The antiproliferative effect of CC-122 is solely mediated by the degradation of IKZF1 and IKZF3 in DLBCL. (A-D) Dose response curves of CC-122 in KARPAS-422 (A) and SU-DHL-4 (B) cells ectopically expressing NanoLuc luciferase (NLuc) or degradation-resistant mutants of IKZF1, IKZF3, or ZFP91. Cells were treatment with DMSO or CC-122 at the indicated concentrations for 5 days, followed by cell viability assessment using CellTiter-Glo (CTG). Shown are percentages of CTG luminescence signals relative to DMSO controls. Data are shown as the mean ± standard error of the mean (SEM). KARPAS-422, 2 technical replicates; SU-DHL-4, 3 technical replicates. (C-D) Immunoblot analysis of KARPAS-422 (C) and SU-DHL-4 (D) cells ectopically expressing NLuc or degradation-resistant mutants of IKZF1, IKZF3, or ZFP91. Cells were treated with CC-122 at the indicated concentrations for 24 hours. (E-F) Cell fitness assessment of KARPAS-422 (E) or SU-DHL-4 (F) cells with knockout of IKZF1 alone, IKZF3 alone, or both, compared with control cells using a flow cytometry–based CRISPR competition assay. KARPAS-422 or 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 targeting IKZF1, IKZF3, or both. Three days after infection, RFP and GFP cells were mixed at a 1:1 ratio, and the change in the RFP⁺/GFP⁺ ratio was monitored by flow cytometry every 3 days thereafter. 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 the sgNT-1/sgNT-2 controls at each time point. Data are shown as the mean ± SEM; 3 biological replicates. (G-H) Immunoblot analysis of KARPAS-422 (G) or SU-DHL-4 (H) cells used for the completion assay described in panels E-F.