Rainey Michael D, Quachthithu Huong, Gaboriau David, Santocanale Corrado
Centre for Chromosome Biology, School of Natural Sciences, National University of Ireland Galway H91 TK33, Ireland.
ACS Chem Biol. 2017 Jul 21;12(7):1893-1902. doi: 10.1021/acschembio.7b00117. Epub 2017 Jun 7.
The CDC7 kinase, by phosphorylating the MCM DNA helicase, is a key switch for DNA replication initiation. ATP competitive CDC7 inhibitors are being developed as potential anticancer agents; however how human cells respond to the selective pharmacological inhibition of this kinase is controversial and not understood. Here we have characterized the mode of action of the two widely used CDC7 inhibitors, PHA-767491 and XL-413, which have become important tool compounds to explore the kinase's cellular functions. We have used a chemical genetics approach to further characterize pharmacological CDC7 inhibition and CRISPR/CAS9 technology to assess the requirement for kinase activity for cell proliferation. We show that, in human breast cells, CDC7 is essential and that CDC7 kinase activity is formally required for proliferation. However, full and sustained inhibition of the kinase, which is required to block the cell-cycle progression with ATP competitor compounds, is problematic to achieve. We establish that MCM2 phosphorylation is highly sensitive to CDC7 inhibition and, as a biomarker, it lacks in dynamic range since it is easily lost at concentrations of inhibitors that only mildly affect DNA synthesis. Furthermore, we find that the cellular effects of selective CDC7 inhibitors can be altered by the concomitant inhibition of cell-cycle and transcriptional CDKs. This work shows that DNA replication and cell proliferation can occur with reduced CDC7 activity for at least 5 days and that the bulk of DNA synthesis is not tightly coupled to MCM2 phosphorylation and provides guidance for the development of next generation CDC7 inhibitors.
细胞周期蛋白依赖性激酶7（CDC7激酶）通过磷酸化微小染色体维持蛋白（MCM）DNA解旋酶，成为DNA复制起始的关键开关。ATP竞争性CDC7抑制剂正作为潜在的抗癌药物进行研发；然而，人类细胞如何响应这种激酶的选择性药理学抑制存在争议且尚不明确。在此，我们已对两种广泛使用的CDC7抑制剂PHA - 767491和XL - 413的作用模式进行了表征，它们已成为探索该激酶细胞功能的重要工具化合物。我们采用化学遗传学方法进一步表征药理学上的CDC7抑制作用，并利用CRISPR/CAS9技术评估激酶活性对细胞增殖的必要性。我们表明，在人乳腺细胞中，CDC7是必需的，并且CDC7激酶活性对于细胞增殖正式来说是必需的。然而，要使用ATP竞争性化合物阻断细胞周期进程，就需要对该激酶进行完全且持续的抑制，而这很难实现。我们确定MCM2磷酸化对CDC7抑制高度敏感，并且作为一种生物标志物，它缺乏动态范围，因为在仅轻微影响DNA合成的抑制剂浓度下它就很容易消失。此外，我们发现，细胞周期和转录周期蛋白依赖性激酶（CDK）的同时抑制可改变选择性CDC7抑制剂的细胞效应。这项工作表明，在CDC7活性降低的情况下，DNA复制和细胞增殖至少可持续5天，并且大部分DNA合成与MCM2磷酸化没有紧密耦合，为下一代CDC7抑制剂的开发提供了指导。
