Etoposide (VP-16)-related secondary myeloid leukemias (t-AML) are most frequently associated with MLL gene translocations at 11q23. Our central hypothesis is that redox cycling of VP-16 initiated by myeloperoxidase (MPO) found prominently in myeloid precursors amplifies the genotoxicity and carcinogenicity of this otherwise clinically effective DNA topoisomerase II (topo ll)-targeted anticancer agent. We propose that MPO converts VP-16 to free radical species and oxidized metabolites that induce oxidative DNA damage and initiate recombinogenic events in myeloid precursor stem cells leading to the chromosomal translocations responsible for t-AML. Specifically, it is proposed: 1) that oxidative damage and abasic DNA sites formed as a consequence of peroxidative activation of VP-16 result in loci that will increase topo II poisoning;and/or: 2) that electrophilic VP-16-ortho-quinone formed in MPO-rich progenitors will poison topo II by adduction to sulfhydryl groups on the enzyme. We further posit that nutritional antioxidants such as vitamin C and vitamin E homologs will prevent VP-16-induced AML by reducing or preventing production of peroxidase-dependent free radical and electrophilic metabolites. We propose to determine the mechanism(s) by which peroxidative activation of VP-16 to phenoxyl radical and ortho-quinone metabolites enhances its DNA damaging and recombinogenic activities in genomic regions of the MLL gene known to contain breakpoints associated with t-AML. In order to test the stated hypotheses four specific aims will be pursued to: 1) determine the role of MPO-mediated VP-16 oxidative activation in MLL gene translocations in MPO-rich HL-60 cells and normal human CD34+ progenitor cells. 2) establish that VP-16-induced oxidative DNA damage and consequent abasic sites on DNA result in enhanced DNA topo ll-mediated strand breaks and increased MLL gene rearrangements. 3) identify functionally important modifications in DNA topo IIa caused by VP-16 oxidative activation in vitro and in intact cells utilizing biochemical and protein mass spectrometric techniques. 4) determine the in vivo effects of ascorbate supplementation on VP-16-induced MLL gene arrangements in CD34+ cells using a unique ascorbate-dependent rat model. Relevance: The current proposal will investigate whether the free radical forms of VP-16 produced in certain normal blood cells are responsible for specific DNA aberrations causal for myeloid leukemia. This work will also examine whether nutritional antioxidants can prevent VP-16-induced leukemia.
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