Combined inhibition of Wee1 and Chk1 gives synergistic DNA damage in S-phase due to distinct regulation of CDK activity and CDC45 loading

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.

Augmentation of the therapeutic efficacy of WEE1 kinase inhibitor AZD1775 by inhibiting the YAP–E2F1–DNA damage response pathway axis

“…Among them, CHEK1 depletion is known to result in the increased sensitivity to AZD1775 [25]. Consistent with this, combination of CHK1 inhibitor with AZD1775 synergistically reduces cancer cell growth [33]. Anyhow, the control of the expression FA components by YAP is a novel finding in this study.…”

Exploiting DNA Replication Stress for Cancer Treatment

“…In addition to driving tumor cells into unscheduled mitosis (68), the cytotoxicity of AZD1775 could also be attributed to the effects WEE1 has on stabilizing stalled replication forks and ensuring timely origin firing, further exacerbating the replication stress in tumor cells when WEE1 is inhibited (69,70). WEE1 inhibitors act synergistically with ATR and CHK1 inhibitors in preclinical studies (71,72), extending the catalogue of possible combinations with other targeted inhibitors that could be implemented. Similar to ATR and CHK1 inhibitors, although AZD1775 has shown single-agent activity in preclinical studies, its efficacy is potentiated when integrated into combination regimens.…”

Inhibition of checkpoint kinase 1 following gemcitabine-mediated S phase arrest results in CDC7- and CDK2-dependent replication catastrophe

“…Other reports suggest that checkpoint inhibition can cause replication catastrophe in a few hours, but those reports rely on cell lines that are sensitive to single-agent CHK1i (e.g. U2OS) (23,24,27,51). The U2OS cells are similar to AsPC-1 (Fig.…”