The objective of this work is to understand the mechanisms of epipodophyllotoxin-induced leukemogenesis. The epipodophyllotoxins etoposide and teniposide, as well as other DNA topoisomerase II inhibitors, are anticancer drugs associated with treatment-related leukemias with translocations of the MLL gene at chromosome band 11q23 with one of many partner genes as a treatment complication. Current evidence suggests that both antineoplastic and leukemogenic epipodophyllotoxin effects are due to chromosomal breakage and that breakage resolved by translocation causes the leukemia. Epipodophyllotoxins stabilize a DNA topoisomerase II-DNA covalent complex, decrease DNA topoisomerase II-mediated religation and cause chromosomal breakage. Epidophyllotoxins are metabolized to several compounds that may contribute to the breakage. The hypotheses are that epipodophyllotoxin as well as its metabolites contribute to leukemogenic breakage, and that secondary genetic changes in addition to the translocations may also be required for leukemia to emerge. Since cytochrome P-450(CYP)3A4 converts epipodophyllotoxin to a genotoxic catechol metabolite, recent data that CYP3A4 genotype modulates the risk of leukemia supports the first hypothesis. Latency to the onset of disease supports secondary changes.
Aim 1 is to clone the translocation breakpoints, identify new partner genes and examine natural history.
Aim 2 is to compare the effects of etoposide with effects of its metabolites on the MLL gene in experimental systems. Here we will perform in vitro DNA topoisomerase II cleavage assays and analyze damage to the MLL gene in normal marrow cells in culture.
In Aim 3 we return to patients to perform phamacokinetic and molecular analyses. Here we will analyze etoposide and its metabolites and assay damage to the MLL gene in patients while undergoing treatment. Using cDNA microarray technology, we will appraise secondary genetic changes in treatment-related leukemias with MLL gene translocations in experiments of aim 4. Successful execution of these specific aims will provide new knowledge of the etiology, pathogenesis, natural history and evolution of this treatment complication. This will inform the rational design of leukemia preventive strategies that preserve the antineoplastic benefit of these important drugs. Already we have shown that the MLL translocation can be detected early in the treatment. We also showed that several MLL translocation breakpoints correspond with functional DNA topoisomerase II cleavage sites in in vitro assays and that cleavage is increased by etoposide metabolites compared to parent drug. Collaboratively, we developed sensitive mass spectrometric methods to synthesize and quantitate etoposide metabolites. We have made progress toward developing a strategy to examine damage to the MLL gene in the context of the cell.
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