Inactivation of NF-κB repressors confers resistance to CC-122. (A) Cell fitness assessment of SU-DHL-4 cells with knockout of CRBN, CYLD, NFKBIA, TRAF2, or TRAF3 in the presence or absence of CC-122, compared with control cells using a flow cytometry–based CRISPR competition assay. SU-DHL-4 cells stably expressing Cas9 were transduced with a lentiviral vector coexpressing GFP and sgNT-1, or with lentiviral vectors coexpressing RFP and the indicated sgRNAs. Three days after infection, RFP and GFP cells were mixed at a 1:1 ratio and treated with DMSO or CC-122 at the indicated concentrations. The change in the RFP⁺/GFP⁺ ratio was then monitored by flow cytometry at the indicated time points. RFP⁺/GFP⁺ ratios of cells after transduction with the indicated sgRNA were first normalized to their RFP⁺/GFP⁺ ratios on day 0 and then to DMSO controls at each time point. Data are shown as mean ± standard deviation (SD); 2 biological replicates. Data were analyzed by 2-tailed unpaired Student t test. *P < .05; **P < .01; ***P < .001; NS (not significant), P > .05. (B) Immunoblot analysis of SU-DHL-4-Cas9 cells transduced with lentiviral vectors expressing the indicated sgRNAs. Three days after transduction, cells were treated with 2 µg/mL puromycin for 3 additional days to select sgRNA-expressing cells. (C) Immunoblot analysis of nuclear protein extractions of SU-DHL-4 cells transduced with the indicated sgRNAs. The histone H3 level was used as the loading control. (D) Heat map showing the CC-122–induced enrichment of DLBCL cells with loss of CRBN or NF-κB repressors in the CRISPR competition assay. The enrichment score is calculated as the FC of the RFP⁺/GFP⁺ ratios between cells treated with 50 µM CC-122 vs their DMSO controls on day 10. (E) Immunoblot analysis of SU-DHL-4-Cas9 cells transduced with lentiviral vectors expressing the indicated sgRNAs. Cells were treated with either DMSO or 10 µM CC-122 for 24 hours. (F) Flow cytometry analysis of SU-DHL-4 cells with expression of the indicated sgRNA. Cells were continuously treated with DMSO or 50 µM CC-122 for 10 days, followed by staining with annexin V and 7-AAD.