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L. Mazzera et al.
ing cIAP1/2-mediated NIK proteasomal degradation,2-7 we investigated the effects of pan-AKI on the protein expres- sion of these NIK negative regulators. We found that pan- AKI treatment induced a significant reduction in the pro- tein levels of TRAF2 but not TRAF3 in all the tested HMCL, either cultured alone (Figure 1A) or co-cultured with HS-5 stromal cells (Figure 1B). Furthermore, pan-AKI treatment did not modulate TRAF2 mRNA levels in MM cells (Figure 1C), thereby indicating that its protein expres- sion is not regulated at transcriptional levels by these inhibitors.
Furthermore, small interfering RNA (siRNA)-mediated knockdown of Aurora-A and -B recapitulated the ability of pan-AKI to down-regulate the negative regulator of NIK, TRAF2, and increase NIK protein levels (Figure 1D), there- by confirming the significant role of Aurora kinases in
modulating NIK stability through TRAF2 in MM cells. On the other hand, siRNA-mediated knockdown of TRAF2 led to NIK accumulation in all the HMCL studied (Figure 1E and Online Supplementary Figure S1), including those with deletion or inactivating mutations of TRAF3 (U266, 8226 and 8226/R5) or bearing alterations in the TRAF3-binding domain of NIK (JJN3),2,3 thereby confirm- ing the important role of TRAF2 in regulating NIK degra- dation in MM.2,4
NF-κB-inducing kinase attenuates the anti-tumor activity of pan-AKI in multiple myeloma cells
We found that NIK inhibition by either the NIK small- molecule inhibitor 4H-isoquinoline-1,3-dione (NIK-in)36 or the NIK-specific siRNA significantly enhanced pan-AKI- induced cell death in all the HMCL tested, either cultured
A
B
Figure 2. NF-κB-inducing kinase (NIK) inhibi- tion sensitizes multiple myeloma (MM) cells to pan-AKI-induced cell death. (A) MM cell lines were incubated with the NIK inhibitor (NIK-in) at 10 μM or were transfected with NIK siRNA and after 3 hours (h) MM cell lines were treated with MK-0457 (0.4 μM) or PHA- 680632 (1 μM) in absence or presence of HS- 5 cells (+HS-5) (see Online Supplementary Methods). After 48 h, cell death was meas- ured by annexin-V labeling. Values represent means±Standard Deviation (SD) of four inde- pendent experiments. (Dunnett and Tukey- Kramer tests, *P<0.05; **P<0.01 vs. MK- 0457 treatment). (B) MM cell lines were treat- ed sequentially with escalating doses of the NIK inhibitor (NIK-in) (1-20 μM) for 3 h and subsequently with MK-0457 (0.1-1 μM) or PHA-680632 (0.1-2 μM) alone or in combina- tion with the NIK inhibitor at a fixed ratio indi- cated in Online Supplementary Table S1. After 48 h, cell death was measured by annexin V labeling and the Combination Index values (CI) were calculated using the Chou-Talalay method and Calcusyn software, and the isobologram plots were constructed. CI<1.0 indicate synergism, CI=1.0 indicate additive effect, and CI>1.0 indicate an antagonistic effect. (C) CD138-purified plasma cells from ten patients with MM seeded in presence of HS-5 cells and peripheral blood mononuclear cells (PBMC) from five healthy volunteers were incubated with the NIK inhibitor (NIK-in) at 10 μM for 3 h and then were treated with MK- 0457 (0.4 μM) or PHA-680632 (1 μM). After 24 h, cell death was measured by annexin-V staining or sub-G1 DNA content. Because of heterogeneous levels of basal cell death, the data of all ten primary samples and PBMC tested are expressed as % of specific cell death with the formula % Specific cell death = 100 x (induced cell death−basal cell death)/(100−basal cell death) and are shown in box plot format (median line in box delimit- ed by 25th and 75th) (*P<0.005 vs. either treat- ment alone; Dunnett test).
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haematologica | 2019; 104(12)