WEE1 inhibitors trigger GCN2-mediated activation of the integrated stress response.
The WEE1 kinase negatively regulates CDK1/2 to control DNA replication and mitotic entry. Genetic factors that determine sensitivity to WEE1 inhibition (WEE1i) are largely unknown. A genome-wide insertional mutagenesis screen revealed that mutation of the alternative translation initiation factor EIF2A sensitized to WEE1i. Mechanistically, WEE1i triggers a translational shut-down, which is lethal in combination with the reduced translation of EIF2AKO cells. A genome-wide CRISPR-Cas9 screen revealed that inactivation of integrated stress response (ISR) kinases GCN1/2 rescued WEE1i-mediated cytotoxicity. Accordingly, WEE1i induced GCN2 activation, ATF4 upregulation, and altered ribosome dynamics. Accordingly, loss of the collided ribosome sensor ZNF598 increased sensitivity to WEE1i. Notably, the ISR was not required for WEE1i to induce DNA damage, premature mitotic entry nor sensitization to DNA-damaging chemotherapeutics. Conversely, ISR activation was independent of CDK1/2 activation. WEE1i-mediated ISR activation was independent of WEE1 presence, pointing at off-target effects, which are shared by multiple chemically distinct WEE1 inhibitors. This response was also observed in peripheral blood monocytic cells. Importantly, WEE1 inhibition at doses that do not induce ISR activation still synergized with PKMYT1 inhibition. Taken together, WEE1i triggers cytotoxic ISR activation and translational shutdown, which can be prevented by low-dose treatments, while retaining the cell cycle checkpoint-perturbing effects. Overall design: mRNA-seq  was performed on RPE1 TP53.KO PAC.KO cells after 48 h of treatment with DMSO or 250nM AZD1775 for differential expression analysis.