GZD824 inhibits GCN2 and sensitizes cancer cells to amino acid starvation stress.

Eukaryotic initiation factor 2α kinase general control non-derepressible 2 (GCN2) drives cellular adaptation to amino acid limitation by activating the integrated stress response that induces activating transcription factor 4 (ATF4). Here, we found that a multi-kinase inhibitor, GZD824, which we identified using a cell-based assay with ATF4 immunostaining, inhibited the GCN2 pathway in cancer cells. Indeed, GZD824 suppressed GCN2 activation, eIF2α phosphorylation, and ATF4 induction during amino acid starvation stress. However, at lower non-suppressive concentrations, GZD824 paradoxically stimulated eIF2α phosphorylation and ATF4 expression in a GCN2-dependent manner under unstressed conditions. Such dual properties conceivably arose from a direct effect on GCN2, as also observed in a cell-free GCN2 kinase assay and shared by a selective GCN2 inhibitor. Consistent with the GCN2 pathway inhibition, GZD824 sensitized certain cancer cells to amino acid starvation stress similarly to ATF4 knockdown. These results establish GZD824 as a multi-kinase GCN2 inhibitor and may enhance its utility as a drug under development. Significance Statement GZD824, as a direct GCN2 inhibitor, suppresses activation of the integrated stress response during amino acid limitation, while it paradoxically stimulates this stress-signaling pathway at lower non-suppressive concentrations. The pharmacological activity, we identified herein, will provide the basis for the use of GZD824 to elucidate the regulatory mechanisms of GCN2 and to evaluate the potential of the GCN2-ATF4 pathway as a target for cancer therapy.