The epipodophyllotoxins, teniposide and etoposide, are among the most effective drugs for the treatment of childhood acute lymphoblastic leukemia (ALL) and other cancers. Although ALL is curable in 70% of patients, therapy is complicated by the development of a secondary acute myeloid leukemia (AML) in up to 12% of children who have been cured of their primary ALL. Currently, it is not possible to identify those patients at highest risk for this fatal secondary AML. Epipodophyllotoxin- induced AML is a distinct clinical and biologic entity, characterized by a unique mechanism: illegitimate site-specific recombination of the MLL (mixed lineage leukemia) gene on chromosome 11 (region q23) with one of a number of partner genes, resulting in the leukemic phenotype and accompanied by translocations of 11q23. The distinctive molecular signature of illegitimately acting V(D)J recombinase has been found at the translocation breakpoints, indicating an ill-fated cellular attempt at inappropriate DNA recombination secondary to the epipodophyllotoxins' inhibition of normal recombination. There have been no prior attempts to determine whether epipodophyllotoxins or their metabolites induce site- specific illegitimate V(D)J-recombination, either in vivo or in vitro. We will define the quantitative relationship between epipodophyllotoxins or their metabolites and illegitimate recombination. We demonstrated that cytochrome P450 3A4 (CYP3A4) O-demethylates teniposide and etoposide in human liver. O-demethylation is important not only because it may represent a major route of elimination, but also because it indicates oxidative activation to reactive catechols. We have demonstrated 10-fold interpatient variability in the systemic clearance of the epipodophyllotoxins and in the formation of catechol metabolites in children. Our hypotheses are that this interpatient variability in CYP3A4- mediated O-demethylation accounts for variability in the overall disposition of epipodophyllotoxins, and that high systemic exposure to etoposide, its catechol, or both is the principal determinant of the mutagenic recombination caused by the epipodophyllotoxins. We will use in vivo pharmacokinetic studies in children with ALL to determine whether O- demethylation determines the clearance of the epipodophyllotoxins. We will evaluate whether high systemic exposure to etoposide, its catechol, or both are significantly related to the frequency of illegitimate V(D)J recombinase-mediated mutations in serial samples of children's leukocyte HPRT DNA, a specific marker of the biologically relevant mechanism of leukemogenesis in vivo. We have also developed in vitro model systems to determine whether illegitimate recombinase activity in specific cell lines is related to epipodophyllotoxin parent drug or metabolite concentrations and/or to the duration of exposure. Together, these laboratory and clinical studies will elucidate the contribution of pharmacokinetic and metabolic variability as determinants of the disposition and leukemogenic effects of the epipodophyllotoxins in children with cancer.
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