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 2

Auranofin disrupts the interaction of TOPBP1 with PHF8 and FANCJ.

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Auranofin disrupts the interaction of TOPBP1 with PHF8 and FANCJ. (A) An...
(A) Analysis of the interaction of BRCT 7–8 with PHF8/C and FANCJ/C by the NanoBiT system in HeLa cells treated with auranofin for 2 hours at the indicated concentrations. Data are shown as mean ± SD. (B) IP followed by immunoblotting with cellular extracts from FLAG-TOPBP1-expressing HeLa cells under 2 hours of treatment with auranofin at the indicated doses. (C) Co-IP analysis of the interactions of TOPBP1-PHF8 and TOPBP1-FANCJ with cellular extracts from HeLa cells treated with auranofin (2 μM, 2 hours). (D) Co-IP analysis of the interactions of TOPBP1-PHF8 and TOPBP1-FANCJ in HeLa cells cultured in the presence of auranofin (2 μM, 2 hours) or different doses of Calcein-AM (4 hours). (E) Co-IP analysis of the interactions of TOPBP1-PHF8 and TOPBP1-FANCJ in HeLa cells treated by auranofin (2 μM, 2 hours), PMX464 (10 μM, 4 hours), or piperlongumine (10 μM, 4 hours). (F) Predicted structure of auranofin docking to the hydrophobic pocket on BRCT 7–8. The hydrophobic pocket (dashed circle) and critical hydrophobic interactions based on crystal structures of BRCT 7–8-PHF8/APS (PDB ID: 7CMZ) and BRCT 7–8-pFANCJ (PDB ID: 3AL3) are shown. (G) BLI analysis of auranofin binding to recombinant BRCT 7–8/WT and BRCT 7–8/F1411A (n = 2). BLI sensorgrams and the KD for each group are shown. Black lines are fitted curves; color traces are raw data. (H) Schematic representation of the prime editing system and the representative sequencing results of F1411A-edited genomes from HeLa cells. The sequences of critical elements of the epegRNA are shown. (I) Co-IP analysis of the association of TOPBP1 with PHF8 and FANCJ in control and F1411A-edited HeLa cells treated with vehicle or auranofin (2 μM, 2 hours). All immunoblots were repeated at least twice and one of them is shown.