Chemoproteomic analysis reveals RECQL4 as a mediator of nitroalkene-dependent double-strand break repair inhibition in cancer.

Nitroalkenes are endogenous products generated by the metabolism of unsaturated fatty acids. They are generated under oxidative stress conditions, mediating important anti-inflammatory signaling activities through covalent modification of protein cysteine thiols. Despite being cytoprotective in benign tissue, nitroalkenes display single-agent anti-proliferative activity in breast cancer cells and sensitize them to multiple DNA-damaging agents. Initial mechanistic evidence suggested that nitroalkene anti-cancer activities are partially mediated by inhibition of homologous recombination (HR) through the recombinase RAD51 at Cys319. However, nitroalkenes are multi-target agents, and thus, it is likely that other important DNA repair targets beyond RAD51 are modified by nitroalkenes, contributing to their anti-cancer effects. We, therefore, conducted a global proteomics analysis to address this question. This analysis led to the identification of the recQ helicase RECQL4 with a nitro-alkylation at Cys1052. This modification was further confirmed by click chemistry-based chemoproteomics and determined to be DNA damage-dependent. Functional analyses demonstrated that nitroalkene modification inhibits RECQL4 ATP-dependent helicase activity and disrupts DSB end resection and downstream homology-dependent repair. Furthermore, experiments with C1052S mutant RECQL4 revealed that RECQL4 is a major mediator of nitroalkene effects on end resection, DSB formation, and repair. The evidence presented here denotes RECQL4 as an important nitroalkene target conferring DSB repair inhibition and supports further evaluation of nitroalkenes as therapeutic agents in RECQL4-amplified cancers.