The present study defined diverse molecular mechanisms activating NF-κB in MM. Most primary MM cases had high expression of the NF-κB signature. This observation suggests frequent engagement of the NF-κB pathway in myeloma for several reasons. First, the NF-κB signature genes were defined based on IKKβ inhibition and coexpression in myeloma cell lines, yet were nevertheless highly correlated in expression across primary patient samples. Second, the presence of the NF-κB p65 subunit in the nucleus of primary MM cells correlated with NF-κB signature expression. Finally, the genetic and epigenetic alterations that we defined were significantly skewed towards high NF-κB signature expression, in both MM patient samples (p=1.01 × 10−9) and cell lines (p=1.08 × 10−17) ().
Some NF-κB pathway activation is likely related to signals that PCs receive in the bone marrow microenvironment. Indeed, among B cell subsets, normal PCs expressed the highest level of the NF-κB signature, presumably due to BAFF- and APRIL-mediated signaling (Hideshima et al., 2002
; Marsters et al., 2000
; Moreaux et al., 2005
; O’Connor et al., 2004
). Blockade of BAFF and APRIL decreases the number of bone marrow PCs in normal mice (O’Connor et al., 2004
), raising the possibility that similar pharmacological inhibition might prove toxic to those MM cells that retain a microenvironmental dependence for NF-κB pathway activation.
However, the most compelling evidence for a critical role of the NF-κB pathway in myeloma pathogenesis was provided by the multiple, recurrent genetic abnormalities that we uncovered in the pathway. In cell lines and primary patient samples, NIK overexpression arose due to amplification or translocation of the NIK locus or due to enhanced NIK protein stability. An additional NF-κB-activating mechanism involved loss of functional TRAF3, either by homozygous deletion, inactivating mutations or epigenetic silencing. Other genetic events associated with high NF-κB activity in MM included CYLD or BIRC2/BIRC3 deletion, CD40 or NFKB1 amplification, and NFKB2 C-terminal truncation. Most of these genetic events increased classical and alternative NF-κB activity in MM cell lines, leading to pathway addiction and sensitivity to IKKβ inhibition. Taken together, these myriad genetic abnormalities strongly support the development of IKKβ inhibitors for the treatment of multiple myeloma.
NIK overexpression in MM cell lines activated both the classical and alternative NF-κB pathways. In normal mouse hematopoietic cells, loss of NIK does not affect activation of the classical pathway by inflammatory stimuli (Yin et al., 2001
). However, NIK is required for the activation of the classical NF-κB pathway by certain TNF receptor family members, including CD40 (Ramakrishnan et al., 2004
). Further, experimental overexpression of NIK can activate IKKβ and the classical pathway via phosphorylation of IKKβ in its activation loop (Delhase et al., 1999
; O’Mahony et al., 2000
). Indeed, NIK can form a complex with IKKα and IKKβ (Woronicz et al., 1997
), perhaps by virtue of its ability to directly bind IKKα (Regnier et al., 1997
). Studies conflict on the exact mechanism of IKKβ activation by NIK (Delhase et al., 1999
; O’Mahony et al., 2000
), but concur that NIK overexpression can activate IKKβ, supporting our finding that high NIK levels activate the classical NF-κB pathway in some myelomas.
We propose that the major mechanism by which NIK promotes tumor cell survival in MM is by stimulating IKKβ and activating the classical NF-κB pathway for several reasons. First, the toxicity of MLN for NIK-expressing MM cells argues for an essential role of IKKβ signaling: MLN is highly selective for IKKβ, with more than 1000-fold greater inhibitory activity for IKKβ than for 30 other cellular kinases, including IKKα (Nagashima et al., 2006
). The IkBα-super-repressor, which also specifically turns off the classical NF-kB pathway, resulted in similar toxicity and gene expression changes in these MM cells. Second, NIK depletion by shRNA inhibited IkBα kinase activity. Since IKKβ is a far more potent IkBα kinase than IKKα, this result suggests that NIK is activating IKKβ in these cells. Third, NIK knock down decreased nuclear DNA binding activity of the classical NF-κB subunits p50 and p65, consistent with an effect on IKKβ activity. Fourth, two shRNAs that effectively decreased IKKα protein expression were not toxic for NIK-expressing cell lines. These data support the argument that NIK acts through IKKβ to stimulate the classical NF-κB pathway, thereby promoting MM survival. Nonetheless, the alternative NF-κB pathway was also affected by NIK knockdown since p52 and RelB DNA binding activity was reduced. It is therefore conceivable that activity of the alternative NF-κB pathway could contribute to the effect of NIK on cell survival.
Multiple mechanisms lead to high NIK protein expression in MM. The wild type NIK protein had a very rapid turnover, and proteasome inhibition caused accumulation of NIK protein, consistent with a ubiquitin-mediated degradative pathway involving TRAF3, as described (Liao et al., 2004
). Nevertheless, when wild type NIK
mRNA was expressed at extraordinarily high levels due to chromosomal aberrations, as in L363 and EJM cells, wild type NIK protein accumulated. Cells with intermediate NIK
mRNA levels engage various mechanisms to stabilize NIK protein. In JJN3 cells, a chromosomal translocation precisely removed the region of NIK that TRAF3 requires to destabilize NIK protein (Liao et al., 2004
), highlighting the interrelationship of TRAF3 and NIK in MM pathogenesis. KMS28PE and KMS18 had enhanced stability of NIK protein without evident alterations in the TRAF3 binding domain of NIK or in TRAF3 itself. Both KMS18 and KMS28PE had genomic deletions of BIRC2
genomic locus, which encodes the ubiquitin ligases cIAP1 and cIAP2. cIAP1 attenuates NF-κB signaling from TNFR2 (Li et al., 2002
), raising the possibility that this protein or cIAP2 may also inhibit NF-κB signaling downstream of NIK by decreasing NIK protein stability.
Another recurrent mechanism of NF-κB activation in myeloma was silencing, homozygous deletion, or somatic mutation of TRAF3. In primary MM patient samples, 4.4% had a TRAF3 abnormality, and these cases had higher NF-κB signature expression than other myelomas. Homozygous TRAF3 deletions occurred in 2 primary cases and in the OCI-My1 cell line. In addition, 2 NF-κB-dependent cell lines and 4 primary patient samples carried homozygous mutations creating truncated TRAF3 proteins that should be incapable of interacting with NIK or TNFR superfamily members. In cases lacking deletion or mutation of TRAF3, low TRAF3 mRNA expression may be due to epigenetic silencing, a frequent event in cancer.
Experimental re-expression of TRAF3 in cell lines lacking functional TRAF3 decreased IKKβ activity and both classical and alternative NF-κB signaling, resulting in cell death. The precise mechanism by which TRAF3 modulates IKK activity in these cell lines remains to be elucidated. The cell lines with inactive TRAF3 do not have high NIK protein expression. Moreover, one of the TRAF3 mutant cells, LP1, was not affected by the NIK shRNA. In this cell line, therefore, TRAF3 must affect IKKβ activity by a NIK-independent mechanism.
In primary MM, the full consequence of TRAF3 inactivation may only be evident in cells dependent on NF-κB signaling from a TNFR family member, that can be negatively regulated by TRAF3 (Hauer et al., 2005
). The most logical candidate cytokines to propagate TRAF3-deficient MM are BAFF and APRIL, which are abundant in the bone marrow and can signal through TACI and BCMA, two TNFRs that are highly expressed in a subset of MM (Moreaux et al., 2005
It is also notable that extreme overexpression of CD40, a TNFR family member, was associated with NF-κB activation in MM. CD40 and other members of the TNFR superfamily can activate NF-κB when overexpressed, even in the absence of ligand (Lee et al., 1996
; Rothe et al., 1995b
; Yamamoto et al., 1998
). It is therefore plausible that the 34-fold average overexpression of CD40 in the CD40 outlier cases is sufficient to activate NF-κB in a cell-autonomous fashion.
A key negative regulator of NF-κB signaling is CYLD, which was homozygously deleted in primary MM cases with high NF-κB signature expression. CYLD is a deubiquitinating enzyme that removes ubiquitin moieties from activated TRAF2, TRAF6, IKKγ and BCL3, thereby interfering with NF-κB signaling at multiple regulatory levels (Brummelkamp et al., 2003
; Kovalenko et al., 2003
; Massoumi et al., 2006
; Regamey et al., 2003
; Trompouki et al., 2003
). Of note, CYLD was ineffective in inhibiting NF-κB activity induced by NIK overexpression (Kovalenko et al., 2003
It therefore appears that diverse upstream signaling pathways activate IKKβ in MM. Further, some myelomas may bypass the requirement for IKK altogether by amplifying and overexpressing NFKB1, encoding the p50 subunit of the classical NF-κB pathway, or by creating truncated and constitutively active NFKB2 proteins. The recurrent yet varied nature of these genetic abnormalities suggests strongly that activity of the NF-κB pathway is the phenotype that is selected rather than any particular genetic lesion.
Overall, we documented genetic abnormalities in the NF-κB pathway in 13 (28%) MM cell lines and 41 (9%) of the primary MM samples. Considering that we resequenced TRAF3 in only 10% of the patient samples, a larger number of TRAF3 mutations probably exist within our sample set. Therefore, the total number of genetic abnormalities in the genes we studied can be predicted to be ~16%. Given the large number and variety of genetic abnormalities discovered in the present report, there may well be genetic aberrations in other components of the NF-κB pathway that we have not studied. As already mentioned, however, many MM cases with high NF-κB signature expression may not have genetic abnormalities in the NF-κB pathway but rather may derive activating signals from the microenvironment. Irrespective of whether the activation of the NF-κB pathway is cell-intrinsic or cell-extrinsic, the MM cell may nonetheless depend upon this pathway for survival.
A clear message from these recurrent yet varied genetic abnormalities is that the NF-κB pathway plays a pervasive role in the pathogenesis of MM, providing a strong impetus to the development of NF-κB pathway inhibitors for the therapy of this malignancy. Currently, IKKβ inhibitors are under development for clinical use and are likely to have manageable on-target side effects (Nagashima et al., 2006
). That the majority of MM cell lines tested retained a dependence on NF-κB signaling for survival is encouraging and consistent with previous results (Hideshima et al., 2006
). The NF-κB signature provides a basis to develop biomarkers of response to NF-κB pathway inhibitors. A gauge of NF-κB activity, such as quantitative RT-PCR to measure the 11-gene NF-κB signature, could be assessed in bone marrow MM samples both pre-treatment and during administration of the drug. Coupling biomarkers with pathway-targeted therapeutics will ultimately allow treatment of MM patients to be tailored to the genetic abnormalities of their cancers.