Inhibition of topoisomerase 1 and BRD4-mediated pause release selectively kills tumours by inducing readthrough transcription [SLAM-seq]

Transcription inhibition has become an attractive strategy for anticancer therapy because many cancers arise from dysregulated transcription programs. Topoisomerase 1 (TOP1) and bromodomain containing protein 4 (BRD4) coordinate each other to regulate transcriptional elongation. Using a collection of patient-derived xenograft mouse models of pancreatic cancer we show that the combined inhibition of TOP1 and BRD4 is synergistic in arresting tumor growth. We link this effect to the ability of TOP1 and BRD4 to control pause-release, a rate-limiting step in early elongation. By comparing nascent transcripts with the recruitment of elongation and termination factors along genes, we found that combined inhibition of TOP1 and BRD4, while globally impairing RNA production, also disturbs the recruitment of proteins involved in termination. Thus, RNA polymerases stay engaged with the DNA downstream of polyadenylation sites for hundreds of kilobases leading to readthrough-transcription. Because this pervasive transcription occurs also during replication, it perturbs replisome progression leading to increased DNA damage and cell cycle arrest. The synergistic effect of TOP1 and BRD4 inhibition is specific for the tumor cells as negligible transcriptional defects are observed in non-tumor cells. This work provides a mechanistic understanding of the benefit of combining TOP1 and BRD4 inhibitors in the treatment of highly transcription- and replication-driven tumors. RNA-seq with extracts from Bo103 cells.

转录抑制已成为颇具前景的抗癌治疗策略，因为诸多癌症均源于转录程序失调。拓扑异构酶1（Topoisomerase 1，简称TOP1）与含溴结构域蛋白4（bromodomain containing protein 4，简称BRD4）相互协同，共同调控转录延伸过程。本研究借助一系列胰腺癌患者来源的异种移植小鼠模型（patient-derived xenograft mouse models）证实，联合抑制TOP1与BRD4可协同抑制肿瘤生长。我们将该效应与二者调控暂停释放（pause-release）的能力相关联——这是转录早期延伸中的限速步骤。通过比较新生转录本（nascent transcripts）与基因上延伸因子及终止因子的招募情况，我们发现，联合抑制TOP1与BRD4不仅会全面损害RNA生成，还会干扰参与转录终止的蛋白质招募。因此，RNA聚合酶会持续结合聚腺苷酸化位点下游长达数千碱基对的DNA区域，进而引发通读转录（readthrough-transcription）。由于这种广泛转录同样发生在细胞复制过程中，它会扰乱复制体（replisome）的行进，导致DNA损伤加剧与细胞周期阻滞。联合抑制TOP1与BRD4的协同效应仅特异性作用于肿瘤细胞，在非肿瘤细胞中仅观察到可忽略的转录缺陷。本研究为联合使用TOP1与BRD4抑制剂治疗高度依赖转录与复制的肿瘤提供了机制层面的阐释。本研究使用Bo103细胞的提取物开展了RNA测序（RNA-seq）实验。