Compensatory IKKα activation of classical NF-κB signaling during IKKβ inhibition identified by an RNA interference sensitization screen

A subtype of diffuse large B- cell lymphoma (DLBCL), termed activated B- cell- like (ABC) DLBCL, depends on constitutive nuclear factor-kappa B (NF-kappa B) signaling for survival. Small molecule inhibitors of I kappa B kinase beta(IKK beta), a key regulator of the NF-kappa B pathway, kill ABC DLBCL cells and hold promise for the treatment of this lymphoma type. We conducted an RNA interference genetic screen to investigate potential mechanisms of resistance of ABC DLBCL cells to IKK beta inhibitors. We screened a library of small hairpin RNAs (shRNAs) targeting 500 protein kinases for shRNAs that would increase the killing of an ABC DLBCL cell line in the presence of a small molecule IKK beta inhibitor. Two independent shRNAs targeting IKK alpha synergized with the IKK beta inhibitor to kill three different ABC DLBCL cell lines but were not toxic by themselves. Surprisingly, IKK alpha shRNAs blocked the classical rather than the alternative NF-kappa B pathway in ABC DLBCL cells, as judged by inhibition of I kappa B alpha phosphorylation. IKK alpha shRNA toxicity was reversed by coexpression of wild-type but not kinase inactive forms of IKK alpha, suggesting that IKK alpha may directly phosphorylate I kappa B alpha under conditions of IKK beta inhibition. In models of physiologic NF-kappa B pathway activation by CARD11 or tumor necrosis factor-alpha, compensatory IKK alpha activity was also observed with IKK alpha inhibition. These results suggest that therapy for ABC DLBCL may be improved by targeting both IKK alpha and IKK alpha, possibly through CARD11 inhibition.