The objective of this work is to better understand the mechanisms of epipodophyllotoxin-induced leukemogenesis. Epipodophyllotoxins result in treatment-related leukemias with translocations of the MLL gene at chromosome band 11q23. Current evidence suggests that both antineoplastic and leukemogenic epipodophyllotoxin effects are due to chromosomal breakage and that breakage resolved by chromosomal translocation is associated with leukemia. Epipodophyllotoxin parent compounds stabilize a DNA topoisomerase II-DNA covalent intermediate, decrease DNA topoisomerase II-mediated religation and cause chromosomal breakage. These events initiate an apoptotic cell death pathway, the desired antineoplastic action. Epipodophyllotoxins are metabolized to several compounds that may also lead to chromosomal breakage by creation of abasic sites or by forming DNA adducts. The applicant hypothesizes that epipodophyllotoxin as well as its metabolites may contribute to the breakage that results in translocations, and that secondary genetic changes, in addition to the translocations may also be required for leukemia to evolve. The first hypothesis is supported by new data that the genotype of cytochrome P-450 (CYP) 3A4, which metabolizes epipodophyllotoxin parent drug, may modulate the risk, while latency to the onset of disease suggests that there are secondary changes.
In Aim 1 the applicant will clone MLL genomic translocation breakpoints by panhandle variant PCR and examine serial pre-leukemic and leukemic marrows to observe establishment of clonality by cells with a particular translocation.
In Aim 2 the applicant will directly compare effects of etoposide and its metabolites on in vitro DNA topoisomerase II cleavage and religation at MLL genomic translocation breakpoints, and on breakage within the MLL gene in bone marrow stem cells.
In Aim 3 the applicant will measure etoposide and potential leukemogenic metabolites in plasma and urine of patients undergoing treatment and concurrently examine chromosomal breakage in MLL and appearance of MLL gene translocations in the treated subjects. Using cDNA microarray technology, the applicant 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 and may advance the rational design of leukemia preventive strategies that preserve the antineoplastic benefit of these important drugs.
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