Research Article Differential Effects of Targeted Disruption of Thiopurine Methyltransferase on Mercaptopurine and Thioguanine Pharmacodynamics Christine Hartford,1,2 Erick Vasquez,1 Matthias Schwab,1,7 Mathew J. Edick,1,6 Jerold E. Rehg,3 Gerard Grosveld,4 Ching-Hon Pui,2,5,6 William E. Evans,1,5,6 and Mary V. Relling1,5,6 Departments of 1Pharmaceutical Sciences, 2Hematology-Oncology, 3Pathology, and 4Genetics, and 5Hematologic Malignancies Program, St. Jude Children’s Research Hospital, 6University of Tennessee, Memphis, Tennessee; and 7Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology, Stuttgart, Germany, and University of Tuebingen, Tuebingen, Germany Abstract TPMT polymorphism. Inherited as an autosomal codominant trait The recessive deficiency in thiopurine methyltransferase (6–8), TPMT activity in erythrocytes (reflective of other tissues as (TPMT), caused by germ-line polymorphisms in TPMT, can well; refs. 7, 9–11) exhibits a trimodal population frequency cause severe toxicity after mercaptopurine. However, the distribution (12–14). TPMT activity is inversely related to the significance of heterozygosity and the effect of the polymor- concentration of active thioguanine metabolites after administra- phism on thioguanine or in the absence of thiopurines is not tion of thiopurines (4). The rare homozygous deficient individuals known. To address these issues, we created a murine knockout develop extreme myelosuppression, which can be fatal when given of Tpmt. Pharmacokinetic and pharmacodynamic studies of usual doses of thiopurines (15), necessitating a 10-fold dosage À À mercaptopurine and thioguanine were done in Tpmt / , decrease to prevent such toxicity (16). Heterozygotes, constituting À Tpmt+/ , and Tpmt+/+ mice and variables were compared 10% of the population, often exhibit an intermediate degree of among genotypes. Methylated thiopurine and thioguanine toxicity following mercaptopurine (17), although the role of nucleotide metabolites differed among genotypes after treat- heterozygosity in dosing thiopurines is somewhat controversial. ment with mercaptopurine (P < 0.0001 and P = 0.044, Patients with homozygous wild-type (high) activity may be at respectively) and thioguanine (P = 0.011 and P = 0.002, increased risk of poor treatment response (e.g., persistence of respectively). Differences in toxicity among genotypes were leukemia; ref. 18). more pronounced following treatment with 10 daily doses Due to the inherent toxicity and possible carcinogenicity of of mercaptopurine at 100 mg/kg/d (0%, 68%, and 100% thiopurines (19–21), conducting randomized clinical trials to fully 50-day survival; P = 0.0003) than with thioguanine at 5 mg/ explore how best to use these agents in humans has ethical kg/d (0%, 33%, and 50% 15-day survival; P = 0.07) in the constraints. For example, there is considerable interest in studying À À À Tpmt / , Tpmt+/ , and Tpmt+/+ genotypes, respectively. Mye- the relative importance of the TPMT polymorphism for thioguanine losuppression and weight loss exhibited a haploinsufficient versus for mercaptopurine, particularly because recent clinical trials phenotype after mercaptopurine, whereas haploinsufficiency have suggested a lower relapse rate with thioguanine treatment in was less prominent with thioguanine. In the absence of childhood acute lymphoblastic leukemia (ALL). TPMT activates the drug challenge, there was no apparent phenotype. The mercaptopurine riboside intermediate metabolite of mercaptopu- murine model recapitulates many clinical features of the rine, but this methylated metabolite is not present after adminis- human polymorphism; indicates that mercaptopurine is more tration of thioguanine (22). Thus, it would be useful to directly affected by the TPMT polymorphism than thioguanine; and compare the effect of the TPMT polymorphism on the pharmaco- provides a preclinical system for establishing safer regimens of kinetics and pharmacodynamics of thioguanine versus mercapto- genetically influenced antileukemic drug therapy. [Cancer Res purine (3, 23). In addition, there has been much speculation about 2007;67(10):4965–72] whether the polymorphism in TPMT has any biological significance in the absence of thiopurine challenge (2, 24). Introduction In humans, the molecular basis of the TPMT polymorphism is largely related to three common nonsynonymous coding single- One of the clearest examples of a pharmacogenetic polymor- nucleotide polymorphisms (13), each of which renders the protein phism affecting drug efficacy and toxicity in humans is that caused unstable (25) and subject to enhanced ubiquitination and by thiopurine methyltransferase (TPMT) deficiency (1–5). Thio- degradation (26, 27). Thus, the homozygous deficiency in humans purines (mercaptopurine, thioguanine, and azathioprine) are is characterized by almost undetectable levels of TPMT protein; commonly used as antineoplastics and immunosuppressants, but heterozygotes have intermediate protein and activity levels; and their proper use remains uncertain and is complicated by the homozygous wild-type individuals have high levels of protein and activity (28, 29). We created a murine model to allow the controlled study of the Note: C-H. Pui is an American Cancer Society Professor. TPMT polymorphism. This model has allowed us to compare the Requests for reprints: Mary V. Relling, Department of Pharmaceutical Sciences, pharmacokinetics and pharmacodynamics of thioguanine and St. Jude Children’s Research Hospital, 332 North Lauderdale, Memphis, TN 38105-2794. mercaptopurine in an in vivo isogenic system and to show that Phone: 901-495-2348; Fax: 901-525-6869; E-mail: [email protected]. I2007 American Association for Cancer Research. biological consequences of the TPMT polymorphism are evident doi:10.1158/0008-5472.CAN-06-3508 only in the presence of drug challenge. www.aacrjournals.org 4965 Cancer Res 2007; 67: (10). May 15, 2007 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2007 American Association for Cancer Research. Cancer Research Materials and Methods Chemicals. Mercaptopurine, thioguanine, 2-deoxyguanosine, 6-thiogua- nosine, 6-mercaptopurine riboside, and 6-methylmercaptopurine riboside were purchased from Sigma. Alkaline phosphatase was purchased from Promega. 6-Thio-2¶deoxyguanosine was purchased from R.I. Chemical, Inc. All other chemicals and reagents were of high-performance liquid chromatography grade. A folic acid–deficient purified diet for mice was purchased from TestDiet. Methotrexate and trimethoprim/sulfamethoxa- zole (Sulfatrim pediatric suspension) were purchased from Alpharma. Targeting construct design and generation. A clone containing the murine Tpmt gene was identified by screening a 129/Ola murine genomic DNA library, packaged in a P1 artificial chromosome vector (Genome Systems), with the assistance of Dr. Evgeny Krynetski and Dr. Mike Fessing (30, 31). The identified clone was verified to contain the Tpmt genomic locus by Southern blotting, sequencing, and fluorescent in situ hybridiza- tion. To generate the targeting construct, an 8.7-kb KpnI restriction endonuclease fragment encompassing part of exon III through a portion of intron 6 was subcloned into the pZERO-2 vector (Invitrogen). A herpes simplex virus-thymidine kinase gene cassette was cloned into the HindIII site of the pZERO vector at the 3¶ end of the Tpmt insert, and a 2.8-kb PacI/ XmaI fragment, internal to the 8.7-kb KpnI fragment and containing exons Figure 1. Erythrocyte TPMT activity differed by genotype. The mean (95% CI) À À À V and VI, was replaced with a neomycin phosphotransferase gene cassette. TPMT activity differed among +/+, +/ , and / genotypes for 129P1/ReJ [15.0 (13.40–16.10) versus 8.33 (7.60–8.80) versus 0.53 (0.10–0.90) units/mL Generation of targeted mice. E14 embryonic stem cells provided by the erythrocytes; P = 0.0273], NMRI-7C10 [15.03 (13.90–16.70) versus 8.03 laboratories of Dr. James Ihle and Dr. Gerard Grosveld (St. Jude Children’s (6.40–10.80) versus 1.47 (0.20–3.20) units/mL erythrocytes; P = 0.0273], Research Hospital) were electroporated in the presence of linearized Tpmt NMRI-5G3 [14.43 (13.40–15.70) versus 6.90 (6.50–7.30) versus 0.53 targeting vector DNA. After selection in G418 for 9 days, three successfully (0.40–0.60) units/mL erythrocytes; P = 0.0265], 129X1/SvJ [10.93 (9.70–12.90) versus 7.13 (5.30–8.96) versus 0.53 (0.15–0.91) units/mL erythrocytes; targeted clones (4B9, 7C10, and 5G3) were identified by PCR and confirmed P = 0.0273], C3H/HeJ [15.3 (14.40–16.80) versus 8.23 (7.60–9.00) units/mL by Southern blotting. C57BL/6 pseudopregnant mice were recipients of erythrocytes versus undetectable; P = 0.0241]. TPMT activity was similar in blastocyst injections and embryo transfers. Four germ-line transmitting patient samples from children with leukemia [18.88 (12.75–25.98) versus 8.97 (3.90–13.45), versus 0.56 (0.37–0.75) units/mL erythrocytes (P < 0.0001) in chimeric mice were produced (three from clone 5G3 and one from clone À À À +/À +/+, +/ , and / , respectively]. P values were determined by the 7C10). Heterozygous Tpmt (Tpmt ) chimeric mice produced offspring in Kruskal-Wallis test. the expected Mendelian distribution, indicating no in utero lethality due to loss of one or both functional Tpmt alleles. Because C57BL/6 have low To evaluate chronic toxicity that partially mimics the exposure conditions constitutive TPMT activity (32, 33), the mixed background 129/C57BL/6 of combination drug therapy given to children with leukemia, six mice of