Aberrant NF-κB activity has been observed in many cancers, including both solid and hematopoietic malignancies, and sustained activation of NF-κB can affect all six hallmarks of cancer 
, including insensitivity to growth inhibitory signals, evasion of apoptosis, induction of angiogenesis, and metastasis 
, thus leading to the concept of NF-κB addiction in cancer cells. However, although the regulation of specific target genes by individual NF-κB subunits has emerged as an important mechanism to achieve the required specificity and selectivity of the NF-κB response 
, the precise contribution of individual NF-κB subunits in MM pathogenesis has not been characterized.
In the present study, we conducted the first extensive analysis of RelB DNA-binding activity on a large cohort of 52 newly diagnosed MM patients. Our data emphasize that constitutive activation of RelB is frequent (approximately 40%) in MM cases, indicating that it represents a major event in MM. Moreover, we uncovered a crucial role for RelB in promoting MM cell survival via the increased expression of a subset of anti-apoptotic NF-κB target genes by a direct transcriptional control. These results have important implications for the role of RelB in MM pathogenesis and therapy.
The alternative NF-κB pathway is dependent on NIK-mediated activation of IKKα, thus leading to the phosphorylation and proteasome-dependent processing of cytoplasmic p100, and resulting in RelB/p52 and RelB/p50 nuclear translocation 
. Two reports have highlighted the relationship between NF-κB gene expression signature and genetic abnormalities in regulators of the alternative NF-κB signaling cascades in 9% 
and 17% 
of patient cohorts with MM, suggesting the biological significance of the alternative NF-κB pathway in MM pathogenesis. However, RelB activation per se
and its physiological significance have not been investigated. Here, we demonstrate that RelB is constitutively activated in approximately 40% of MM cases, a much higher frequency than expected based on evaluation of the alternative NF-κB signaling cascade activation by cancer genomic approaches. One can hypothesized that important regulators of the alternative NF-κB pathway remain to be identified, and thereby their putative mutations being overlooked. Alternatively, additional mechanisms might contribute to the regulation of RelB activity in MM cells. This hypothesis is supported by the observation that knockdown of IKKα, the physiological target of NIK, has no effect on RelB binding activity in MM cells 
. First, Fbxw7α and GSK3-mediated proteosomal degradation of nuclear p100 has recently emerged to account for the control of RelB transcriptional activity and MM cell survival 
. Second, because the MM cell line RPMI 8226 harbors constitutive RelB activity maintained by the recently described proteasome inhibitor-resistant (PIR) pathway 
, it is possible that RelB activation is induced by such atypical proteasome-independent mechanisms in malignant plasma B cells isolated from newly diagnosed MM patients. Third, NF-κB is frequently activated in malignant cells, particularly MM cells, in response to inflammatory stimuli originating from the microenvironment, and not because of intrinsic mutations 
. The adherence of MM cells to bone-marrow stromal cells (BMSCs) induces NF-κB-dependent cytokine transcription and secretion (e.g. IL-6, TNFα and BAFF) by BMSCs, which in turn promote MM cell growth and survival through paracrine mechanisms 
. Although both classical and alternative pathways are activated by coculture of MM cells with BMSCs 
, the relative contribution of the microenvironment in mediating RelB activation in MM tumor cells is poorly understood and worth further investigation. Finally, EMSA experiments have shown that RelB activation in MM cases is not significantly associated with variations in the level of RelA DNA binding (), suggesting that RelB exerts its activity irrespectively of RelA. Nonetheless, it is important to note that RelB activation does not make RelA less critical for the survival of MM cells. Indeed, we have observed that RelB-positive patient samples were also positive for RelA, and knockdown of RelA expression in MM cell lines leads to a marked increase in apoptosis, thus supporting the idea that an efficient anti-NF-κB therapeutic approach should target both RelA and RelB activation in MM.
Our study has revealed that RelB promotes tumor cell survival in MM. In addition, we have demonstrated that RelB activity is required for optimal expression of a subset of anti-apoptotic NF-κB target genes in MM cells, most strikingly cIAP2, and ChIP experiments have shown that RelB is recruited to the region that encompasses the NF-κB binding sequence of the promoter of these anti-apoptotic genes. Altogether, we propose that the pro-survival activity of RelB in MM cells is exerted through κB-site and DNA-binding-dependent recruitment of RelB to anti-apoptotic NF-κB target genes. In support to this hypothesis, we have demonstrated that cIAP2 expression plays a crucial anti-apoptotic function in RelB-positive MM cell lines. Interestingly, cIAP2 belongs to the NF-κB gene signature reported as a marker of NF-κB activation in MM cell lines and patient samples 
. Thus, our results strongly suggest that increased cIAP2 expression is directly controlled by RelB heterodimers in MM tumor cells. Although cIAP2 was originally characterized by its ability to inhibit cell death through suppression of caspase activity 
, it has more recently emerged that cIAP2 is also involved in many aspects of innate and adaptive immune cell function through the ubiquitin-mediated regulation of the NF-κB pathways 
. The role of cIAP1/2 in NF-κB regulation and function is highly complex, and is dependent of the cellular context. In normal cells, cIAP2 acts as a negative regulator of the alternative NF-κB pathway through NIK proteosomal degradation via
its E3 ligase activity. However, in the pathological context of MM cells, it has been proposed that any mutations-which are frequent in MM cells- affecting the binding of cIAP2 to other crucial NF-κB regulators, especially cIAP1, TRAF2/3 and NIK, would result in NIK stabilization. Overexpression of NIK in turn leads to constitutive activation of the classical and alternative pathways, and an increase in B-cell growth and survival. In regards to our results, we propose that RelB-dependent increase in cIAP2 expression constitutes a strong pro-survival signal in MM cells through its anti-apoptotic activity. Beyond MM, close relation between anti-apoptotic gene expression and RelB activity has emerged in other B-cell neoplasms. Inhibition of Notch-induced RelB/p52 activity in Hodgkin lymphoma cell lines is associated with apoptosis and decreased expression of cIAP2 
. Moreover, BMSCs prevent apoptosis of primary B lymphoma cells, at least in part, through RelB-dependent increased expression of NF-κB-dependent anti-apoptotic genes (including cIAP1/2 and XIAP) 
. Thus, it is likely that the prosurvival effects of RelB observed in MM may be generalized to other B-cell neoplasms, especially those addicted to NF-κB.
In conclusion, we established that RelB is a crucial positive regulator of MM cell survival frequently activated in MM patient samples. Our data are of great functional importance because they constitute a significant advance in the understanding of RelB physiological function and provide a strong rationale for the development of new molecules targeting RelB in the treatment of MM.