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 4

Auranofin dissolves TOPBP1 liquid-liquid condensate.

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Auranofin dissolves TOPBP1 liquid-liquid condensate. (A) Liquid droplet ...
(A) Liquid droplet formation of BRCT 6-8/WT observed by confocal and differential interference contrast (DIC) microscopy in the presence of vehicle, auranofin, and 1,6-hexanediol (1,6-HD) as indicated. The occupied areas of the droplets were quantified and shown. The GFP-tagged recombinant proteins purified from bacterial cells were examined by Coomassie brilliant blue (CBB) staining. (B) Representative micrographs and quantitative analysis of BRCT 6-8/WT and BRCT 6-8/F1411A droplet. The GFP-tagged recombinant proteins were examined by CBB staining. (C) Representative micrographs and quantitative analysis of the puncta intensity of mCherry-LacI-TOPBP1 and mCherry-LacI under 1,6-HD treatment (2.5%, 5 minutes) and removal (10 minutes later). U2OS cells stably integrated with 256 × LacO repeats were transfected with mCherry-LacI-TOPBP1 and mCherry-LacI, and the intensity of mCherry-marked puncta at each time point was quantified and shown (n ≥ 4). (D) Representative micrographs and quantitative analysis of the puncta intensity of mCherry-LacI-TOPBP1/WT, mCherry-LacI-TOPBP1/F1411A, and mCherry-LacI under auranofin treatment (n > 17). (E) Quantitative analysis of time-lapse opto-droplet formation of mCherry-Cry2–tagged proteins under vehicle or auranofin treatment after blue light activation (n > 15). A schematic of opto-droplet induction is shown. (F) Droplet formation of endogenous TOPBP1 under the indicated treatment. The number of larger puncta of nucleolar TOPBP1 under nonstressed conditions and nuclear-wide smaller TOPBP1 foci under hydroxyurea (HU) treatment in HeLa cells was quantified and shown (n > 60). (G) Representative micrographs and quantitative analysis of TOPBP1/WT and TOPBP1/F1411A droplets under the indicated treatment (n > 85). Data are shown as mean ± SD (A–G) from biological triplicate experiments. **P < 0.01; NS, not significant; 1-way ANOVA followed by Tukey’s multiple-comparison test (A, D, F, and G); unpaired 2-tailed t test with Welch’s correction for B and C; 2-way ANOVA for E. Scale bars: 5 μm for A and B; 10 μm for C–G.