WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response

  • Nat Commun. 2025 Nov 24;16(1):11598. doi: 10.1038/s41467-025-66514-0.
Rinskje B Tjeerdsma  #  1 ,  Timothy F Ng  #  2  3 ,  Maurits Roorda  #  1 ,  Daniëlle Bianchi  4 ,  Sora Yang  5 ,  Clara Bonnet  2 ,  Michael VanInsberghe  5 ,  Marieke Everts  1 ,  Femke J Bakker  1 ,  H Rudolf de Boer  1 ,  Nathalie Moatti  2 ,  Nicole Hustedt  2 ,  Jay Z Yin  2  3 ,  Lisa Hoeg  2 ,  Matthew Leibovitch  6 ,  Frank Sicheri  2  7 ,  Alexander van Oudenaarden  5 ,  Steven de Jong  1 ,  Jeroen van den Berg  5 ,  Marvin E Tanenbaum  5  8 ,  Thijn R Brummelkamp  4 ,  Daniel Durocher  9  10 ,  Marcel A T M van Vugt  11
Affiliations
  • 1. Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands.
  • 2. The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada.
  • 3. Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.
  • 4. Oncode Institute, Division of Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands.
  • 5. Oncode Institute, Hubrecht Institute-KNAW and University Medical Center Utrecht, Utrecht, The Netherlands.
  • 6. Lady Davis Institute, Sir Mortimer B. Davis Jewish General Hospital, Montréal, QC, Canada.
  • 7. Department of Biochemistry, University of Toronto, Toronto, ON, Canada.
  • 8. Department of Bionanoscience, Kavli Institute of Nanoscience Delft, Delft University of Technology, Delft, the Netherlands.
  • 9. The Lunenfeld-Tanenbaum Research Institute, Mount Sinai Hospital, Toronto, ON, Canada. [email protected].
  • 10. Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada. [email protected].
  • 11. Department of Medical Oncology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands. [email protected].
  • # Contributed equally.
PMID: 41285872 DOI: 10.1038/s41467-025-66514-0
Abstract

The Wee1 kinase negatively regulates CDK1/2 to control DNA replication and mitotic entry. Genetic factors that determine sensitivity to Wee1 inhibitors (WEE1i) are largely unknown. A genome-wide insertional mutagenesis screen revealed that mutation of EIF2A, a translation regulator, sensitized to WEE1i. Additionally, a genome-wide CRISPR-Cas9 screen revealed that inactivation of integrated stress response (ISR) kinase GCN2 or its co-factor GCN1 rescued WEE1i-mediated cytotoxicity. Conversely, loss of the collided ribosome sensor ZNF598 increased sensitivity to WEE1i. Mechanistically, WEE1i induced paradoxical GCN2 activation, ATF4 upregulation, and altered ribosome dynamics. ISR activation was independent of Wee1 presence, pointing at off-target GCN2 engagement by multiple chemically distinct WEE1i. ISR activation was observed in Cancer cells as well as non-transformed cells, and required GCN1 and ongoing translation. Consequently, WEE1i induce multiple independent cellular effects: DNA damage, premature mitotic entry and sensitization to DNA-damaging chemotherapeutics in an ISR-independent fashion, as well as ISR activation independently of CDK1/2 activation. Importantly, low-dose Wee1 inhibition did not induce ISR activation, while it still synergized with PKMYT1 inhibition. Taken together, WEE1i trigger toxic ISR activation and translational shutdown, which can be prevented by low-dose or combination treatments, while retaining the cell cycle checkpoint-perturbing effects.

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