A Mouse Model for Studying the Interaction of Bisdioxopiperazines with Topoisomerase IIα in Vivo

The bisdioxopiperazines such as (+)-( S )-4,4′-propylenedi-2,6-piperazinedione (dexrazoxane; ICRF-187), 1,2-bis(3,5-dioxopiperazin-1-yl)ethane (ICRF-154), and 4,4′-(1,2-dimethyl-1,2-ethanediyl)bis-2,6-piperazinedione (ICRF-193) are agents that inhibit eukaryotic topoisomerase II, whereas their ring-opened hydrolysis products are strong iron chelator. The clinically approved analog ICRF-187 is a pharmacological modulator of topoisomerase II poisons such as etoposide in preclinical animal models. ICRF-187 is also used to protect against anthracycline-induced cardiomyopathy and has recently been approved as an antidote for alleviating tissue damage and necrosis after accidental anthracycline extravasation. This dual modality of bisdioxopiperazines, including ICRF-187, raises the question of whether their pharmacological in vivo effects are mediated through interaction with topoisomerase II or via their intracellular iron chelating activity. In an attempt to distinguish between these possibilities, we here present a transgenic mouse model aimed at identifying the contribution of topoisomerase IIα to the effects of bisdioxopiperazines. A tyrosine 165 to serine mutation (Y165S) in topoisomerase IIα, demonstrated previously to render the human ortholog of this enzyme highly resistant toward bisdioxopiperazines, was introduced at the TOP2A locus in mouse embryonic stem cells by targeted homologous recombination. These cells were used for the generation of transgenic TOP2A Y165S/+ mice, which were demonstrated to be resistant toward the general toxicity of both ICRF-187 and ICRF-193. Hematological measurements indicate that this is most likely caused by a decreased ability of these agents to induce myelosuppression in TOP2A Y165S/+ mice, highlighting the role of topoisomerase IIα in this process. The biological and pharmacological implications of these findings are discussed, and areas for further investigations are proposed.