BACKGROUND The effective treatment of triple-negative breast cancer (TNBC) remains a profound clinical challenge. Despite frequent epidermal growth factor receptor (EGFR) overexpression and reliance on downstream signalling pathways in TNBC, resistance to EGFR-tyrosine kinase inhibitors (TKIs) remains endemic. Therefore, the identification of targeted agents, which synergise with current therapeutic options, is paramount. METHODS Compound-based, high-throughput, proliferation screening was used to profile the response of TNBC cell lines to EGFR-TKIs, western blotting and siRNA transfection being used to examine the effect of inhibitors on EGFR-mediated signal transduction and cellular dependence on such pathways, respectively. A kinase inhibitor combination screen was used to identify compounds that synergised with EGFR-TKIs in TNBC, utilising sulphorhodamine B (SRB) assay as read-out for proliferation. The impact of drug combinations on cell cycle arrest, apoptosis and signal transduction was assessed using flow cytometry, automated live-cell imaging and western blotting, respectively. RNA sequencing was employed to unravel transcriptomic changes elicited by this synergistic combination and to permit identification of the signalling networks most sensitive to co-inhibition. RESULTS We demonstrate that a dual cdc7/CDK9 inhibitor, PHA-767491, synergises with multiple EGFR-TKIs (lapatinib, erlotinib and gefitinib) to overcome resistance to EGFR-targeted therapy in various TNBC cell lines. Combined inhibition of EGFR and cdc7/CDK9 resulted in reduced cell proliferation, accompanied by induction of apoptosis, G2-M cell cycle arrest, inhibition of DNA replication and abrogation of CDK9-mediated transcriptional elongation, in contrast to mono-inhibition. Moreover, high expression of cdc7 and RNA polymerase II Subunit A (POLR2A), the direct target of CDK9, is significantly correlated with poor metastasis-free survival in a cohort of breast cancer patients. RNA sequencing revealed marked downregulation of pathways governing proliferation, transcription and cell survival in TNBC cells treated with the combination of an EGFR-TKI and a dual cdc7/CDK9 inhibitor. A number of genes enriched in these downregulated pathways are associated with poor metastasis-free survival in TNBC. CONCLUSIONS Our results highlight that dual inhibition of cdc7 and CDK9 by PHA-767491 is a potential strategy for targeting TNBC resistant to EGFR-TKIs.

中文翻译：

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激酶抑制剂筛选确定了一种 cdc7/CDK9 双重抑制剂，可使三阴性乳腺癌对 EGFR 靶向治疗敏感。


背景技术三阴性乳腺癌（TNBC）的有效治疗仍然是一个严峻的临床挑战。尽管 TNBC 中经常出现表皮生长因子受体 (EGFR) 过度表达并依赖下游信号通路，但对 EGFR 酪氨酸激酶抑制剂 (TKI) 的耐药性仍然普遍存在。因此，鉴定与当前治疗方案具有协同作用的靶向药物至关重要。方法 使用基于化合物的高通量增殖筛选来分析 TNBC 细胞系对 EGFR-TKI 的反应，使用蛋白质印迹和 siRNA 转染来检查抑制剂对 EGFR 介导的信号转导的影响以及细胞对此类信号转导的依赖性。路径，分别。激酶抑制剂组合筛选用于鉴定在 TNBC 中与 EGFR-TKI 协同作用的化合物，并利用磺罗丹明 B (SRB) 测定作为增殖读数。分别使用流式细胞术、自动活细胞成像和蛋白质印迹评估药物组合对细胞周期停滞、细胞凋亡和信号转导的影响。 RNA测序被用来揭示这种协同组合引起的转录组变化，并允许鉴定对共抑制最敏感的信号网络。结果我们证明，cdc7/CDK9 双重抑制剂 PHA-767491 与多种 EGFR-TKI（拉帕替尼、厄洛替尼和吉非替尼）具有协同作用，可克服各种 TNBC 细胞系对 EGFR 靶向治疗的耐药性。与单一抑制相比，EGFR 和 cdc7/CDK9 的联合抑制导致细胞增殖减少，并伴有细胞凋亡诱导、G2-M 细胞周期停滞、DNA 复制抑制和 CDK9 介导的转录延伸终止。 此外，cdc7 和 RNA 聚合酶 II 亚基 A (POLR2A)（CDK9 的直接靶标）的高表达与乳腺癌患者队列中较差的无转移生存率显着相关。 RNA测序显示，在联合使用EGFR-TKI和cdc7/CDK9抑制剂治疗的TNBC细胞中，控制增殖、转录和细胞存活的通路显着下调。这些下调途径中富集的许多基因与 TNBC 中较差的无转移生存率相关。结论 我们的结果强调，PHA-767491 对 cdc7 和 CDK9 的双重抑制是针对 EGFR-TKI 耐药的 TNBC 的潜在策略。