castrated males. Oestrogen supplementation activated PARP in PBMCs (peripheral blood mononuclear cells) from female mice (P=0.003), but inhibited PARP-1 in their livers by 80%. PARP activity and expression were not dependent on the investigated polymorphisms, but there was a modest correlation of PARP activity with expression. Studies in the HV revealed sex differences in PARP activity, which was confirmed in mice and shown to be associated with sex hormones. Toxic response to treatment was not associated with PARP activity and/or expression.

Increased sensitivity to thiopurines in methylthioadenosine phosphorylase-deleted cancers.

The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG), are used in the treatment of leukemia. Incorporation of deoxythioguanosine nucleotides (dG(s)) into the DNA of thiopurine-treated cells causes cell death, but there is also evidence that thiopurine metabolites, particularly the 6-MP metabolite methylthioinosine monophosphate (MeTIMP), inhibit de novo purine synthesis (DNPS). The toxicity of DNPS inhibitors is influenced by methylthioadenosine phosphorylase (MTAP), a gene frequently deleted in cancers. Because the growth of MTAP-deleted tumor cells is dependent on DNPS or hypoxanthine salvage, we would predict such cells to show differential sensitivity to 6-MP and 6-TG. To test this hypothesis, sensitivity to 6-MP and 6-TG was compared in relation to MTAP status using cytotoxicity assays in two MTAP-deficient cell lines transfected to express MTAP: the T-cell acute lymphoblastic leukemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP(-)) transfected to express MTAP constitutively (A549-MTAP(+)). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intracellularly to MeTIMP, was markedly higher in both cell lines under MTAP(-) conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dG(s) incorporation is the main cause of cytotoxicity with 6-TG. These data suggest that thiopurines, particularly 6-MP, may be more effective in patients with deleted MTAP.

Thiopurines: factors influencing toxicity and response.

Thiopurines are the backbone of current anti-leukemia regimens and have also been effective immunosuppressive agents for the past half a century. Extensive research on their mechanism of action has been undertaken, yet many issues remain to be addressed to resolve unexplained cases of thiopurine toxicity or treatment failure. The aim of this review is to summarize current knowledge of the mechanism of thiopurine action in experimental models and put into context with clinical observations. Clear understanding of their metabolism will contribute to maximizing efficacy and minimizing toxicity by individually tailoring therapy according to the expression profile of relevant factors involved in thiopurine activation pathway.

Thiopurine methyltransferase genetics is not a major risk factor for secondary malignant neoplasms after treatment of childhood acute lymphoblastic leukemia on Berlin-Frankfurt-Münster protocols.

Thiopurine methyltransferase (TPMT)is involved in the metabolism of thiopurines such as 6-mercaptopurine and 6-thioguanine. TPMT activity is significantly altered by genetics, and heterozygous and even more homozygous variant people reveal substiantially decreased TPMT activity. Treatment for childhood acute lymphoblastic leukemia (ALL) regularly includes the use of thiopurine drugs. Importantly, childhood ALL patients with low TPMT activity have been considered to be at increased risk of developing therapy-associated acute myeloid leukemia and brain tumors. In the present study, we genotyped 105 of 129 patients who developed a secondary malignant neoplasm after ALL treatment on 7 consecutive German Berlin-Frankfurt-Münster trials for all functionally relevant TPMT variants. Frequencies of TPMT variants were similarly distributed in secondary malignant neoplasm patients and the overall ALL patient population of 814 patients. Thus, TPMT does not play a major role in the etiology of secondary malignant neoplasm after treatment for childhood ALL, according to Berlin-Frankfurt-Münster strategies.

Chemotherapy individualization.

The current practice of dosing patients with anticancer drugs based on body size, leads to a large degree of interpatient variation in clinical outcome following standard doses of chemotherapy. Some patients may fail to respond to treatment, whilst others experience unacceptable side effects. Recent studies have identified more rational approaches to drug dosing, based on patient characteristics such as renal function, pharmacogenetic factors, and drug metabolizing activity. These can be used together with therapeutic drug monitoring and adaptive dosing to achieve a targeted systemic drug exposure in each patient, which may lead to more consistent clinical outcomes in patients receiving comparable chemotherapy dosing regimens. The purpose of this review is to present some approaches to chemotherapy individualization, examples of how this might be applied, and speculation as to how recent advances in pharmacogenetics may lead to further dose-optimization. Whilst it is hoped that the design of new agents, targeted to specific genes involved in oncogenesis, will lead to increased success in the treatment of cancer patients, it is essential that the drugs currently available are used to their maximum potential.

Molecular diagnostics: a healthcare perspective.

Recent progress in the development of molecular diagnostics in medicine has been rapid and the hope has been expressed by some people that it will soon be possible to have a detailed 'genetic readout' to assist in the diagnosis of treatment of a variety of diseases. However, such an outcome may be neither achievable nor desirable when viewed from the perspective of suppliers of healthcare.

The effect of thiopurine methyltransferase expression on sensitivity to thiopurine drugs.

Although the thiopurine drugs 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are well established agents for the treatment of leukemia, controversies remain regarding their main mode of action. Previous evidence has suggested that although 6-TG exerts a cytotoxic effect through incorporation of 6-thioguanine nucleotides into newly synthesized DNA (DNA-TGN), an important component of the mode of action of 6-MP is inhibition of purine de novo synthesis (PDNS) through the production of S-methyl-thioinosine 5'-monophosphate (MeTIMP), not formed in cells exposed to 6-TG. We have shown that thiopurine methyltransferase (TPMT) modulates this effect. By transfection of the human TPMT gene using an inducible system to produce a 3.8-fold increase in TPMT activity in the ecdysone receptor 293 embryonic kidney cell line, we demonstrated a 4.4-fold increase in sensitivity to 6-MP. This was associated with a rise in intracellular levels of MeTIMP but a decrease in levels of DNA-TGN. In contrast, induction of TPMT produced a 1.6-fold decrease in sensitivity to 6-TG, a decrease in levels of DNA-TGN, and an increase in levels of methylated thioguanosine monophosphate. Exposure of cells to equitoxic doses of drug showed similar incorporation of DNA-TGN for 6-TG but for 6-MP significantly reduced DNA-TGN in TPMT-induced compared with uninduced cells. For equitoxic doses of 6-MP, equivalent levels of MeTIMP correlated with equivalent amounts of PDNS. These observations suggest that intracellular TGN levels do not give an accurate reflection of cytotoxic potential in patients treated with 6-MP, because different levels of DNA-TGN may be associated with equitoxic effects.