Hauge Sissel, Naucke Christian, Hasvold Grete, Joel Mrinal, Rødland Gro Elise, Juzenas Petras, Stokke Trond, Syljuåsen Randi G
Department of Radiation Biology, Institute for Cancer Research, Norwegian Radium Hospital, Oslo University Hospital, Oslo, N-0310, Norway.
Oncotarget. 2017 Feb 14;8(7):10966-10979. doi: 10.18632/oncotarget.14089.
Recent studies have shown synergistic cytotoxic effects of simultaneous Chk1- and Wee1-inhibition. However, the mechanisms behind this synergy are not known. Here, we present a flow cytometry-based screen for compounds that cause increased DNA damage in S-phase when combined with the Wee1-inhibitor MK1775. Strikingly, the Chk1-inhibitors AZD7762 and LY2603618 were among the top candidate hits of 1664 tested compounds, suggesting that the synergistic cytotoxic effects are due to increased S-phase DNA damage. Combined Wee1- and Chk1-inhibition caused a strong synergy in induction of S-phase DNA damage and reduction of clonogenic survival. To address the underlying mechanisms, we developed a novel assay measuring CDK-dependent phosphorylations in single S-phase cells. Surprisingly, while Wee1-inhibition alone induced less DNA damage compared to Chk1-inhibition, Wee1-inhibition caused a bigger increase in S-phase CDK-activity. However, the loading of replication initiation factor CDC45 was more increased after Chk1- than Wee1-inhibition and further increased by the combined treatment, and thus correlated well with DNA damage. Therefore, when Wee1 alone is inhibited, Chk1 suppresses CDC45 loading and thereby limits the extent of unscheduled replication initiation and subsequent S-phase DNA damage, despite very high CDK-activity. These results can explain why combined treatment with Wee1- and Chk1-inhibitors gives synergistic anti-cancer effects.
最近的研究表明，同时抑制Chk1和Wee1具有协同细胞毒性作用。然而，这种协同作用背后的机制尚不清楚。在这里，我们展示了一种基于流式细胞术的筛选方法，用于筛选与Wee1抑制剂MK1775联合使用时会在S期导致DNA损伤增加的化合物。令人惊讶的是，Chk1抑制剂AZD7762和LY2603618是1664种测试化合物中的顶级候选命中物，这表明协同细胞毒性作用是由于S期DNA损伤增加所致。联合抑制Wee1和Chk1在诱导S期DNA损伤和降低克隆形成存活率方面产生了强烈的协同作用。为了探究潜在机制，我们开发了一种新的检测方法，用于测量单个S期细胞中CDK依赖性磷酸化。令人惊讶的是，虽然单独抑制Wee1与抑制Chk1相比诱导的DNA损伤较少，但抑制Wee1会导致S期CDK活性有更大的增加。然而，Chk1抑制后复制起始因子CDC45的加载比Wee1抑制后增加得更多，并且联合处理进一步增加，因此与DNA损伤密切相关。因此，当单独抑制Wee1时，尽管CDK活性非常高，但Chk1会抑制CDC45的加载，从而限制计划外复制起始的程度和随后的S期DNA损伤。这些结果可以解释为什么联合使用Wee1和Chk1抑制剂会产生协同抗癌效果。
