Abstract 6877: Oxidative stress induced lipid peroxidation as a mechanism of topoisomerase II alpha poisoning by chemotherapeutic agents

Abstract Topoisomerase II alpha (TOP2A) is an ATPase essential for maintaining optimal genomic DNA topology in cells. By relaxing DNA supercoils and entanglements, TOP2A relieves torsional stress to facilitate transcription, replication, and mitosis. In cancer cells, TOP2A is often overexpressed, enabling abnormally rapid genomic replication, in turn facilitating cellular hyperproliferation and driving tumor progression. Thus, TOP2A is a key target of several chemotherapeutic drugs, including etoposide and doxorubicin. While etoposide has long been proposed to stabilize the TOP2A-DNA cleavage complex (TOP2Acc) via interfacial inhibition, we have elucidated a complementary oxidative stress mediated pathway involving lipid peroxidation (LPO) and subsequent generation of reactive lipid electrophiles (LDEs). The LDE 4-hydroxynonenal (HNE) covalently modifies TOP2A at cysteine residues in its ATPase domain, in a DNA-dependent manner. HNE modification inhibits TOP2A catalytic activity and traps TOP2Acc, leading to accumulation of lethal DNA double strand breaks (DSBs). Induction of reactive oxygen species (ROS) by etoposide and doxorubicin were shown by flow cytometry assays, and LPO and HNE-protein adducts were imaged using fluorescence microscopy. Several chemotherapeutic TOP2A poisons significantly depleted reduced glutathione, a key antioxidant. In vitro, HNE inhibited DNA decatenation and relaxation activities of purified human TOP2A in kDNA and plasmid DNA assays. Mass spectrometry revealed sites of covalent HNE modification, including Cys216 within the ATPase domain. Malachite green ATPase assays demonstrated that HNE reduced ATP hydrolysis, consistent with disruption of ATP binding. In cell-based studies, HNE directly induced TOP2Acc and DSBs, as detected by trapped in agarose DNA immunostaining (TARDIS) assays and neutral comet assays, respectively. TOP2Acc and DSBs induced by etoposide or doxorubicin were mitigated by a lipid antioxidant, implicating LPO as a mediator of TOP2A targeted drug poisoning. An etoposide resistant cancer cell line exhibited cross resistance to HNE-induced toxicity, further supporting a shared mechanism involving redox metabolism. Proteasome inhibitors including bortezomib exacerbated the persistence of HNE-induced DSBs, indicating that repair of HNE-induced TOP2Acc primarily depends on the proteasome for degradation. Conclusion: Taken together, our data suggest an underappreciated role for TOP2A as a redox sensor in tumor cells, connecting oxidative stress to DNA damage signaling and creating a target for redox active drugs. This work suggests novel opportunities for improving chemotherapeutic strategies, by leveraging proteasome inhibitors or designing covalent inhibitors targeting Cys216 to optimize therapeutic outcomes and minimize off target toxicities. Citation Format: Amy C. Flor, Don Wolfgeher, Stephen J. Kron. Oxidative stress induced lipid peroxidation as a mechanism of topoisomerase II alpha poisoning by chemotherapeutic agents [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 6877.