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"An antineoplastic compound which also has antimetabolite action. The drug is used in the therapy of acute leukemia." (MeSH 2013)

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Web Resources: Tioguanine
Latest Research Publications

Web Resources: Tioguanine (6 links)

Latest Research Publications

This list of publications is regularly updated (Source: PubMed).

Jeanbart L, Kourtis IC, van der Vlies AJ, et al.
6-Thioguanine-loaded polymeric micelles deplete myeloid-derived suppressor cells and enhance the efficacy of T cell immunotherapy in tumor-bearing mice.
Cancer Immunol Immunother. 2015; 64(8):1033-46 [PubMed] Free Access to Full Article Related Publications
Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid cells that suppress effector T cell responses and can reduce the efficacy of cancer immunotherapies. We previously showed that ultra-small polymer nanoparticles efficiently drain to the lymphatics after intradermal injection and target antigen-presenting cells, including Ly6c(hi) Ly6g(-) monocytic MDSCs (Mo-MDSCs), in skin-draining lymph nodes (LNs) and spleen. Here, we developed ultra-small polymer micelles loaded with 6-thioguanine (MC-TG), a cytotoxic drug used in the treatment of myelogenous leukemia, with the aim of killing Mo-MDSCs in tumor-bearing mice and thus enhancing T cell-mediated anti-tumor responses. We found that 2 days post-injection in tumor-bearing mice (B16-F10 melanoma or E.G7-OVA thymoma), MC-TG depleted Mo-MDSCs in the spleen, Ly6c(lo) Ly6g(+) granulocytic MDSCs (G-MDSCs) in the draining LNs, and Gr1(int) Mo-MDSCs in the tumor. In both tumor models, MC-TG decreased the numbers of circulating Mo- and G-MDSCs, as well as of Ly6c(hi) macrophages, for up to 7 days following a single administration. MDSC depletion was dose dependent and more effective with MC-TG than with equal doses of free TG. Finally, we tested whether this MDSC-depleting strategy might enhance cancer immunotherapies in the B16-F10 melanoma model. We found that MC-TG significantly improved the efficacy of adoptively transferred, OVA-specific CD8(+) T cells in melanoma cells expressing OVA. These findings highlight the capacity of MC-TG in depleting MDSCs in the tumor microenvironment and show promise in promoting anti-tumor immunity when used in combination with T cell immunotherapies.

Tedeschi PM, Kathari YK, Johnson-Farley N, Bertino JR
Methylthioadenosine phosphorylase (MTAP)-deficient T-cell ALL xenografts are sensitive to pralatrexate and 6-thioguanine alone and in combination.
Cancer Chemother Pharmacol. 2015; 75(6):1247-52 [PubMed] Free Access to Full Article Related Publications
PURPOSE: To investigate the effectiveness of a combination of 6-thioguanine (6-TG) and pralatrexate (PDX) in methylthioadenosine phosphorylase (MTAP)-deficient T-cell acute lymphoblastic leukemia (T-cell ALL).
METHODS: CCRF-CEM (MTAP(-/-)) and Molt4 (MTAP(+/+)) T-cell ALL cell lines were treated with 6-TG or PDX and evaluated for efficacy 72 h later. NOD/SCID gamma mice bearing CEM or Molt4 xenografts were treated with 6-TG and PDX alone or in combination to evaluate antitumor effects.
RESULTS: CEM cells were more sensitive to 6-TG and PDX in vitro than Molt4. In vivo, CEM cells were very sensitive to PDX and 6-TG, whereas Molt4 cells were highly resistant to 6-TG. A well-tolerated combination of PDX and 6-TG achieved significant tumor regression in CEM xenografts.
CONCLUSIONS: The loss of MTAP expression may be therapeutically exploited in T-cell ALL. The combination of 6-TG and PDX, with the inclusion of leucovorin rescue, allows for a safe and effective regimen in MTAP-deficient T-cell ALL.

Nielsen SN, Frandsen TL, Nersting J, et al.
Pharmacokinetics of 6-Thioguanine and 6-Mercaptopurine Combination Maintenance Therapy of Childhood ALL: Hypothesis and Case Report.
J Pediatr Hematol Oncol. 2015; 37(3):e206-9 [PubMed] Related Publications
Methotrexate/6-mercaptopurine maintenance therapy of childhood acute lymphoblastic leukemia is challenged by treatment-related hepatotoxicity, failure to achieve the myelosuppressive target, and lack of direct parameters for monitoring treatment efficacy or even intensity. Patients with low thiopurine methyltransferase (TPMT) activity have lower levels of hepatotoxic methylated thiopurine metabolites (MeMPs), higher levels of thioguanine nucleotides (TGNs), and reduced relapse rates. Addition of 6-thioguanine to maintenance therapy of a child with ALL and high TPMT activity increased the TGN/MeMP index in erythrocytes 5.5-fold, mimicking the more favorable thiopurine metabolism seen in patients with low TPMT activity.

Munshi PN, Lubin M, Bertino JR
6-thioguanine: a drug with unrealized potential for cancer therapy.
Oncologist. 2014; 19(7):760-5 [PubMed] Free Access to Full Article Related Publications
Sixty years ago, 6-thioguanine (6-TG) was introduced into the clinic. We suggest its full potential in therapy may not have been reached. In this paper, we contrast 6-TG and the more widely used 6-mercaptopurine; discuss 6-TG metabolism, pharmacokinetics, dosage and schedule; and summarize many of the early studies that have shown infrequent but nevertheless positive results with 6-TG treatment of cancers. We also consider studies that suggest that combinations of 6-TG with other agents may enhance antitumor effects. Although not yet tested in man, 6-TG has recently been proposed to treat a wide variety of cancers with a high frequency of homozygous deletion of the gene for methylthioadenosine phosphorylase (MTAP), often codeleted with the adjacent tumor suppressor CDKN2A (p16). Among the cancers with a high frequency of MTAP deficiency are leukemias, lymphomas, mesothelioma, melanoma, biliary tract cancer, glioblastoma, osteosarcoma, soft tissue sarcoma, neuroendocrine tumors, and lung, pancreatic, and squamous cell carcinomas. The method involves pretreatment with the naturally occurring nucleoside methylthioadenosine (MTA), the substrate for the enzyme MTAP. MTA pretreatment protects normal host tissues, but not MTAP-deficient cancers, from 6-TG toxicity and permits administration of doses of 6-TG that are much higher than can now be safely administered. The combination of MTA/6-TG has produced substantial shrinkage or slowing of growth in two different xenograft human tumor models: lymphoblastic leukemia and metastatic prostate carcinoma with neuroendocrine features. Further development and a clinical trial of the proposed MTA/6-TG treatment of MTAP-deficient cancers seem warranted.

Wray L, Vujkovic M, McWilliams T, et al.
TPMT and MTHFR genotype is not associated with altered risk of thioguanine-related sinusoidal obstruction syndrome in pediatric acute lymphoblastic leukemia: a report from the Children's Oncology Group.
Pediatr Blood Cancer. 2014; 61(11):2086-8 [PubMed] Free Access to Full Article Related Publications
Sinusoidal obstruction syndrome is a complication of therapy for pediatric ALL and may be modified by thiopurine methyltransferase activity as well as by MTHFR genotype. We assessed TPMT *3A, *3B, *3C, and MTHFR C677T and A1298C germline genetic polymorphisms among 351 patients enrolled in the thioguanine treatment arm of CCG-1952 clinical trial. TPMT and MTHFR C677T genotypes were not associated with SOS risk. The combination of MTHFR and TPMT variant genotypes was not associated with SOS risk. These suggest that germline genetic variation in TPMT and MTHFR do not significantly alter SOS risk in patients exposed to thioguanine.

Matimba A, Li F, Livshits A, et al.
Thiopurine pharmacogenomics: association of SNPs with clinical response and functional validation of candidate genes.
Pharmacogenomics. 2014; 15(4):433-47 [PubMed] Free Access to Full Article Related Publications
AIM: We investigated candidate genes associated with thiopurine metabolism and clinical response in childhood acute lymphoblastic leukemia.
MATERIALS & METHODS: We performed genome-wide SNP association studies of 6-thioguanine and 6-mercaptopurine cytotoxicity using lymphoblastoid cell lines. We then genotyped the top SNPs associated with lymphoblastoid cell line cytotoxicity, together with tagSNPs for genes in the 'thiopurine pathway' (686 total SNPs), in DNA from 589 Caucasian UK ALL97 patients. Functional validation studies were performed by siRNA knockdown in cancer cell lines.
RESULTS: SNPs in the thiopurine pathway genes ABCC4, ABCC5, IMPDH1, ITPA, SLC28A3 and XDH, and SNPs located within or near ATP6AP2, FRMD4B, GNG2, KCNMA1 and NME1, were associated with clinical response and measures of thiopurine metabolism. Functional validation showed shifts in cytotoxicity for these genes.
CONCLUSION: The clinical response to thiopurines may be regulated by variation in known thiopurine pathway genes and additional novel genes outside of the thiopurine pathway.

Gueranger Q, Li F, Peacock M, et al.
Protein oxidation and DNA repair inhibition by 6-thioguanine and UVA radiation.
J Invest Dermatol. 2014; 134(5):1408-17 [PubMed] Related Publications
Damage to skin DNA by solar UV is largely unavoidable, and an optimal cellular response to it requires the coordinated operation of proteins in numerous pathways. A fully functional DNA repair proteome for removing harmful DNA lesions is a prerequisite for an appropriate DNA damage response. Genetically determined failure to repair UV-induced DNA damage is associated with skin photosensitivity and increased skin cancer risk. Patients treated with immunosuppressant/anti-inflammatory thiopurines are also photosensitive and have high rates of sun-related skin cancer. Their DNA contains the base analog 6-thioguanine (6-TG), which acts as a UVA photosensitizer to generate reactive oxygen species (ROS), predominantly singlet oxygen ((1)O2). ROS damage both DNA and proteins. Here we show that UVA irradiation of cultured human cells containing DNA 6-TG causes significant protein oxidation and damages components of the DNA repair proteome, including the Ku, OGG-1, MYH, and RPA proteins. Assays of DNA repair in intact cells or in cell extracts indicate that this protein damage compromises DNA break rejoining and base and nucleotide excision repair. As these experimental conditions simulate those in the skin of patients taking thiopurines, our findings suggest a mechanism whereby UVA in sunlight may contribute to skin carcinogenesis in immunosuppressed patients.

Karim H, Ghalali A, Lafolie P, et al.
Differential role of thiopurine methyltransferase in the cytotoxic effects of 6-mercaptopurine and 6-thioguanine on human leukemia cells.
Biochem Biophys Res Commun. 2013; 437(2):280-6 [PubMed] Related Publications
The thiopurine antimetabolites, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are inactive pro-drugs that require intracellular metabolism for activation to cytotoxic metabolites. Thiopurine methyltransferase (TPMT) is one of the most important enzymes in this process metabolizing both 6-MP and 6-TG to different methylated metabolites including methylthioinosine monophosphate (meTIMP) and methylthioguanosine monophosphate (meTGMP), respectively, with different suggested pharmacological and cytotoxic properties. While meTIMP is a potent inhibitor of de novo purine synthesis (DNPS) and significantly contributes to the cytotoxic effects of 6-MP, meTGMP, does not add much to the effects of 6-TG, and the cytotoxicity of 6-TG seems to be more dependent on incorporation of thioguanine nucleotides (TGNs) into DNA rather than inhibition of DNPS. In order to investigate the role of TPMT in metabolism and thus, cytotoxic effects of 6-MP and 6-TG, we knocked down the expression of the gene encoding the TPMT enzyme using specifically designed small interference RNA (siRNA) in human MOLT4 leukemia cells. The knock-down was confirmed at RNA, protein, and enzyme function levels. Apoptosis was determined using annexin V and propidium iodide staining and FACS analysis. The results showed a 34% increase in sensitivity of MOLT4 cells to 1μM 6-TG after treatment with TPMT-targeting siRNA, as compared to cells transfected with non-targeting siRNA, while the sensitivity of the cells toward 6-MP was not affected significantly by down-regulation of the TPMT gene. This differential contribution of the enzyme TPMT to the cytotoxicity of the two thiopurines is probably due to its role in formation of the meTIMP, the cytotoxic methylated metabolite of 6-MP, while in case of 6-TG methylation by TPMT substantially deactivates the drug.

Lennard L, Cartwright CS, Wade R, et al.
Thiopurine methyltransferase genotype-phenotype discordance and thiopurine active metabolite formation in childhood acute lymphoblastic leukaemia.
Br J Clin Pharmacol. 2013; 76(1):125-36 [PubMed] Free Access to Full Article Related Publications
AIMS: In children with acute lymphoblastic leukaemia (ALL) bone marrow activity can influence red blood cell (RBC) kinetics, the surrogate tissue for thiopurine methyltransferase (TPMT) measurements. The aim of this study was to investigate TPMT phenotype-genotype concordance in ALL, and the influence of TPMT on thiopurine metabolite formation.
METHODS: We measured TPMT (activity, as units ml(-1) packed RBCs and genotype) at diagnosis (n = 1150) and TPMT and thioguanine nucleotide (TGN) and methylmercaptopurine nucleotide (MeMPN) metabolites (pmol/8 × 10(8) RBCs) during chemotherapy (n = 1131) in children randomized to thioguanine or mercaptopurine on the United Kingdom trial ALL97.
RESULTS: Median TPMT activity at diagnosis (8.5 units) was significantly lower than during chemotherapy (13.8 units, median difference 5.1 units, 95% confidence interval (CI) 4.8, 5.4, P < 0.0001). At diagnosis genotype-phenotype was discordant. During chemotherapy the overall concordance was 92%, but this fell to 55% in the intermediate activity cohort (45% had wild-type genotypes). For both thiopurines TGN concentrations differed by TPMT status. For mercaptopurine, median TGNs were higher in TPMT heterozygous genotype (754 pmol) than wild-type (360 pmol) patients (median difference 406 pmol, 95% CI 332, 478, P < 0.0001), whilst median MeMPNs, products of the TPMT reaction, were higher in wild-type (10 650 pmol) than heterozygous patients (3868 pmol) (P < 0.0001). In TPMT intermediate activity patients with a wild-type genotype, TGN (median 366 pmol) and MeMPN (median 8590 pmol) concentrations were similar to those in wild-type, high activity patients.
CONCLUSIONS: In childhood ALL, TPMT activity should not be used to predict heterozygosity particularly in blood samples obtained at disease diagnosis. Genotype is a better predictor of TGN accumulation during chemotherapy.

Wareham NE, Heilmann C, Abrahamsson J, et al.
Outcome of poor response paediatric AML using early SCT.
Eur J Haematol. 2013; 90(3):187-94 [PubMed] Related Publications
BACKGROUND: Children with poor response acute myeloid leukaemia (AML) generally have a very poor outcome. Allogeneic stem cell transplantation (SCT) is often recommended for these children but the benefit is unclear. The aim of this study was to investigate survival for poor response AML patients treated with SCT.
MATERIAL AND METHODS: Treatment was given according to the NOPHO-AML 2004 protocol. All patients received AIET (Cytarabine, Idarubicin, Etoposide, Thioguanine) and AM (Cytarabine, Mitoxantrone) as induction. We included poor response defined as > 15% blasts on day 15 after AIET (n = 17) or > 5% blasts after AM (n = 14, refractory disease). Poor response patients received intensively timed induction and proceeded to SCT when a donor was available.
RESULTS: Thirty-one of 267 evaluable patients (12%) had a poor response. SCT was performed in 25; using matched unrelated donors in 13, matched sibling donors in 6, cord blood donor in 4, and haploidentical donor in two. The median follow-up for the 31 poor responding patients was 2.6 years (range 0.4 - 8.1 years) and 3-year probability of survival 70% (95% CI 59-77%).
CONCLUSIONS: The poor responders in the NOPHO-AML 2004 protocol had a favourable prognosis treated with time-intensive induction followed by SCT.

van der Vlies AJ, Hasegawa U, Hubbell JA
Reduction-sensitive tioguanine prodrug micelles.
Mol Pharm. 2012; 9(10):2812-8 [PubMed] Related Publications
Colloidal drug and prodrug conjugates have unique targeting characteristics for tumor vasculature from the blood and for the lymphatics draining a tissue injection site. Tioguanine and tioguanine-generating prodrugs have been investigated as anticancer and immunosuppressive agents, including use in cancer immunotherapy. Recently we developed block copolymers of poly(ethylene glycol)-bl-poly(propylene sulfide) that self-assemble in aqueous solutions to form micellar structures. Since the polymers carry a free terminal thiol group resulting from the ring-opening polymerization of the propylene sulfide monomer, we sought to prepare prodrug block copolymers with tioguanine linked by a reduction-sensitive disulfide bond. The synthesis involved a disulfide exchange between the oxidized form of tioguanine and the polymer. Spectroscopic data is presented to support the proposed reaction. The polymers self-assembled when dispersed in water to form tioguanine prodrug micelles with a size range between 18 and 40 nm that released tioguanine in response to cysteine and serum as shown spectroscopically. In comparison with a poly(ethylene glycol) prodrug polymer, we show that the rate of tioguanine release can be controlled by changing the poly(propylene sulfide) block length and that the tioguanine remains bioactive with cultured cells.

Tang B, Testa JR, Kruger WD
Increasing the therapeutic index of 5-fluorouracil and 6-thioguanine by targeting loss of MTAP in tumor cells.
Cancer Biol Ther. 2012; 13(11):1082-90 [PubMed] Free Access to Full Article Related Publications
Methylthioadenosine phosphorylase (MTAP), a key enzyme in the catabolism of 5'-deoxy-5'-methylthioadenosine (MTA), catalyzes the formation of adenine and 5-methylthioribose-1-phosphate. MTAP is expressed in all cells throughout the body, but a significant percentage of human tumors have lost MTAP expression, thereby making MTAP-loss a potential therapeutic target. Here, we have tested an MTAP-targeting strategy based on the idea that MTAP-expressing cells can be protected from toxic purine and uracil analogs by addition of MTA, but MTAP-deleted tumor cells cannot. Addition of as little as 10 μM MTA could entirely protect isogenic MTAP (+) , but not MTAP (-) , HT1080 cells from toxicity caused by the chemotherapy agents 6-thioguanine (6TG) or 5-fluorouracil (5FU). Inhibitor studies showed that MTA protection requires functional MTAP activity. Addition of adenine protected both MTAP (+) and MTAP (-) cells from 6TG and 5FU, consistent with the idea that adenine produced from the MTAP reaction competes with 6TG and 5FU for a rate limiting pool of phosphoribosyl-1-pyrophosphate (PRPP), which is required for the conversion of purine and uracil bases into nucleotides. Extracellular MTA can also protect mouse mesothelioma cells from killing by 6-TG or the drug L-alanosine in an MTAP-dependent manner. In addition, MTA can protect non-transformed MTAP (+) mouse embryo fibroblasts from 6TG toxicity. Taken together, our data suggest that the addition of MTA to anti-purine-based chemotherapy may greatly increase the therapeutic index of this class of drugs if used specifically to treat MTAP (-) tumors.

Jacobsen JH, Schmiegelow K, Nersting J
Liquid chromatography-tandem mass spectrometry quantification of 6-thioguanine in DNA using endogenous guanine as internal standard.
J Chromatogr B Analyt Technol Biomed Life Sci. 2012; 881-882:115-8 [PubMed] Related Publications
Thiopurines are S-substituted antimetabolites that are widely used in the treatment of hematological malignancies and as immunosuppressants. Because of extensive inter-individual variation in drug disposition and the significant toxicity associated with thiopurine therapy, there is a need for improved individualized treatment. We here present a fast and sensitive method for quantifying the pharmacological end-point of thiopurines, 6-thioguanine (TG) in chromosomal DNA. Purine nucleobases are released from DNA, etheno-derivatized with chloroacetaldehyde, separated by HILIC and quantified by tandem mass spectrometry using endogenous chromosomal guanine as internal standard. The method is linear up to at least 10 pmol TG/μg DNA and the limit of detection and quantification are 4.2 and 14.1 fmol TG/μg DNA, respectively. The matrix (DNA) had no effect upon quantification of TG. SPE recovery was estimated at 63% (RSD 26%), which is corrected for by the internal standard resulting in stable quantification. The TG levels found were above the LOQ in 18 out of 18 childhood leukemia patients on 6-mercaptopurine/methotrexate maintenance therapy (median 377, range 45-1190 fmol/μg DNA) with intra- and inter-day RSDs of less than 11%. The method uses 2 μg DNA/sample, which can easily be obtained from these patients.

Attard NR, Karran P
UVA photosensitization of thiopurines and skin cancer in organ transplant recipients.
Photochem Photobiol Sci. 2012; 11(1):62-8 [PubMed] Related Publications
The thiopurines azathioprine, 6-mercaptopurine and 6-thioguanine (6-TG) are important medications for cancer and inflammatory disorders. They are also widely prescribed as immunosuppressants in organ transplant patients. Their metabolism results in the incorporation of 6-TG into patients' DNA, and this increases skin sensitivity to incident UVA. Unlike the canonical DNA bases, which do not absorb UVA to a significant degree, DNA 6-TG is a strong UVA chromophore. It acts as a Type II UVA photosensitizer, and the combination of 6-TG and UVA treatment induces a synergistic toxicity in cultured human cells. Here, we review some of the damage that this interaction causes. Photochemical activation of DNA 6-TG triggers DNA and protein oxidation; it induces DNA breakage, DNA crosslinking, oxidation of DNA bases and the covalent attachment of proteins to DNA. Many of these photochemical DNA lesions are difficult for cells to deal with, and we review the evidence linking thiopurine immunosuppression with genome instability and the high incidence of skin cancer in organ transplant recipients.

Karim H, Hashemi J, Larsson C, et al.
The pattern of gene expression and gene dose profiles of 6-Mercaptopurine- and 6-Thioguanine-resistant human leukemia cells.
Biochem Biophys Res Commun. 2011; 411(1):156-61 [PubMed] Related Publications
Exposure of MOLT4 human T-cell leukemia cells to 6-Mercaptopurine (6-MP) and 6-Thioguanine (6-TG) resulted in acquired resistance associated with attenuated expression of the genes encoding concentrative nucleoside transporter 3 (CNT3) and equilibrative nucleoside transporter 2 (ENT2). To identify other alterations at the RNA and DNA levels associated with 6-MP- and 6-TG resistance, we compared here the patterns of gene expression and DNA copy number profiles of resistant sublines to those of the parental wild-type cells. The mRNA levels for two nucleoside transporters were down-regulated in both of the thiopurine-resistant sublines. Moreover, both of these cell lines expressed genes encoding the enzymes of purine nucleotide composition and synthesis, including adenylate kinase 3-like 1 and guanosine monophosphate synthetase at significantly lower levels than wild-type cells. In addition, expression of the mRNA for a specialized DNA polymerase, human terminal transferase encoded by the terminal deoxynucleotidyl transferase (DNTT) gene, was 122- and 93-fold higher in 6-TG- and 6-MP-resistant cells, respectively. The varying responses to 6-MP- and 6-TG observed here may help identify novel cellular targets and modalities of resistance to thiopurines, as well as indicating new potential approaches to individualization therapy with these drugs.

Rawat D, Gillett PM, Devadason D, et al.
Long-term follow-up of children with 6-thioguanine-related chronic hepatoxicity following treatment for acute lymphoblastic leukaemia.
J Pediatr Gastroenterol Nutr. 2011; 53(5):478-9 [PubMed] Related Publications
6-Thioguanine (6-TG) therapy in childhood acute lymphoblastic leukaemia results in chronic hepatotoxicity and portal hypertension. We report follow-up data in a cohort of 10 children with acute lymphoblastic leukaemia and 6-TG-related hepatotoxicity described initially in 2006. Clinically significant portal hypertension was present in the majority of patients several years after cessation of 6-TG treatment. These data reflect the natural history of noncirrhotic portal hypertension and emphasises the need to incorporate ongoing surveillance in the transition arrangement to adult services in this select group of patients.

Escherich G, Richards S, Stork LC, et al.
Meta-analysis of randomised trials comparing thiopurines in childhood acute lymphoblastic leukaemia.
Leukemia. 2011; 25(6):953-9 [PubMed] Free Access to Full Article Related Publications
Mercaptopurine has been used in continuing treatment of childhood acute lymphoblastic leukaemia since the mid 1950s. Recent advances in the understanding of thiopurine pharmacology indicated that thioguanine (TG) might be more effective than mercaptopurine (MP). The US and UK cooperative groups began randomised thiopurine trials and agreed prospectively to a meta-analysis. All randomised trials of TG versus MP were sought, and data on individual patients were analysed by standard methods. Combining three trials (from US, UK and Germany), the overall event-free survival (EFS) was not significantly improved with TG (odds ratio (OR)=0.89; 95% confidence interval 0.78-1.03). Apparent differences in results between trials may be partly explained by the different types of patients studied. The larger treatment effect reported in males in the US trial was confirmed in the other trials. There was heterogeneity between sex/age subgroups (P=0.001), with significant EFS benefit of TG only observed for males aged <10 years old (OR=0.70; 0.58-0.84), although this did not result in a significant difference in overall survival (OR=0.83; 0.62-1.10). Additional toxicity occurs with TG. Mercaptopurine remains the standard thiopurine of choice, but further study of TG may be warranted to determine whether it could benefit particular subgroups.

Coulthard SA, Redfern CP, Vikingsson S, et al.
Increased sensitivity to thiopurines in methylthioadenosine phosphorylase-deleted cancers.
Mol Cancer Ther. 2011; 10(3):495-504 [PubMed] Free Access to Full Article Related Publications
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.

Yuan B, Zhang J, Wang H, et al.
6-Thioguanine reactivates epigenetically silenced genes in acute lymphoblastic leukemia cells by facilitating proteasome-mediated degradation of DNMT1.
Cancer Res. 2011; 71(5):1904-11 [PubMed] Free Access to Full Article Related Publications
Thiopurines including 6-thioguanine ((S)G), 6-mercaptopurine, and azathioprine are effective anticancer agents with remarkable success in clinical practice, especially in effective treatment of acute lymphoblastic leukemia (ALL). (S)G is understood to act as a DNA hypomethylating agent in ALL cells, however, the underlying mechanism leading to global cytosine demethylation remains unclear. Here we report that (S)G treatment results in reactivation of epigenetically silenced genes in T leukemia cells. Bisulfite genomic sequencing revealed that (S)G treatment universally elicited demethylation in the promoters and/or first exons of the genes that were reactivated. (S)G treatment also attenuated the expression of histone lysine-specific demethylase 1 (LSD1), thereby stimulating lysine methylation of the DNA methylase DNMT1 and triggering its degradation via the ubiquitin-proteasomal pathway. Taken together, our findings reveal a previously uncharacterized but vital mechanistic link between (S)G treatment and DNA hypomethylation.

Yuan B, O'Connor TR, Wang Y
6-Thioguanine and S⁶-methylthioguanine are mutagenic in human cells.
ACS Chem Biol. 2010; 5(11):1021-7 [PubMed] Free Access to Full Article Related Publications
Thiopurines are effective immunosuppressants and anticancer agents. However, the long-term use of thiopurines was found to be associated with a significantly increased risk of various types of cancer. To date, the specific mechanism(s) underlying the carcinogenicity associated with thiopurine treatment remain(s) unclear. Herein, we constructed duplex pTGFP-Hha10 shuttle vectors carrying a 6-thioguanine ((S)G) or S⁶-methylthioguanine (S⁶mG) at a unique site and allowed the vectors to propagate in three different human cell lines. Analysis of the replication products revealed that although neither thionucleoside blocked considerably DNA replication in any of the human cell lines, both (S)G and S⁶mG were mutagenic, resulting in G→A mutation at frequencies of ~8% and ~39%, respectively. Consistent with what was found from our previous study in E. coli cells, our data demonstrated that the mutagenic properties of (S)G and S⁶mG provided significant evidence for mutation induction as a potential carcinogenic mechanism associated with chronic thiopurine intervention.

Issaeva N, Thomas HD, Djureinovic T, et al.
6-thioguanine selectively kills BRCA2-defective tumors and overcomes PARP inhibitor resistance.
Cancer Res. 2010; 70(15):6268-76 [PubMed] Free Access to Full Article Related Publications
Familial breast and ovarian cancers are often defective in homologous recombination (HR) due to mutations in the BRCA1 or BRCA2 genes. Cisplatin chemotherapy or poly(ADP-ribose) polymerase (PARP) inhibitors were tested for these tumors in clinical trials. In a screen for novel drugs that selectively kill BRCA2-defective cells, we identified 6-thioguanine (6TG), which induces DNA double-strand breaks (DSB) that are repaired by HR. Furthermore, we show that 6TG is as efficient as a PARP inhibitor in selectively killing BRCA2-defective tumors in a xenograft model. Spontaneous BRCA1-defective mammary tumors gain resistance to PARP inhibitors through increased P-glycoprotein expression. Here, we show that 6TG efficiently kills such BRCA1-defective PARP inhibitor-resistant tumors. We also show that 6TG could kill cells and tumors that have gained resistance to PARP inhibitors or cisplatin through genetic reversion of the BRCA2 gene. Although HR is reactivated in PARP inhibitor-resistant BRCA2-defective cells, it is not fully restored for the repair of 6TG-induced lesions. This is likely to be due to several recombinogenic lesions being formed after 6TG. We show that BRCA2 is also required for survival from mismatch repair-independent lesions formed by 6TG, which do not include DSBs. This suggests that HR is involved in the repair of 6TG-induced DSBs as well as mismatch repair-independent 6TG-induced DNA lesion. Altogether, our data show that 6TG efficiently kills BRCA2-defective tumors and suggest that 6TG may be effective in the treatment of advanced tumors that have developed resistance to PARP inhibitors or platinum-based chemotherapy.

Wang H, Wang Y
LC-MS/MS coupled with stable isotope dilution method for the quantification of 6-thioguanine and S(6)-methylthioguanine in genomic DNA of human cancer cells treated with 6-thioguanine.
Anal Chem. 2010; 82(13):5797-803 [PubMed] Free Access to Full Article Related Publications
Thiopurines, including mercaptopurine (MP), 6-thioguanine ((S)G) and azathioprine, are widely used for the treatment of many human diseases including acute lymphoblastic leukemia (ALL). To exert their cytotoxic effect, these prodrugs need to be metabolically activated to (S)G nucleotides and incorporated into nucleic acids. (S)G in DNA can be methylated spontaneously to S(6)-methylthioguanine (S(6)mG) in the presence of S-adenosyl-l-methionine. It was proposed that S(6)mG, owing to its high miscoding potential (pairing preferentially with thymine), may induce cell death by triggering the postreplicative mismatch repair pathway. Understanding the implications of this pathway in the cytotoxic effect of thiopurine drugs necessitates an accurate measurement of the level of S(6)-methylthio-2'-deoxyguanosine (S(6)mdG) in DNA of cells treated with thiopurine drugs. Here we developed a sensitive HPLC coupled with tandem mass spectrometry (LC-MS/MS) method and measured the level of 6-thio-2'-deoxyguanosine ((S)dG) and S(6)mdG in genomic DNA of four human leukemia cell lines and one human colorectal carcinoma cell line. Our results revealed that, upon treatment with 3 muM (S)G for 24 h, approximately 10, 7.4, 7, and 3% of guanine was replaced with (S)G in Jurkat T, HL-60, CCRF-CEM and K-562 cells, respectively. However, only less than 0.02% of (S)dG was converted to S(6)mdG in the above cell lines. HCT-116 cells had the lowest level (0.2%) of guanine being replaced with (S)G in DNA, and approximately 5 out of 10(4 S)G was converted to its methylated counterpart. This is the first report of the simultaneous and accurate quantification of (S)dG and S(6)mdG in genomic DNA of cultured human cells treated with (S)G. In addition, our results suggested that DNA (S)G might trigger mismatch repair (MMR) pathway without being converted to S(6)mG.

Heo J, Hong I
Ras-targeting action of thiopurines in the presence of reactive nitrogen species.
Biochemistry. 2010; 49(18):3965-76 [PubMed] Related Publications
Thiopurine drugs are commonly used in the treatment of certain cancers, autoimmune disorders, organ transplant rejection, and bowel disease. Because long-term treatment with thiopurines for certain diseases is common, the cytotoxic effects associated with chronic exposure to thiopurine drugs are inevitable. The results shown in this study indicate that the oncogenic Ras in model cancer cell lines forms a complex with thioguanine nucleotide that is derived from long-term treatment with thiopurines. This study also showed that the Ras thioguanine nucleotide binary complex is likely to be a direct target of a redox agent, resulting in downregulation of the oncogenic Ras. This study proposes a radical-based molecular mechanism for the path of Ras-targeting thiopurines used in conjunction with redox agents. Given that Ras plays a central role in cellular signaling pathways, any interference with Ras activity by thiopurines and redox agents has the potential for devastating cytotoxic effects.

Stork LC, Matloub Y, Broxson E, et al.
Oral 6-mercaptopurine versus oral 6-thioguanine and veno-occlusive disease in children with standard-risk acute lymphoblastic leukemia: report of the Children's Oncology Group CCG-1952 clinical trial.
Blood. 2010; 115(14):2740-8 [PubMed] Free Access to Full Article Related Publications
The Children's Cancer Group 1952 (CCG-1952) clinical trial studied the substitution of oral 6-thioguanine (TG) for 6-mercaptopurine (MP) and triple intrathecal therapy (ITT) for intrathecal methotrexate (IT-MTX) in the treatment of standard-risk acute lymphoblastic leukemia. After remission induction, 2027 patients were randomized to receive MP (n = 1010) or TG (n = 1017) and IT-MTX (n = 1018) or ITT (n = 1009). The results of the thiopurine comparison are as follows. The estimated 7-year event-free survival (EFS) for subjects randomized to TG was 84.1% (+/- 1.8%) and to MP was 79.0% (+/- 2.1%; P = .004 log rank), although overall survival was 91.9% (+/- 1.4%) and 91.2% (+/- 1.5%), respectively (P = .6 log rank). The TG starting dose was reduced from 60 to 50 mg/m(2) per day after recognition of hepatic veno-occlusive disease (VOD). A total of 257 patients on TG (25%) developed VOD or disproportionate thrombocytopenia and switched to MP. Once portal hypertension occurred, all subjects on TG were changed to MP. The benefit of randomization to TG over MP, as measured by EFS, was evident primarily in boys who began TG at 60 mg/m(2) (relative hazard rate [RHR] 0.65, P = .002). The toxicities of TG preclude its protracted use as given in this study. This study is registered at http://clinicaltrials.gov as NCT00002744.

Lee DH, Chung NG, Cho B, et al.
Idarubicin plus behenoyl cytarabine and 6-thioguanine compares favorably with idarubicin plus cytarabine-based regimen for children with previously untreated acute myeloid leukemia: 10-year retrospective, multicenter study in Korea.
J Korean Med Sci. 2010; 25(1):9-15 [PubMed] Free Access to Full Article Related Publications
We investigated the outcome of idarubicin plus N(4)-behenoyl-1-beta-D-arabinofuranosyl cytosine (BHAC)-based chemotherapy (BHAC group, n=149) compared to idarubicin plus cytarabine-based chemotherapy (cytarabine group, n=191) for childhood acute myeloid leukemia (AML). Between January 1996 and December 2005, 340 children with AML from 5 university hospitals in Korea received the BHAC-based or cytarabine-based chemotherapy, with or without hematopoietic stem cell transplantation. After induction therapy, 264 (77.6%) of 340 children achieved a complete remission (CR) and 43 (12%) achieved a partial remission (PR). The CR rate in the BHAC group was higher than in the cytarabine group (85.2% vs. 71.7%, P=0.004). However, the overall response rate (CR+PR) was not different between the two groups (93.3% vs. 87.9%, P=0.139). The 5-yr estimates of overall survival (OS) of children in the two groups were similar (54.9% for the BHAC group vs. 52.4% for the cytarabine group, P=0.281). Although the results were analyzed according to the treatment type and cytogenetic risk, the OS showed no significant difference between the BHAC group and the cytarabine group. In the present study, the clinical outcomes of the BHAC-based chemotherapy, consisting of BHAC, idarubicin, and 6-TG, are comparable to that of the cytarabine-based chemotherapy for childhood AML.

Gefen N, Brkic G, Galron D, et al.
Acquired resistance to 6-thioguanine in melanoma cells involves the repair enzyme O6-methylguanine-DNA methyltransferase (MGMT).
Cancer Biol Ther. 2010; 9(1):49-55 [PubMed] Related Publications
O(6)-methylguanine-DNA methyltransferase (MGMT), is a DNA repair enzyme that recognizes O(6)-alkylated guanine, a base analog resulting from treatment with alkylating agents. O(6)-6-thioguanine (6-TG) is used clinically to treat malignant as well as inflammatory diseases. Although MGMT participates in resistance to alkylating agents, it has not been shown to be involved in resistance of tumors to 6-TG. In this study we used a human melanoma cell line (GA) and its selected 6-TG drug resistant variant (GA-6-TG) to investigate whether MGMT plays a role in determining the drug resistant phenotype of GA-6-TG cells. We showed that GA-6-TG resistant cells express about three fold more MGMT protein and mRNA than GA cells. Treatment with 6-TG diminishes significantly MGMT amounts in both cell lines. Increased amounts of MGMT in resistant cells, are consistent with hypermethylation of the MGMT gene coding-region. Pretreatment of cells with the MGMT inhibitor O6 benzyl guanine, resulted in sensitization of GA-6-TG cells to 6-TG. Taken together, our data suggests that MGMT is associated with 6-TG drug resistance. In analogy to patients treated with alkylating agents, patients with tumors containing increased MGMT amounts, may be more resistant to 6-TG and therefore may benefit from treatment with MGMT inhibitors.

Erikci AA, Ozturk A, Tekgunduz E, Sayan O
Acute myeloid leukemia complicating multiple myeloma: a case successfully treated with etoposide, thioguanine, and cytarabine.
Clin Lymphoma Myeloma. 2009; 9(4):E14-5 [PubMed] Related Publications
BACKGROUND: The association of acute leukemia and multiple myeloma (MM) has been usually described not only as a complication of chemotherapy but also in the absence of chemotherapy or together at the time of diagnosis. Such leukemias are typically acute myeloid leukemia (AML). The myelomonocytic subtype is particularly found.
CASE REPORT: We report a case of a 68-year-old female who developed AML 2 years after the diagnosis of light chain (kappa) myeloma. She had been treated with oral melphalan and prednisone for MM. The patient was treated with an anthracycline-lacking therapy consisting of etoposide 120 mg/m2, thioguanine 100 mg/m2 orally twice daily on 1-5 days, and cytarabine 40 mg/m2 subcutaneously on day 1 (ETC) because of poor cardiac performance.
CONCLUSION: Following ETC therapy our particular patient has been in complete hematologic remission for 29 months. This therapy might be a safe alternative in secondary leukemia especially for elderly patients.

Ding X, Mohd AB, Huang Z, et al.
MLH1 expression sensitises ovarian cancer cells to cell death mediated by XIAP inhibition.
Br J Cancer. 2009; 101(2):269-77 [PubMed] Free Access to Full Article Related Publications
BACKGROUND: The X-linked inhibitor of apoptosis protein (XIAP), an endogenous apoptosis suppressor, can determine the level of caspase accumulation and the resultant response to apoptosis-inducing agents such as cisplatin in epithelial ovarian cancer (EOC). In addition, the mismatch repair protein, hMLH1, has been linked to DNA damage-induced apoptosis by cisplatin by both p53-dependent and -independent mechanisms.
METHODS: In this study, hMLH1 expression was correlated with clinical response to platinum drugs and survival in advanced stage (III-IV) EOC patients. We then investigated whether MLH1 loss was a determinant in anti-apoptosis response to cisplatin mediated by XIAP in isogenic and established EOC cell lines with differential p53 status.
RESULTS: The percentage of cells undergoing cisplatin-induced cell killing was higher in MLH1-proficient cells than in MLH1-defective cells. In addition, the presence of wild-type hMLH1 or hMLH1 re-expression significantly increased sensitivity to 6-thioguanine, a MMR-dependent agent. Cell-death response to 6-thioguanine and cisplatin was associated with significant proteolysis of MLH1, with XIAP destabilisation and increased caspase-3 activity. The siRNA-mediated inhibition of XIAP increased MLH1 proteolysis and cell death in MLH1-proficient cells but not in MLH1-defective cells.
CONCLUSION: These data suggest that XIAP inhibitors may prove to be an effective means of sensitising EOC to MLH1-dependent apoptosis.

Palle J, Josefine P, Frost BM, et al.
Thioguanine pharmacokinetics in induction therapy of children with acute myeloid leukemia.
Anticancer Drugs. 2009; 20(1):7-14 [PubMed] Related Publications
We studied the pharmacokinetics of 6-thioguanine (6TG) in 50 children treated for newly diagnosed acute myeloid leukemia, four of them with Down syndrome (DS). They received oral 6TG 100 mg/m2 body surface area twice daily for 4 days. Etoposide, 100 mg/m2/24 h, and cytarabine, 200 mg/m2/24 h, were administered concomitantly by intravenous infusion. On day 5, doxorubicin 75 mg/m2 was given as an 8-h infusion. The concentration of thioguanine nucleotides (TGN) in erythrocytes, the active metabolites of 6TG, was determined by high-performance liquid chromatography. The mean TGN concentration from 72, 95, and 106-h samples was used as a measure of drug exposure for each individual. The median TGN concentration in non-DS children above 2 years of age was 2.30 micromol/mmol Hb (range 0.57-25.3). The TGN concentrations varied widely (30-fold) also after dose normalization. We found no correlation with demographic, clinical, or biochemical parameters, and differences in bioavailability might be the most important explanation to interpatient variability. Children with high TGN concentration tended to have longer treatment interval to the next course, but we found no correlation with our predefined parameters for clinical response, that is, remission and relapse rate. Therefore, 6TG does not seem to be a candidate for therapeutic drug monitoring by TGN measurement, at least not in the setting of short multidrug treatment courses. Children with DS had significantly higher TGN concentrations, indicating that dose reduction might be considered to reach the same drug exposure as in non-DS children.

Schmiegelow K, Forestier E, Kristinsson J, et al.
Thiopurine methyltransferase activity is related to the risk of relapse of childhood acute lymphoblastic leukemia: results from the NOPHO ALL-92 study.
Leukemia. 2009; 23(3):557-64 [PubMed] Free Access to Full Article Related Publications
Myelotoxicity during thiopurine therapy is enhanced in patients, who because of single nucleotide polymorphisms have decreased activity of the enzyme thiopurine methyltransferase (TPMT) and thus more thiopurine converted into 6-thioguanine nucleotides. Of 601 children with acute lymphoblastic leukemia (ALL) who were treated by the NOPHO ALL-92 protocol, 117 had TPMT genotype determined, whereas for 484 patients only erythrocyte TPMT activity was available. The latter were classified as heterozygous, if TPMT activity was <14 IU/ml, or deficient (<1.0 IU/ml). 526 patients had TPMT wild type, 73 were presumed heterozygous, and two were TPMT deficient. Risk of relapse was higher for the 526 TPMT wild type patients than for the remaining 75 patients (18 vs 7%, P=0.03). In Cox multivariate regression analysis, sex (male worse; P=0.06), age (higher age worse, P=0.02), and TPMT activity (wild type worse; P=0.02) were related to risk of relapse. Despite a lower probability of relapse, patients in the low TPMT activity group did not have superior survival (P=0.82), possibly because of an excess of secondary cancers among these 75 patients (P=0.07). These data suggest that children with ALL and TPMT wild type might have their cure rate improved, if the pharmacokinetics/-dynamics of TPMT low-activity patients could be mimicked without a concurrent excessive risk of second cancers.

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