Delineation of Canonical and Non-Canonical NF-κB Pathways in Multiple Myeloma: Therapeutic Implications.
Authors: Hideshima Teru , Raje Noopur , Carrasco Ruben , Ikeda Hiroshi , Okawa Yutaka , Kiziltepe Tanyel , Valet Sonia , Pozzi Samantha , Chauhan Dharminder , Mitsiades Constantine , Podar Klaus , Tai Yu-Tzu , Munshi Nikhil C. , Richardson Paul G. , Anderson Kenneth C. 
Published: October 2019

Abstract
NF-κB pathway plays a crucial role in the pathogenesis in cancer cells including multiple myeloma (MM). The NF-κB complex is dimer in different combinations of Rel family proteins, including p65 (RelA), RelB, c-Rel, p50 (NF-κB1), and p52 (NF-κB2). Recent studies have revealed that NF-κB activity is mediated via two distinct pathways. In the canonical pathway, NF-kB is typically a heterodimer composed of p50 and p65 subunits. In the non-canonical pathway, NF-kB is typically a heterodimer composed of RelB and p100 subunits. We have shown anti-MM activities of IKKβ inhibitors (PS-1145, MLN120B); however, effects of these agents were modest. Our studies therefore suggest that baseline NF-kB activity in MM cells is not totally dependent on the canonical pathway, and that inhibition of only canonical NF-κB pathway may not be sufficient to block total NF-kB activity. In this study, we therefore hypothesized whether non-canonical inhibitors significantly enhanced NF-κB inhibition induced by canonical inhibitors in MM cells. We first examined baseline NF-κB activity using electrophoretic mobility shift assay (EMSA). NF-κB activity varied between cell lines; for example MM.1S, MM.1R and H929 cells have higher level of NF-κB activity than in RPMI8226, INA6 and OPM2 cells. To define the role of canonical and non-canonical pathway, we next examined protein expression of p50, p65 and p52 NF-κB in these cell lines: p65 was highly expressed in all MM cell lines; however, expression of p50 and 52 is variable. Surprisingly, no detectable or weak expression of p50 was observed in U266, RPMI8226, LR5, H929 and OPM2 cell lines, suggesting that baseline NF-kB activity in these cell lines is not maintained only by the canonical pathway. We then attempted to block non-canonical NF-κB pathway in MM cell lines. Specifically since IKKα and IKKβ are client proteins of heat shock protein (Hsp) 90, we examined whether 17AAG could inhibit expression and/or function of IKKα and IKKβ in MM cells. Importantly, both IKKα and IKKβ were significantly downregulated by 17AAG in MM cell lines. To determine whether downregulation of these IKK proteins by 17AAG was due to inhibition of transcription, we next performed real-time quantitative PCR and no significant inhibition of relative expression of IKKβ was observed by 17AAG treatment, suggesting that downregulation of these proteins was a post transcription event. We further examined whether 17AAG enhanced the effect of IKKβ inhibitor MLN120B on NF-κB activity. Although the inhibitory effect by either MLN120B or 17AAG alone on phosphorylation (p) of IκBα triggered by TNFα was marginal, combination treatment of MLN120B with 17AAG almost completely blocked IκBα, suggesting that this combination synergistically inhibit canonical NF-κB activity in MM cells. Importantly, the combination of MLN120B with 17AAG also significantly blocked baseline and TNFα-triggered NF-κB activity, assessed by EMSA, in MM cells. Finally, 17AAG augmented the growth inhibitory effect of MLN120B in the context of bone marrow stromal cells. Taken together, these results showed that baseline and TNFα-triggered NF-κB activities were completely blocked by this combination treatment, and provide the rationale for its clinical evaluation to induce maximum inhibition of NF-κB activity and improve patient outcome in MM.