ANTINEOPLASTIC v LEUKEMOGENIC EPIPODOPHYLLOTOXIN EFFECTS

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DESCRIPTION (provided by applicant): The objective of this work is to understand the nature of the DNA damage leading to MLL translocations in leukemias following anticancer treatment with DNA topoisomerase II inhibitors. The CYP3A4 promoter is polymorphic and CYP3A4 genotype confers susceptibility. CYP3A4 converts etoposide to etoposide catechol; the catechol is readily oxidized to a quinone. These metabolites are genotoxins. MLL joins with one of many partner genes to form the translocations. The genomic breakpoint junction sequences contain evidence of DNA damage and repair. Several genomic breakpoint junction sequences indicate precise or near-precise interchromosomal DNA recombinations, but the cloning of additional breakpoints is essential to discern the damage spectrum. Etoposide and its metabolites induce DNA topoisomerase II cleavage at the translocation breakpoints in MLL and in its partner genes in vitro. We propose that etoposide and its metabolites can stimulate a series of different DNA lesions, which are repaired to form the breakpoint junctions, and that the heterogeneity in genomic breakpoint junction sequences reflects heterogeneity in the damage and its resolution. The DNA lesions to be tested include the direct induction of DNA topoisomerase II cleavage by etoposide parent drug, induction of DNA topoisomerase II cleavage from DNA adduct formation by etoposide quinone or reactive oxygen species, replication fork collisions with DNA topoisomerase II covalent complexes and DNA topoisomerase II-independent damage. Aim 1 will examine the spectrum and quantify the relative importance of DNA adducts from etoposide metabolites in an MLL bcr DNA substrate using mass spectrometry. Aim 2 will investigate the induction of functional DNA topoisomerase II covalent complexes in MLL and in the genome by etoposide and etoposide metabolites in human CD34+ hematopoietic progenitor cells using DNA arrays. To answer whether, how often and to what degree precise recombinations, exonucleolytic nibbling, large deletions, insertions, inversions, duplications and nonhomologous end-joining have occurred in creation of the breakpoint junctions, Aim 3 will characterize the genomic sequences of both derivative chromosomes in the leukemias in patients. Solving the mechanism of leukemogenesis of the DNA topoisomerase II inhibitors is highly relevant to the targeted prevention of this usually fatal complication of anticancer treatment.