Disruption of IκB Kinase (IKK)-mediated RelA Serine 536 Phosphorylation Sensitizes Human Multiple Myeloma Cells to Histone Deacetylase (HDAC) Inhibitors

Post-translational modifications of RelA play an important role in regulation of NF-kappa B activation. We previously demonstrated that in malignant hematopoietic cells, histone deacetylase inhibitors (HDACIs) induced RelA hyperacetylation and NF-kappa B activation, attenuating lethality. We now present evidence that I kappa B kinase(IKK) beta-mediatedRelASer-536phosphorylation plays a significant functional role in promoting RelA acetylation, inducing NF-kappa B activation, and limiting HDACI lethality in human multiple myeloma (MM) cells. Immunoblot profiling revealed that although basal RelA phosphorylation varied in MM cells, Ser-536 phosphorylation correlated with IKK activity. Exposure to the pan-HDACIs vorinostat or LBH-589 induced phosphorylation of IKK alpha/beta(Ser-180/Ser-181) and RelA (Ser-536) in MM cells, including cells expressing an I kappa B alpha super-repressor, accompanied by increased RelA nuclear translocation, acetylation, DNA binding, and transactivation activity. These events were substantially blocked by either pan-IKK or IKK beta-selective inhibitors, resulting in marked apoptosis. Consistent with these events, inhibitory peptides targeting either the NF-kappa B essential modulator (NEMO) binding domain for IKK complex formation or RelA phosphorylation sites also significantly increased HDACI lethality. Moreover, IKK beta knockdown by shRNA prevented Ser-536 phosphorylation and significantly enhanced HDACI susceptibility. Finally, introduction of a nonphosphorylatable RelA mutant S536A, which failed to undergo acetylation in response to HDACIs, impaired NF-kappa B activation and increased cell death. These findings indicate that HDACIs induce Ser-536 phosphorylation of the NF-kappa B subunit RelA through an IKK beta-dependent mechanism, an action that is functionally involved in activation of the cytoprotective NF-kappa B signaling cascade primarily through facilitation of RelA acetylation rather than nuclear translocation.