Abstract
Wee1 kinase is an inhibitor of cyclin-dependent kinase (cdk)s, crucial cell cycle progression drivers. By phosphorylating cdk1 at tyrosine 15, Wee1 inhibits activation of cyclin B-cdk1 (Cdk1), preventing cells from entering mitosis with incompletely replicated or damaged DNA. Thus, inhibiting Wee1, alone or in combination with DNA damaging agents, can kill cancer cells by mitotic catastrophe, a tumor suppressive response that follows mitosis onset in the presence of under-replicated or damaged DNA. AZD1775, an orally available Wee1 inhibitor, has entered clinical trials for cancer treatment following this strategy, with promising results. Recently, however, AZD1775 has been shown to inhibit also the polo-like kinase homolog Plk1 in vitro, casting doubts on its mechanism of action. Here we asked whether, in the clinically relevant concentration range, AZD1775 inhibited Wee1 or Plk1 in transformed and non-transformed human cells. We found that in the clinically relevant, nanomolar, concentration range AZD1775 inhibited Wee1 rather than Plk1. In addition, AZD1775 treatment accelerated mitosis onset overriding the DNA replication checkpoint and hastened Plk1-dependent phosphorylation. On the contrary selective Plk1 inhibition exerted opposite effects. Thus, at therapeutic concentrations, AZD1775 inhibited Wee1 rather than Plk1. This information will help to better interpret results obtained by using AZD1775 both in the clinical and experimental settings and provide a stronger rationale for combination therapies.
Highlights
Wee1 is a crucial cell cycle kinase that inhibits activation of cyclin-dependent kinases by phosphorylating the cyclin-dependent kinase moiety at inhibitory sites
Cells relyprobed on Wee1 activity implement re separated on isSDS/PAGE, and forto the indicated an important checkpoints ensuring that mitosis onset is delayed until completion of DNA replication or in case ofof
MO, USA), A549 cells were grown in RPMI 1640 medium (Sigma-Aldrich), HTERT-RPE1 cells were grown in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium (Gibco, Thermo Fisher Scientific, Waltham, MA USA), all media supplemented with 10% fetal bovine serum (FBS; GE Healthcare Life Sciences, Pittsburgh, PA, USA), 1% penicillin/streptomycin (Euroclone, Pero, MI, Italy) and incubated in a humidified incubator at 37 ◦ C with 5% CO2
Summary
Wee is a crucial cell cycle kinase that inhibits activation of cyclin-dependent kinases by phosphorylating the cyclin-dependent kinase (cdk) moiety at inhibitory sites. Checkpoint; DRC) or delay mitosis onset in case of DNA damage (DNA Damage Checkpoint; DDC), rely on the action of Wee to inhibit Cdk activation and the onset of mitosis before DNA replication completion or DNA damage repair [2]. If these checkpoints fail, cells enter mitosis with incompletely or damaged DNA. Cells enter mitosis with incompletely or damaged DNA These conditions result in aberrant mitosis that often ends up in cell death or senescence due to a still poorly defined pathway called mitotic catastrophe.
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