CC-90009 activates the GCN2-mediated integrated stress response and subsequent apoptosis in AML. Characterization of the role of (A) GCN2, (B) GCN1, (C) ATF4, and (D) DDIT4 in mediating CC-90009 response using a flow cytometry-based CRISPR competition assay. U937 cells stably expressing Cas9 were infected with a lentiviral vector constitutively coexpressing GFP and sgNT-1, or with lentiviral vectors constitutively coexpressing RFP and sgNT-1, sgNC-8, or 1 of the gene-specific sgRNAs as indicated. Three days after infection, RFP and GFP cells were mixed at a 1:1 ratio and treated with DMSO or 10 μM CC-90009. The change of RFP⁺/GFP⁺ ratio was monitored by flow cytometry every 2 days thereafter. The RFP⁺/GFP⁺ ratios of U937-Cas9 cells coexpressing RFP and sgNT-1, sgNC-8, or 1 of 3 sgRNAs targeting GCN2 (A), GCN1 (B), ATF4 (C), or DDIT4 (D) mixed with cells coexpressing GFP and sgNT-1 at each indicated timepoint were normalized to the RFP⁺/GFP⁺ ratio of the cell mixtures on day 0. (E-F) Immunoblot (E) or quantitative RT-PCR analysis (F) of U937 parental and GCN2^−/− cells treated with DMSO or CC-90009 at the indicated concentrations for 24 hours. The U937 GCN2^−/− cell line is derived from a single clone of U937 parental cells stably infected with a lentiviral CRISPR vector targeting GCN2 (see “Methods and materials”). (G) Immunoblot analysis of U937 parental cells and GCN2^−/− cells with or without a stably transduced lentiviral vector expressing HA-tagged GCN2 wild-type or mutants as indicated. Cells were treated with DMSO or CC-90009 for 24 hours. (H) The effect of CC-90009 on proliferation of cells shown in panel G. On day 3 after CC-90009 treatment, cell proliferation was assessed by CTG. Data in panels F and G are shown as mean ± standard deviation (SD), n = 3 or 4 technical replicates. Result shown in all figure panels is representative of 3 biological replicates.