Abstract 4738: Lipid peroxidation mediates the effects of topoisomerase poisons on their targets
Authors: Flor Amy C. , Li Jing , Hanakahi Leslyn A. , Kron Stephen J. 
Published: August 2019

Our studies characterizing responses of cancer cells to oxidative stress have implicated the topoisomerases TOP1 and TOP2 as key mediators linking reactive oxygen species to DNA damage. Normally, TOP1 and TOP2 maintain chromosome integrity by transiently cleaving and rejoining DNA, thereby releasing topological strain and facilitating transcription, replication, and other processes critical for cancer cell proliferation. The topoisomerase poisons are a structurally diverse group of chemotherapy agents such as camptothecin, doxorubicin and etoposide that trap topoisomerases as cleavage complexes, TOP1cc and/or TOP2cc, where the enzymes remain covalently bound at single or double strand breaks, respectively. Prior studies identified a common mechanism of interfacial inhibition, but the constraints of locking protein onto DNA are hard to reconcile with the diversity of topoisomerase poisons, including simple compounds like H2O2 and 1,4-benzoquinone. Pointing to an alternative mechanism, camptothecin, doxorubicin, etoposide, and other topoisomerase poisons share the potential to markedly increase cellular oxidative stress. Among the most toxic consequences of oxidative stress is lipid peroxidation, leading to formation of lipid aldehydes such as 4-hydroxynonenal that modify and crosslink proteins at nucleophilic residues. In prior work, we showed that sequestering aldehydes blocked cellular effects of topoisomerase poisons while treating cells with 4-hydroxynonenal recapitulated many of their effects. Here, we have examined whether lipid aldehydes also mediate effects of topoisomerase poisons on their protein targets. Thus, we modulated oxidative stress in cells along with directly assaying formation TOP1cc and/or TOP2cc by detection in situ, flow cytometry, and Western blotting. We confirmed that camptothecin, doxorubicin, and etoposide induce lipid peroxidation, leading to production of aldehydes and other electrophiles. Under conditions where TOP1cc and/or TOP2cc are normally formed, blocking lipid peroxidation with butylated hydroxytoluene (BHT) was sufficient to protect cells from topoisomerase poisons. Among candidate mediators, 4-hydroxy-2-nonenal, 1- and E-2-hexadecanal, and 15-deoxy-Δ12, 14-prostaglandin J2 could each induce TOP1cc and TOP2cc. The toxicity of the lipid-derived electrophiles was also dependent on the activity of TDP1, which serves a key role in removing TOP1cc and TOP2cc from DNA. Our results suggest a distinct, shared mechanism of action of topoisomerase poisons that depends on oxidative stress, lipid peroxidation, accumulation of lipid aldehydes and covalent modification of surface nucleophiles on topoisomerase enzymes. These covalent modifications may be sufficient to poison topoisomerases on their own and drive formation of TOP1cc and TOP2cc, independent of whether the cleaved conformation is stabilized by interfacial inhibition.

Citation Format: Amy C. Flor, Jing Li, Leslyn A. Hanakahi, Stephen J. Kron. Lipid peroxidation mediates the effects of topoisomerase poisons on their targets [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4738.