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L. Mazzera et al.
bination with other agents for the treatment of MM.47 Instead, by inducing a NIK-dependent cytoplasmic relocal- ization and activation of c-Abl, pan-AKI switch it from a pro-apoptotic to a pro-survival factor, thereby turning it into a potentially effective target for MM. In accordance with this, we demonstrate here that c-Abl inhibitors con- sistently increase the sensitivity of MM cells to pan-AKI in different experimental settings and in patient-derived cells.
Our data identify NIK as a kinase responsible for phos- phorylation of c-Abl at Thr735, which is critical for its cytoplasmic retention, thereby indicating that NIK influ- ences the subcellular localization of c-Abl in MM cells. NIK, stabilized by pan-AKI, forms a trimeric complex
with c-Abl and STAT3 and, together with c-Abl, con- tributes to the serine 727 and tyrosine 705 phosphoryla- tion of STAT3. NIK is also involved in the tyrosine-phos- phorylation/ activation of c-Abl observed after pan-AKI treatment, as supported by our genetic perturbation exper- iments of NIK in MM cells. This recalls the fact that also serine/threonine kinases, in addition to SRC family kinas- es, may regulate the catalytic activity of c-Abl via direct protein-protein interactions and/or by promoting phos- phorylation of c-Abl on serine and/or threonine residues.16,17 Moreover, pan-AKI failed to induce c-Abl and STAT3 tyrosine phosphorylation in those HMCL (U266 and JJN3) in which the high basal activity of Src kinase
Figure 11. NF-κB-inducing kinase (NIK) accumulation promotes pro-survival signals by inducing PIM kinases. (A) Western Blot analysis of NIK, Bcl-xL, A1/Bfl-1, Mcl-1, XIAP, PIM2, PIM1, phos- pho-Bad (Ser112) and Actin proteins in stable clones of RPMI- 8226 and 8226/R5 transfected with empty vector or with plas- mid expressing NIK; bands were subjected to densitometric scan- ning and normalized relative fold change in protein levels are reported below each lane. Relative protein levels of each PIM2 isoform at 34, 37, and 40 kDa are reported. (B) NIK expression in RPMI-8226-NIK and 8226/R5-NIK cells was inhibited using the NIK inhibitor (NIK-in) at 10 μM or by siRNA silencing; after 3 hours (h) cells were treated with MK-0457 (0.4 μM) and PHA- 680632 (1 μM). The cytotoxic effects of NIK inhibition of 8226- NIK and 8226/R5-NIK cells were compared to those of 8226 and 8226/R5 transfected with empty vector. After 72 h, apoptosis was measured by sub-G1 DNA content. Values represent means±Standard Deviation (SD) of three independent experi- ments. (*P<0.01 vs. Pan-AKI-treated NIK-expressing cells; Dunnett and Tukey-Kramer test). (C) Western blot analysis of NIK, PIM2, PIM1, phospho-Bad (Ser112) and Actin proteins in multiple myeloma (MM) cell lines transfected with NIK siRNA or treated with the NIK inhibitor (NIK-in) in presence or absence of pan-AKI. All western blotting results were evaluated by densito- metric scanning and normalized to the untreated control set as 1. The histogram below shows combined densitometric values of the 34, 37, and 40 kDa PIM2 bands. Histogram represents the mean±SD of three independent experiments. (°P<0.05, #P<0.01, *P<0.005, **P<0.001 vs. untreated control, Dunnett test). (D) MM cell lines cells were transfected with non-specific control siRNA (Cont) or STAT3 siRNA and after 3 h MM cell lines were treated with MK-0457 (0.4 μM). After 48 h whole cell lysates were immunoblotted against STAT3, PIM2, PIM1 and Actin. PIM2 and PIM1 bands were subjected to densitometric scanning and the number below each lane represents the rela- tive amount of the indicated proteins normalized to Actin expres- sion. In Figure are reported combined densitometric values of the 34, 37, and 40 kDa PIM2 bands. At the same time point cell death was measured by flow cytometry analysis of Annexin V- FITC/PI or Annexin V-PE/7-AAD staining. Values in the graph rep- resent means±SD of three independent experiments. (*P<0.005 vs. either treatment alone; Dunnett and Tukey-Kramer tests).
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haematologica | 2019; 104(12)