Auranofin attenuates TOPBP1-mediated ATR replication stress response and improves chemotherapeutic response in breast tumor models
Shuai Ma, Yingying Han, Rui Gu, Qi Chen, Qiushi Guo, Yuan Yue, Cheng Cao, Ling Liu, Zhenzhen Yang, Yan Qin, Ying Yang, Kai Zhang, Fei Liu, Lin Liu, Na Yang, Jihui Hao, Jie Yang, Zhi Yao, Xiaoyun Mao, Lei Shi
Shuai Ma, Yingying Han, Rui Gu, Qi Chen, Qiushi Guo, Yuan Yue, Cheng Cao, Ling Liu, Zhenzhen Yang, Yan Qin, Ying Yang, Kai Zhang, Fei Liu, Lin Liu, Na Yang, Jihui Hao, Jie Yang, Zhi Yao, Xiaoyun Mao, Lei Shi
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Research Article Cell biology Oncology

Auranofin attenuates TOPBP1-mediated ATR replication stress response and improves chemotherapeutic response in breast tumor models

Abstract

Genome instability is most commonly caused by replication stress, which also renders cancer cells extremely vulnerable once their response to replication stress is impeded. Topoisomerase II binding protein 1 (TOPBP1), an allosteric activator of ataxia telangiectasia and Rad3-related kinase (ATR), coordinates ATR in replication stress response and has emerged as a potential therapeutic target for tumors. Here, we identify auranofin, the FDA-approved drug for rheumatoid arthritis, as a lead compound capable of binding to the BRCT 7–8 domains and blocking TOPBP1 interaction with PHF8 and FANCJ. The liquid-liquid phase separation of TOPBP1 is also disrupted by auranofin. Through targeting these TOPBP1-nucleated molecular machineries, auranofin leads to an accumulation of replication defects by impairing ATR activation and attenuating replication protein A loading on perturbed replication forks, and it shows significant anti–breast tumor activity in combination with a PARP inhibitor. This study provides mechanistic insights into how auranofin challenges replication integrity and expands the application of this FDA-approved drug in breast tumor intervention.

Authors

Shuai Ma, Yingying Han, Rui Gu, Qi Chen, Qiushi Guo, Yuan Yue, Cheng Cao, Ling Liu, Zhenzhen Yang, Yan Qin, Ying Yang, Kai Zhang, Fei Liu, Lin Liu, Na Yang, Jihui Hao, Jie Yang, Zhi Yao, Xiaoyun Mao, Lei Shi

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Figure 1

Auranofin directly targets TOPBP1 BRCT 7–8.

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Auranofin directly targets TOPBP1 BRCT 7–8. (A) Schematic illustration o...
(A) Schematic illustration of drug screening based on the NanoBiT system. HeLa cells stably expressing LgBiT-BRCT 7–8 and SmBiT-PHF8/C were treated with DMSO and 2,579 FDA-approved drugs (10 μM) for 4 hours followed by luminescent signal detection in live cells. (B) Summary of the initial drug screening. The luminescent signals in cells treated with different compounds were normalized with that of cells treated with DMSO; 47 compounds with normalized values lower than 0.4 (black line) were considered as potential candidates. (C) Secondary small-scale drug screening in HeLa cells stably expressing LgBiT-BRCT 7–8-SmBiT-PHF8/C and LgBiT-BRCT 7–8-SmBiT-FANCJ/C. These cells were cultured in the presence of 47 candidate compounds followed by an assessment of the luminescent signals. Calcein-AM was included as a positive control. Data are shown as mean ± SD. (D) Quantitation of the binding affinity between 4 candidate drugs and the His-tagged recombinant BRCT 7–8 purified from E coli cells by biolayer interferometry (BLI). BLI sensorgrams and the KD for each group are shown. Black lines are fitted curves; color traces are raw data. (E) BLI analysis of the inhibitory effect of auranofin on BRCT 7–8 binding to PHF8/APS peptide and phosphorylated Thr1133 containing peptide of FANCJ (pFANCJ). His-tagged BRCT 7–8 (1 μM) was preincubated with auranofin at the indicated concentrations before examining peptide-protein interactions. The experimental schemes and BLI sensorgrams are shown. Black lines are fitted curves; color traces are raw data.