WEE1’s established role is well documented. It phosphorylates and inhibits cyclin-dependent kinases, preventing entry into mitosis and establishing the G2 DNA damage checkpoint. Because tumours with TP53 mutations become reliant on this WEE1-mediated checkpoint, WEE1 inhibitors have been designed to be synthetically lethal in those cancers, and such inhibitors are currently undergoing clinical trials. Yet the picture has never been tidy. WEE1 inhibitors also show efficacy in tumours with wild-type TP53, which the synthetic lethality logic does not predict, and many trials have been terminated because of high levels of toxic side effects. The authors of the new study argue that both anomalies point in the same direction: WEE1 must have functions that the field has not accounted for.

The new function emerged from experiments in which the team removed WEE1’s activity while quiescent cells were trying to re-enter the cycle. Cells with loss of WEE1 function during G0 and G1 showed delayed entry into S phase, the period of DNA replication, and an impaired G1/S transition. They also accumulated the cyclin-dependent kinase inhibitor p21^Cip1/Kip1 abnormally during S phase, and this led to a p21-dependent arrest in G2.

The mechanism the researchers uncovered is molecularly precise. WEE1 phosphorylates CDH1, the protein encoded by the FZR1 gene, at key residues that mediate the interaction between CDH1 and the APC/C core. Using in vitro kinase assays with recombinant proteins and mass spectrometry, the team identified two tyrosine phosphorylation sites on the N-terminal region of human CDH1, at residues Y91 and Y130, both confirmed in peptides analysed by data-independent acquisition mass spectrometry. The Y130 site sits directly adjacent to threonine 129, a residue that the growth-regulating kinase mTOR phosphorylates to transiently inactivate APC/C^CDH1, and serine 131 phosphorylation has also been detected in cells. The authors suggest this motif may be a key regulatory hotspot, probably involving stabilisation of the interaction between the C-box of CDH1 and the APC8 subunit of the APC/C, and they propose that mTOR phosphorylation of Thr129 may precede WEE1 phosphorylation of Tyr130.

To test what those phosphorylations actually do, the team turned to cell-free systems derived from Xenopus eggs. Wild-type human CDH1 and tyrosine mutant variants were produced and mixed with interphase extracts, after which endogenous APC/C was immunoprecipitated and CDH1-mediated substrate destruction was measured using radiolabelled APC/C substrates, with full-length cyclin B as a positive control and a D-box-deleted cyclin B as a negative control. Complementing these biochemical assays, the researchers engineered human cell lines in which endogenous WEE1 was tagged with GFP and an auxin-inducible degron, allowing acute, doxycycline- and IAA-triggered degradation of WEE1 precisely when cells were released from a G0/G1 arrest induced by CDK4/6 inhibition with palbociclib.

Single-cell live imaging then traced the consequences. The experimental data fit best with a model in which WEE1’s inhibition of APC/C^CDH1 is independent of the better-known CDK2-mediated inhibition of the complex. The logic of the overall circuit, as the authors lay out in their discussion, resolves an apparent conundrum: WEE1 inhibits CDK2 to prevent S phase entry, yet it also inhibits APC/C^CDH1 to promote S phase entry. By restraining APC/C^CDH1, WEE1 permits the phosphatase CDC25A to accumulate, and CDC25A in turn removes WEE1’s inhibitory phosphorylation from CDK2, tipping the balance toward S phase. WEE1 may be required for the initial inhibition of APC/C^CDH1 before CDK2 itself takes over CDH1 phosphorylation and degradation.

The team also built a mathematical model of the G1/S regulatory network, a system of coupled nonlinear differential equations describing the biochemical species that control the G1 and S states, and compared its predictions with the data. Most predictions held, but two discrepancies were candidly reported. The model expects CDC25A levels to rise after S phase entry as APC/C^CDH1 switches off at the G1/S transition, but this post-G1/S increase does not occur in cells, most likely because CDC25A is degraded through CHK1-mediated phosphorylation in S phase. The model also predicts high CDK2 activity and Rb inhibition in cells arrested at very low WEE1 activity, which was not observed, possibly because cyclin-activating kinase activity is reduced in arrested cells. These gaps, the authors note, mark questions for future work, including mutation of the WEE1 and CDK phosphorylation sites in CDH1 and identification of the tyrosine phosphatases that act on CDH1.

The findings have direct implications for WEE1 inhibitor drugs such as MK-1775, the inhibitor used throughout the study. In dose-response experiments across cell lines including hTert-RPE1, MCF7, KU1919 and HBLAK, reducing the expression of APC/C^CDH1 or of p21 made cells more sensitive to acute WEE1 inhibition. Conversely, when the researchers generated MCF7 cells resistant to the drug by chronic exposure with escalating doses up to 0.6 micromolar, RNA sequencing revealed that both the p53-p21 pathway and APC/C^CDH1 pathway components were downregulated in the resistant cells, suggesting adaptation to chronic drug pressure. The authors therefore propose that CDKN1A, the gene encoding p21, and APC/C^CDH1 pathway activity merit investigation as biomarkers for WEE1 inhibitor-based therapies, including in CDKN1A-mutant bladder cancer.

The toxicity question may also find an answer in quiescence. Side effects reported in clinical trials of WEE1 inhibitors, including myelosuppression, gastrointestinal problems and fatigue, have often been attributed to off-target effects on the kinase PLK1. The authors offer an alternative explanation: if WEE1 is needed for quiescent cells to re-enter proliferation, then inhibiting it in healthy tissues could raise APC/C^CDH1 activity in quiescent stem cells and impair their ability to resume proliferation and maintain tissue function. That mechanism, they suggest, could account for at least part of the observed toxicity in proliferative tissues such as bone marrow and gut.

It is worth being clear about the scope of the evidence. This is preclinical work conducted in cultured human cell lines, engineered reporter lines, in vitro kinase assays and cell-free extracts from Xenopus eggs, supported by computational modelling; it does not establish clinical outcomes in patients. The biomarker proposals and the toxicity hypothesis are framed by the authors as directions for further investigation, not as demonstrated clinical results. Even so, the study reframes a drug target that oncology has been attacking for a decade. WEE1 is not simply a brake that cancers exploit; it is also, paradoxically, a gatekeeper that resting cells need in order to start dividing again. Drugs designed to remove the brake may, in some tissues, also remove the gatekeeper, and understanding that dual role, the authors argue, should shape how WEE1 inhibitors are used in the clinic.

Subject of Research: How the WEE1 kinase promotes S phase entry from quiescence by inhibiting the APC/C^CDH1 ubiquitin ligase, with implications for WEE1 inhibitor toxicity and biomarkers

Article Title: WEE1 opposes APC/CCDH1 activity to promote S phase entry

Article References: Vuillemenot, L.-A., Morales, L. G., Dragoi, C.-M., Pennycook, B. R., Harris, E., Lin, L. C., Weston, W. A., Brown, J.-E., Graham, E., Moulin, L., Andrew, I., Roumelioti, G., Montoya, A., Game, L., Young, G. R., Datta, K. K., Shliaha, P., Novák, B., Yamano, H., & Barr, A. R. (2026). WEE1 opposes APC/CCDH1 activity to promote S phase entry. Nature. https://doi.org/10.1038/s41586-026-11188-x

Image Credits: AI Generated

DOI: 10.1038/s41586-026-11188-x

Keywords: WEE1, APC/C^CDH1, CDH1, cell cycle, quiescence, S phase entry, p21, CDC25A, WEE1 inhibitors, MK-1775, biomarkers, CDK2