ALCLs are addicted to IRF4. (A) IRF4 shRNA #1 and #2 downregulate IRF4 mRNA in K299, JB6, DEL, and Mac-2A cells 48 hours after shRNA induction measured by quantitative PCR. IRF4 mRNA levels were normalized to expression of GAPDH. Error bars indicate standard deviation. (B) IRF4 shRNA #1 and #2 downregulate IRF4 protein in K299, JB6, DEL, and Mac-2A cells 96 hours after shRNA induction measured by western blotting. (C) IRF4 knockdown by two independent shRNAs is toxic to IRF4-positive ALCL cell lines. In contrast, the IRF4 low expressing ALCL cell line SU-DHL-1, the SS cell line HuT 78, and IRF4-negative T-ALL cell lines are not affected by IRF4 downregulation. Representative results from at least 2 independent replicates are shown. Baseline expression of IRF4 in the investigated cell lines is indicated based on western blotting (Figure 1C) and the ALK translocation status is indicated. (D) Exogenous expression of IRF4 cDNA rescues K299, FE-PD, and Mac-2A cells from IRF4 shRNA-induced toxicity. Representative results from 3 independent replicates are shown. (E) IRF4 and MYC knockdown is detectable by western blotting in mouse xenograft (K299 and JB6) tumor biopsies from cells transduced with IRF4 shRNA #1 compared with control shRNA transduced cells (shRNA directed against MSMO1). (F) Tumor growth curve of K299 and JB6 xenograft mouse models that inducibly express IRF4 shRNA #1 (blue) or a control shRNA directed against MSMO1 (red). IRF4 knockdown significantly reduced in vivo tumor growth (P = .0007 for K299, IRF4 shRNA vs control shRNA; P = .03 for JB6, IRF4 shRNA vs control shRNA; paired Student t test). Error bars indicate standard error of the mean. (G) Treatment of ALK⁺ (K299 and JB6) and ALK⁻ (FE-PD and Mac-2A) ALCL cell lines with 150 nM crizotinib for 24 hours leads to IRF4 and MYC downregulation as measured by western blotting. *P < .05, **P < .01, ***P < .001.