Wee1 Rather Than Plk1 Is Inhibited by AZD1775 at Therapeutically Relevant Concentrations

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.

Antitumor effect of a WEE1 inhibitor and potentiation of olaparib sensitivity by DNA damage response modulation in triple-negative breast cancer

“…MDA-MB-231 and BT-549 cells, which had IC 50 values of <0.5 µmol/L were deemed sensitive, and MDA-MB-468 cells, which had an IC 50 value of >0.5 were deemed moderately sensitive. According to previous report 23 , diminished phosphorylations of CDK1 and CDK2 (direct targets of WEE1) confirmed AZD1775 effectively downregulated target kinase activity ( Supplementary Fig. S1a,b).…”

Compartmentalized control of Cdk1 drives mitotic spindle assembly

“…Indeed, addition of RO-3306 at 0.5 mM (low-RO), from 60 to 80 min post G2 release, substantially restored spindle assembly (Figure 1A). Defects in spindle assembly were also induced by cdk1AF overexpression under similar conditions of G2-arrest and release, paralleling what was described by Krek and Nigg in 1991, as well as in Wee1-siRNA-treated HeLa cells or by chemical inhibition of Wee1, and in all cases, defects were substantially reversed by low-RO (Krek and Nigg, 1991;Serpico et al, 2019;Figures S2, S3 and S4A). In another set of experiments, non-targeting-siRNA-, as control, and Wee1-siRNA-treated hTERT-RPE1 cells were arrested at prometaphase by the reversible microtubule inhibitor nocodazole added shortly after siRNA treatments.…”

Upregulation of Myt1 Promotes Acquired Resistance of Cancer Cells to Wee1 Inhibition

“…In addition to Wee1, adavosertib also exhibits activity against Polo-like kinse-1 (Plk1) in some cell types including small-cell lung carcinoma cells (44,45). However, adavosertib treatment induces premature mitosis in cells consistent with the inhibition of Wee1, whereas inhibition of Plk1 induces a G 2 arrest (46).…”