Etoposide Quinone Is a Covalent Poison of Human Topoisomerase IIβ

Etoposide is a topoisomerase II poison that is utilized to treat a broad spectrum of human cancers. Despite its wide clinical use, 2-3% of patients treated with etoposide eventually develop treatment-related acute myeloid leukemias (t-AMLs) characterized by rearrangements of the MLL gene. The molecular basis underlying the development of these t-AMLLs is not well understood; however, previous studies have implicated etoposide metabolites (i.e., etoposide quinone) and topoisomerase II beta in the leukemogenic process. Although interactions between etoposide quinone and topoisomerase II alpha have been characterized, the effects of the drug metabolite on the activity of human topoisomerase II beta have not been reported. Thus, we examined the ability of etoposide quinone to poison human topoisomerase II beta. The quinone induced similar to 4 times more enzyme-mediated DNA cleavage than did the parent drug. Furthermore, the potency of etoposide quinone was similar to 2 times greater against topoisomerase II beta than it was against topoisomerase II alpha, and the drug reacted similar to 2-4 times faster with the beta isoform. Etoposide quinone induced a higher ratio of double- to single-stranded breaks than etoposide, and its activity was less dependent on ATP. Whereas etoposide acts as an interfacial topoisomerase II poison, etoposide quinone displayed all of the hallmarks of a covalent poison: the activity of the metabolite was abolished by reducing agents, and the compound inactivated topoisomerase II beta when it was incubated with the enzyme prior to the addition of DNA. These results are consistent with the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis through an interaction with topoisomerase II beta.