Figure 6. IRF4-SPIB and CARD11 form an essential oncogenic loop in ABC DLBCL.
(A) UCSC browser depiction of ChIP-seq data from the HBL1 ABC DLBCL line showing IRF4 (in triplicate) and SPIB-biotag binding at the CARD11 locus, with an evolutionarily conserved EICE binding motif indicated. Control (IRF4): OCI-Ly19 (IRF4-). Control (SPIB): HBL1 with empty biotag vector. TSS (arrow). (B) ChIP analysis in HBL1 cells for IRF4 and SPIB binding at the CARD11 peak identified in (A) or at a negative control (ctrl.) locus. IRF4 or control shRNAs were induced for 4 days. GCB DLBCL line: OCI-Ly19 (IRF4-) is IRF4-negative. Error bars show SEM of triplicates. (C) Relative CARD11 mRNA expression, depicted according to the color scale shown, from gene expression profiling of ABC DLBCL lines after induction of shIRF4 or the IRF4-SPIB chimeric repressor for 4 days. (D) IKK activity measured by an IκBα-luciferase reporter in TMD8 (ABC DLBCL) after induction of various shRNAs for 3 days (left) or the IRF4-SPIB chimeric repressor for 1 day (right). Also shown is the effect of 1 day exposure to an IKKβ inhibitor (MLN120B) or DMSO. (E) Western blot analysis of the indicated proteins following treatment of OCI-Ly10 cells with lenalidomide (10μM) for the indicated times. (F) IKK activity measured by an IκBα-luciferase reporter after treatment of TMD8 cells with lenalidomide at the indicated concentrations for 48 h. Error bars show the SEM of triplicates. See also Figure S5.