Abstract
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.
Highlights
Inhibitors of Wee1 kinase are currently in clinical trials for cancer treatment as single agents and in combination with radiation or chemo-therapy [1]
Our results suggest that Checkpoint kinase 1 (Chk1) limits the induction of DNA damage after Wee1 inhibition by suppressing CDC45 loading
We previously found that siRNA mediated partial depletion of either CDK1 or CDK2 reduced the S-phase DNA damage upon Wee1, as well as Chk1, inhibition, suggesting that both CDK1 and CDK2 activities contribute to the effects [7]
Summary
Inhibitors of Wee kinase are currently in clinical trials for cancer treatment as single agents and in combination with radiation or chemo-therapy [1]. Wee inhibition leads to abnormally high CDK1 activity, resulting in G2 checkpoint abrogation followed by mitotic catastrophe [4, 5]. Inhibition of Wee leads to high CDK1 and CDK2 activity in S-phase followed by unscheduled replication initiation This results in shortage of replication factors such as nucleotides and replication factor A (RPA), and subsequent replication stalling and endonuclease-induced DNA breakage [7, 9, 10]. Such S-phase damage has been termed “replication catastrophe” [10] and is most likely the major cause behind single-agent antitumor activity of Wee inhibitors [11]
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