DRUG METABOLISM IN CHILDHOOD CANCER

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Despite great progress in the past 20 years, cancer remains the leading cause of death by disease in US children between 1 and 15 years of age. In acute lymphoblastic leukemia (ALL), the most common childhood cancer, 30% of patients fail current therapy, and many patients fail current therapy, and many patients who are cured experience significant morbidity from chemotherapy. The long-term objective of this competitive renewal is to develop more effective yet less toxic therapy, by understanding how leukemia cells metabolize and activate mercaptopurine (MP), an extensively used antileukemic agent. MP metabolism in normal tissues is highly variable,a nd methylation by thiopurine methyltransferase (TPMT) is subject to genetic polymorphism. Patients who inherit TPMT-deficiency as an autosomal recessive trait, and heterozygots, have significantly different patterns of MP metabolism in RBCs, which affects MP's toxicity to normal hematopoietic cells. However, there have been no in vivo studies of MP metabolism in leukemia cells or how this relates to MP's antileukemic effects. Our recent work established that metabolism of another antimetabolite (methotrexate) in ALL blasts differed among patients and was related to the lineage and ploidy (chromosomal number) of leukemia cells. The studies supported by the continuation of this grant will characterize the in vivo polymorphic metabolism of MP in leukemia cells isolated from children given Mp, and assess the pharmacodynamics of blast concentrations of methylmercaptopurine nucleotides and thioguanine nucleotides (TGN) on MP's mechanisms of cytotoxicity (e.., TGN incorporation into DNA, inhibition of de novo purine synthesis), and decrease in ALL blast count. The activity of polymorphic TPMT will be determined in these ALL blasts and in normal erythrocytes, to assess its relation to mP's metabolism and effects. To determine the molecular mechanisms of the TPMT polymorphism, RNA and DNA will be isolated from normal leukocytes of children with TPMT- deficiency and heterozygous genotypes, from which inactivating mutations will be identified in the gene encoding TPMT. Once inactivating mutations have been defined (the first of which we have recently characterized), PCR- based tests will be developed to determine TPMT genotype and prospectively identify patients with aberrant MP metabolism and effects. As the first human studies to examine the in vivo metabolism of MP in leukemia cells, they will provide important new insights on the molecular mechanisms underlying interindividual differences in antileukemic response to mercaptopurine.