<i>In Vivo</i> Tumor Growth Control by General Control Nonderepressible 2–Targeting Agents Results from Kinase Activation

General control nonderepressible 2 (GCN2; EIF2AK4) is a serine-threonine kinase in the integrated stress response signaling pathway that initiates adaptive responses during nutrient stress conditions. Although pharmacologic inhibition of GCN2 under nutrient stress conditions induces apoptosis and inhibits tumor growth, GCN2 inhibition without nutrient stress has been reported to have no effect on tumor growth. By exploring an array of GCN2 inhibitors, we demonstrate that multiple agents in fact activate GCN2 in biochemical and cell-based assays at low concentrations and inhibit GCN2 at higher concentrations. Unexpectedly, it is this activation, and not inhibition, of the GCN2 pathway that is associated with decreased viability in vitro and tumor growth inhibition in vivo across multiple models. Knockdown and knockout experiments show that activation of the integrated stress response by GCN2-targeting agents is dependent on GCN2. ISRIB, a modulator of eIF2B, ablates the viability effect, demonstrating the dependence on translation initiation. Activating doses result in the induction of cleaved caspase 3 and cleaved PARP. In contrast, a nonactivating GCN2-targeting agent does not affect viability. These results provide a clearer understanding of the challenges and opportunities for the clinical development of compounds targeting GCN2.