The lethality of the histone deacetylase inhibitors SAHA and NVP-LAQ824 is enhanced by interrupting NF-kappaB activation through a mechanism involving inhibition of IKK-mediated RelA/p65 phosphorylation (serine 536) in human multiple myeloma cells

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We previously reported that histone deacetylase inhibitors (HDACIs) induce RelA acetylation and NF-κB activation, while interruption of these events leads to a marked enhancement of HDACI lethality (Mol Cell Biol 13:5429-44, 2005). Recently, it has been found that RelA phosphorylation is also involved in regulation of RelA acetylation and NF-κB activation. In this study, we have examined whether RelA phosphorylation is associated with HDACI-mediated NF-κB activation and lethality in human multiple myeloma (MM) cells. Exposure of U266, 8226, MM.1S, or MM.1R cells to subtoxic doses of SAHA or NVP-LAQ824 resulted in NF-κB activation, an event diminished by the NF-κB inhibitor Bay 11-7082, resulting in markedly increased apoptosis. The latter phenomenon was also observed in primary CD138+ MM cells. Exposure to HDACIs led to phosphorylation of IKKα/β, accompanied by RelA phosphorylation at Ser536 but not Ser276 or Ser468, while did not affect phosphorylation of p100 and p52 (RelB) which is mediated by IKKα. Bay 11-7082 blocked phosphorylation of both IKKs and RelA, associated with inhibition of RelA acetylation. Because RelA phosphorylation at Ser536 is primarily mediated by IKKs, attempts were made to examine the specific effects of IKK inhibition on those HDACI-mediated events. Using 8226 cell lines stably transfected with wild-type or mutant 3xκB luciferase reporter constructs, it was found that TPCA-1, a selective IKKβ inhibitor, diminished HDACI-induced NF-κB activation and markedly increased apoptosis. Similar interactions between TPCA-1 and either SAHA or LAQ-NVP-LAQ824 were noted in multiple human MM cell lines. Western blot demonstrated that TPCA-1 blocked RelA phosphorylation (Ser536) mediated by HDACIs. Moreover, downregulation of IKKβ by stable transfection with an IKKβ siRNA expression plasmid dramatically increased the sensitivity of MM cells to apoptosis induced by HDACIs. Lastly, specific inhibitory peptides, designed to interfere with either formation of the IKK complex (NBD peptide) or phosphorylation of RelA (PTD peptide), significantly increased the lethality of HDACIs. Finally, co-administration of SAHA or NVP-LAQ824 with Bay 11-7082 markedly downregulated expression of certain NF-κB-responsive antiapoptotic genes, including cIAP2, XIAP, Bcl-xL and FLIP, but not cIAP1 and A1. Collectively, these findings indicate that HDACI-induced NF-κB activation is associated with IKK-mediated RelA phosphorylation, whereas blockade of this event by either specific IKKβ inhibitors or genetic downregulation of IKKβ leads to a pronounced increase in HDACI lethality in MM cells. The strategy of combining IKKβ inhibitors and HDACIs warrants exploration in MM and other hematopoietic malignancies.