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L. Mazzera et al.
2 cells that have low NF-κB index3,4,25), NIK knockdown reduced the basal phosphorylation/activation status of IKKa and IKKβ (p-IKKa/β) and their respective down- stream direct targets NF-κB2/p100 and IκB-a (Figure 3A), thus confirming that NIK affects not only the non-canoni- cal but also the canonical NF-κB pathway in MM cells.2-4 Notably, pan-AKI were ineffective (OPM-2) or only par- tially effective (all the other HMCL analyzed) in attenuat- ing NF-κB signaling25 (Figure 3A), and their reduced inhibitory activity on NF-κB signaling was closely linked to NIK induction because its knockdown by siRNA com- pletely abrogated the pan-AKI-induced NF-κB activation in OPM-2 as well as greatly enhanced the pan-AKI- induced NF-κB inhibition in all the other HMCL analyzed (Figure 3A).
In support of these data, we found that experimental overexpression of NIK in MM cells (Figure 3B) caused enhanced phosphorylation of IKKa/β, NF-κB2/p100 and IκB-a, and increased nuclear localization and DNA bind- ing activities of the NF-κB p65, p50, p52, and RelB sub- units (Figure 3C). In contrast, NIK knockdown in these NIK-over-expressing MM cells consistently and signifi- cantly decreased their basal NF-κB activity (Figure 3D), thus confirming the important role of NIK in controlling NF-κB signaling in MM.3,4
NF-κB-inducing kinase induction by pan-AKI activates the STAT3 signaling pathway in multiple myeloma cells
Because NIK induction by pan-AKI was not associated with an increased activation of NF-κB pathways in 4 of 5 HMCL tested (except OPM-2), and yet NIK signaling has been demonstrated to crosstalk at different levels with other important prosurvival signaling pathways including MEK-ERK and STAT3 pathways,8-10 we explored whether NIK induction by pan-AKI affected these pathways in MM cells.
Because NIK can phosphorylate MEK1 and thereby cause activation of downstream MAPK ERK,9 we investigated whether NIK induction by pan-AKIs is associated with increased phosphorylation/activation of ERK in MM cells.
We found no significant change (U266, R5) or even a decrease (OPM-2, JJN3) in ERK activity (p-ERK1/2) in the pan-AKIs-treated HMCL (Figure 4A), thereby indicating that NIK, stabilized by pan-AKI, does not act through this pathway. Because STAT3 activity is regulated by two independent phosphorylations, one occurring at Tyr705 and one at Ser727, which are both required for it to be fully functional,37 we specifically analyzed the STAT3 (Tyr705) and STAT3 (Ser727) phosphorylation patterns alongside with the overall protein expression levels. We found that treatment with pan-AKI significantly increased both Ser727 and Tyr705 STAT3 phosphorylation in OPM- 2, RPMI-8226 and 8226/R5, but not in U266 and JJN3 HMCL where no significant changes in p-Ser-STAT3 or a decrease in p-Tyr-STAT3 phosphorylation were observed (Figure 4A).
Notably, NIK knockdown in MM cells completely abro- gated both Ser727 and Tyr705 STAT3 phosphorylation induced by pan-AKI (Figure 4B), which would suggest that NIK is involved in the pan-AKI-mediated STAT3 activa- tion. Confirming these data, we found that ectopic expres- sion of NIK in MM cells caused enhanced phosphoryla- tion of STAT3 in both serine and tyrosine residues (Figure 4C), whereas its depletion in these NIK-over-expressing MM cells consistently and significantly (P<0.001)
decreased their basal STAT3 activity levels (Figure 4D).
In the light of evidence supporting reciprocal regulatory mechanisms and crosstalk between the NIK and STAT3 proteins,10 we examined whether NIK exists in a complex with STAT3 in MM cells. Co-immunoprecipitation showed that STAT3 was associated with NIK and that this association was significantly enhanced by pan-AKI treat-
ment of the cells (Figure 4E).
We also examined the Ser727 and Tyr705 phosphoryla-
tion state of STAT3 that co-immunoprecipitated with NIK and found that treatment with pan-AKI promoted a strong increase in the phosphorylation of NIK-associated STAT3 in both serine and tyrosine residues (Figure 4E and F), stressing the putative function of NIK in controlling STAT3 activation.
Aurora kinases inhibitors induce a NF-κB-inducing kinase dependent cytoplasmic relocalization and activation of c-Abl and promote the formation of the NIK-c-Abl-STAT3 ternary complex in multiple myeloma
Given the high levels of tyrosine-phosphorylated STAT3 that co-immunoprecipitates with the serine/threo- nine kinase NIK in response to pan-AKI treatment, we explored whether pan-AKI affect the Stat3 upstream tyro- sine kinases JAK2, Src and/or c-Abl38 activity/expression.
We found that, depending on the HMCL examined, pan-AKI caused a decrease or no significant changes in the Tyr-phosphorylation/activity of JAK2 (p-JAK2) and Src (p- SRC) kinases (Figure 5A), whereas they were able to sig- nificantly activate c-Abl in 4 of 6 HMCL tested (except U266 and JJN3 in which no significant changes or a decrease in c-Abl tyrosine-phosphorylation levels were observed, respectively) (Figure 5A). A significant increase (>3-fold) in p-c-Abl, but not in p-JAK2 and p-Src, was also observed in untreated 8226-NIK as compared to untreated 8226 HMCL (Figure 5A, lane 5 vs. lane 3). This finding links NIK to c-Abl signaling and, indeed, experimental overexpression of NIK in MM cells causes enhanced phos- phorylation of endogenous c-Abl on Tyr245 and Tyr412 residues (both commonly used as Abl activation markers),16,17 as well as Tyr705 phosphorylation of STAT3. Conversely, knockdown of NIK in these NIK-over- expressing MM cells consistently and significantly decreased their basal tyrosine-phosphorylation levels (Figure 5B). Accordingly, abrogation of Aurora-A and -B induced c-Abl phosphorylation at both Tyr245 and Tyr412 residues in MM cells (Figure 5C).
As c-Abl may exhibit both pro- and antiapoptotic func- tions depending on the subcellular localization (nuclear or cytoplasmic),15-18 and its intracellular localization is regulat- ed by phosphorylation of its Thr735 residue promoting cytoplasmic sequestration by the 14-3-3 protein,19 we explored whether pan-AKI affect Thr735 phosphorylation and/or subcellular localization of c-Abl in MM cells in which the pervasive DNA damage leads to a predomi- nantly nuclear localization of c-Abl.21,22 As shown in Figure 6 and Online Supplementary Figure S5, endogenous c-Abl was predominantly accumulated in the nucleus of the MM cells,21 while pan-AKI were able to cause a signif- icant translocation of c-Abl from the nucleus to cytoplasm, thus elevating its cytoplasm/nucleus ratio in 4 of 5 HMCL tested (except JJN3) (Figure 6). Notably, the pan-AKI- induced cytoplasmic accumulation of c-Abl was associat- ed with increased Thr735 phosphorylation of the cyto- plasmic fraction of c-Abl (Figures 6 and 7A).
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haematologica | 2019; 104(12)