DNA Replication Dynamics and Cellular Responses to ATP Competitive CDC7 Kinase Inhibitors

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.