CD27-Mediated Apoptosis Is Dependent on Siva-Induced Caspase Activation in Human Multiple Myeloma.
Authors: Tai Yu-Tzu , Li Xian-Feng , Coffey Rory , Breitkreutz Iris , Catley Laurence , Schlossman Robert L. , Richardson Paul G. , Munshi Nikhil C. , Treon Steven P. , Anderson Kenneth C. 
Published: April 2020

Abstract
CD27, a member of tumor necrosis factor receptor superfamily that lacks a death domain in its cytoplasmic region, and its interaction with its ligand, CD70, is crucial for differentiation into plasma cells. In malignant B cells, aberrant expression and reverse signaling of CD70 might contribute to disease progression. Recent studies showed that CD27 is heterogeneously expressed on multiple myeloma (MM) plasma cells and the expression is reduced with the progression of MM. However, a possible role for the loss of CD27-CD70 interaction in myelomagenesis was never defined. In this study, we identify functional significance of CD27-CD70 interaction in 4 CD27-expressing MM lines and define mechanisms regulating CD27-mediated MM cell death. Using RT-PCR and flow cytometric analysis, we first found that all of MM lines highly express CD70 (n=10) and 4 MM lines 12BM, 12PE, 28BM, 28PE express CD27 on the cell surface. We next evaluated the effect of CD27 ligation, by CD70-transfected NIH3T3 cells (CD70 transfectant), on [3H] thymidine incorporation by CD27-expressing MM lines. CD27 ligation by CD70 transfectants inhibited DNA synthesis in these 4 CD27-expressing MM lines, but not the control transfectants. Conversely, a blocking anti-CD70 mAb blocked CD27-mediated growth inhibition in a dose-dependent manner, indicating induced growth inhibition specific triggered by CD27-CD70 interaction. Using MTT assay, CD27 ligation by CD70 transfectant also inhibited MM cell survival. IL-6 (20 ng/ml) could overcome the inhibitory effect triggered by CD27 ligation on MM cell growth and survival. In addition, CD27 ligation further enhanced Dex-induced MM cell death. Importantly, CD27-mediated MM cell death was also observed in 2 CD27-expressing patient MM cells. Since Siva is a death domain-containing proapoptotic protein identified as an intracellular ligand of CD27, we investigated its role in CD27-mediated apoptosis in MM cells. Overexpression of Siva by transducing adenovirus-expressing Siva (Ad-Siva-GFP) in 12BM MM line is sufficient to induce cell death whereas control adenovirus (Ad-GFP) transduction did not alter 12BM cell growth and survival. CD27 ligation by CD70 transfectants on Siva-overexpressing 12BM cells further enhanced Siva-induced apoptosis, as evidenced by increased subG0 fraction in cell cycle analysis. Thus, the apoptosis triggered by Siva overexpression was related to the CD27-mediated apoptotic pathway. We further determined caspase involvement in the Siva-induced apoptosis in the absence and presence of CD70 transfectants. Caspase 8 and caspase 9 activities were detected 24h following Ad-Siva-GFP transduction in 12BM cells, whereas caspas-3 activity was detected 48h after transduction. Coculture of Ad-Siva-GFP-transduced 12BM cells with CD70 transfectant further enhanced caspase activities. Therefore, overexpression of Siva is sufficient to induce apoptosis and CD27-mediated apoptosis is mediated by Siva-dependent caspase activation in MM. Furthermore, these results suggest that lack of CD27 may lead to evasion of apoptosis in human MM.