Forced Mitotic Entry of S-Phase Cells as a Therapeutic Strategy Induced by Inhibition of WEE1

Abstract

Inhibition of the protein kinase WEE1 synergizes with chemotherapy in preclinical models and WEE1 inhibitors are being explored as potential cancer therapies. Here, we investigate the mechanism that underlies this synergy. We show that WEE1 inhibition forces S-phase-arrested cells directly into mitosis without completing DNA synthesis, resulting in highly abnormal mitoses characterized by dispersed chromosomes and disorganized bipolar spindles, ultimately resulting in mitotic exit with gross micronuclei formation and apoptosis. This mechanism of cell death is shared by CHK1 inhibitors, and combined WEE1 and CHK1 inhibition forces mitotic entry from S-phase in the absence of chemotherapy. We show that p53/p21 inactivation combined with high expression of mitotic cyclins and EZH2 predispose to mitotic entry during S-phase with cells reliant on WEE1 to prevent premature cyclin-dependent kinase (CDK)1 activation. These features are characteristic of aggressive breast, and other, cancers for which WEE1 inhibitor combinations represent a promising targeted therapy. SIGNIFICANCE:Here, we describe a novel mechanism of inducing cancer cell death by WEE1 inhibition, forcing mitotic entry directly from S-phase. This mechanism represents a potential therapeutic approach for aggressive breast cancers, and in particular triple-negative and basal-like breast cancers, as WEE1 inhibition specifi cally targets the features inherent in these cancers: frequent TP53 mutation and high expression of mitotic cyclins and the polycomb protein EZH2. Cancer Discov; 2(6); 524-39.

Cytokinetic effects of Wee1 disruption in pancreatic cancer

“…2B). Flow cytometry revealed dramatic changes, similar to those reported by Aorts et al, 19 with the appearance of cells positively labeled for Cyclin A2, Cyclin B1, and phosphorylated H3S10 in early S-phase (Fig. 2C).…”

“…The effects of MK-1775 on OCIP23 cells were much more dramatic than Wee1 knockdown, with the accumulation of cells in early S-phase showing H3S10 phosphorylation, associated with high levels of Cyclin A2 and Cyclin B1 that are normally highly expressed in late S-phase and G2. These effects have been previously observed, and attributed to premature entry of S-phase cells into mitosis, 19,20 which is consistent with the confocal microscopy features seen in Figure 2B. However, we did not observe them in the OCIP23 xenografts, even at the high dose of 80mg/kg (for comparison, the human maximum tolerated dose of 225mg twice daily 13 is roughly 6mg/kg), and it remains unclear if forced mitotic entry is a significant drug effect in humans.…”

“…The CDK2/CyclinA2 results are consistent with previous results, where depletion of WEE1 in the presence of roscovitine has been shown to rescue the WEE1-depletion phenotype. 19 Thus, CDK2/CyclinA2 is required for the observed phenotype and sensitivity in the presence of MK-1775 (Fig. 7F).…”

“…18 Subsequently it was reported that treatment with the clinical Wee1 inhibitor MK-1775 (AZD-1775) likewise had striking cytokinetic effects, including features suggestive of premature entry into mitosis during early S-phase. 19,20 Of the Wee1 inhibitors, MK-1775 is currently the most advanced in clinical development, and it is reported to be highly selective toward Wee1 (21). As a single agent it appears to be well tolerated, but has little anti-cancer activity.…”

Strong antitumor synergy between DNA crosslinking and HSP90 inhibition causes massive premitotic DNA fragmentation in ovarian cancer cells

“…The extent of chromosome fragmentation per cell was analyzed by counting the fragments in 10 randomly chosen metaphases. Significance was determined by the Student's t-test and the P-value is shown by asterisks (***P-valueo0.001) 25,41,42 In this scenario,…”