Etoposide Quinone Is a Covalent Poison of Human Topoisomerase IIβ

Abstract

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-AMLs is not well understood; however, previous studies have implicated etoposide metabolites (i.e., etoposide quinone) and topoisomerase IIβ in the leukemogenic process. Although interactions between etoposide quinone and topoisomerase IIα have been characterized, the effects of the drug metabolite on the activity of human topoisomerase IIβ have not been reported. Thus, we examined the ability of etoposide quinone to poison human topoisomerase IIβ. The quinone induced ∼4 times more enzyme-mediated DNA cleavage than did the parent drug. Furthermore, the potency of etoposide quinone was ∼2 times greater against topoisomerase IIβ than it was against topoisomerase IIα, and the drug reacted ∼2–4 times faster with the β 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 IIβ 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 IIβ.

Myeloperoxidase Enhances Etoposide and Mitoxantrone-Mediated DNA Damage: A Target for Myeloprotection in Cancer Chemotherapy

“…3, C and D). Etoposide quinone displays enhanced TOP2-mediated DNA cleavage activity in vitro compared with the parent compound (Jacob et al, 2011), and this is more pronounced for TOP2B (Smith et al, 2014b). The observation that SA pretreatment, which reduces MPO-mediated etoposide phenoxy radical production, partially suppressed etoposide-induced TOP2-CC formation in MPO-expressing NB4 cells supports the conclusion that oxidative metabolism of etoposide plays a role in TOP2-mediated DNA damage in vivo.…”

“…2A). Etoposide phenoxy radicals have been observed in vitro and in cell culture systems (Haim et al, 1986; Kalyanaraman et al, 1989; Yalowich et al, 1996; Kagan et al, 2001; Vlasova et al, 2011) and can be further oxidized to etoposide quinone (Haim et al, 1986; Fan et al, 2006), which exhibits enhanced TOP2-dependent DNA cleavage activity compared with etoposide, especially for TOP2B (Jacob et al, 2011; Smith et al, 2014b). Etoposide ortho-quinone reacts spontaneously with GSH in solution (Fan et al, 2006), and so the effect of MPO expression on TOP2-DNA complex accumulation may be limited by GSH titration of etoposide quinone within cells.…”

“…Etoposide quinone is more effective at inducing TOP2-mediated DNA breaks than etoposide in vitro (Jacob et al, 2011) and this is particularly true for TOP2B (Smith et al, 2014b). This increased potency of etoposide quinone as a TOP2 poison has been attributed to covalent modification of the enzyme by the quinone (Jacob et al, 2011; Smith et al, 2014b). Furthermore, etoposide metabolites have the potential to form adducts with DNA and protein (Haim et al, 1987; Lovett et al, 2001), which may also impact on genotoxicity.…”

Mechanisms of Action and Reduced Cardiotoxicity of Pixantrone; a Topoisomerase II Targeting Agent with Cellular Selectivity for the Topoisomerase II Isoform

“…The preparation of the full-length human topoisomerase IIa has been described in a previous publication (Hasinoff et al, 2005). The human topoisomerase IIb, prepared as described (Smith et al, 2014), was a gift of N. Osheroff (Vanderbilt University School of Medicine, Nashville, TN).…”

Modulation of therapy-induced senescence by reactive lipid aldehydes

“…Although etoposide is known to inhibit topoisomerase II activity by binding at the protein–DNA interface, 30 there is also evidence that its mode of action may require metabolism to a radical and covalent protein modification. 31,32 This raised the consideration whether the induction of senescence might reflect dual activities of this agent not only as a topoisomerase inhibitor but also as a source of free radicals.…”