Role of BAFF in Adhesion and Growth of Human Multiple Myeloma Cells in the Bone Marrow Microenvironment.
Authors: Tai Yu-Tzu , Li Xian-Feng , Coffey Rory , Breitkreutz Iris , Catley Laurence Catley Laurence , Podar Klaus , Hideshima Teru , Chauhan Dharminder , Raje Noopur , Schlossman Robert , Richardson Paul , Munshi Nikhil C. , Anderson Kenneth C. 
Published: April 2020

Abstract
Recent studies have underscored the role of B-cell activating factor (BAFF), a member of the tumor necrosis factor superfamily, in promoting the survival of malignant B cells, including human MM. Since the tumor bone marrow microenvironment plays a crucial role in MM cell growth and survival, we here characterize the functional significance of BAFF in the interaction between MM and bone marrow stromal cells (BMSCs), and further defined molecular mechanisms regulating these processes. We first confirmed the expression of BAFF and its receptors (BCMA, TACI, BAFF-R) on majority of MM cell lines and CD138-purified patient MM cells. Significantly, gene expression profiling revealed increased BCMA expression on newly diagnosed and relapsed MM versus normal plasma cells (p&lt;0.0001, student T test, Abstract # 552872). Although BAFF and its 3 receptors are expressed on CD138+patient MM cells (n=10) by RT-PCR analysis, the pattern of expression of TACI and BAFF-R receptors was heterogeneous, assessed by flow cytometric analysis. We next examined BAFF expression in BMSC lines (n=4) and BMSCs derived from patient BM (n=5). Baff expression is easily detected in BMSCs, and Baff levels are approximately 3–10-fold higher in supernatants of BMSCs than equivalent numbers of MM cells. Thus BMSCs are the main source of BAFF in MM patients. We then asked whether MM adhesion to BMSCs further upregulates BAFF secretion from BMSCs. Adhesion of 5 MM cell lines to BMSCs augments BAFF secretion by 2–5 fold, using BAFF ELISA. Immunoblotting using anti-BAFF Ab confirmed markedly higher expression of BAFF in BMSCs than MCCAR MM cells, as well as greater BAFF upregulation induced by MM adhesion. Since NF-kappaB (NF-κB) is crucial for MM adhesion-induced cytokine secretion from BMSCs and the BAFF gene promoter contains at least six NF-κB-binding sites, we next transfected KM104 BMSC line with a luciferase reporter vector carrying the BAFF gene promoter (BAFF-LUC) and then allowed MM cells to adhere to BMSCs for 24 hr, followed by measurement of luciferase activity. NF-κB-LUC reporter was used as positive and pGL3 plasmid as a negative control. Adhesion of MCCAR and MM1S MM lines to KM103 BMSC line with BAFF-LUC and NF-κB-LUC, but not control reporters, significantly enhanced luciferase activity, suggesting that NF-κB activation induced by MM adhesion to BMSCs mediates BAFF upregulation. In parallel, we also asked whether BAFF induces MM adhesion to BMSCs. BAFF (0–100 ng/ml) increases adhesion of 5 MM lines to BMSCs in a dose-dependent manner; conversely, TACI-Ig or blocking anti-BCMA Ab inhibited BAFF stimulation, indicating increased adhesion specific triggered by BAFF. Using adenoviruses expressing dominant-negative and constitutively expressed AKT as well as IkappaB kinase (IKK) inhibitor (PS-1145), we further showed that BAFF-induced MM cell adhesion is primarily mediated via activation of AKT and NF-κB signaling. Finally, BAFF significantly increased adhesion of CD138-expressing patient MM cells to BMSCs. These studies suggest a role for BAFF in localization and growth of MM cells in the BM microenvironment and strongly support novel important therapeutics targeting the interaction between BAFF and its receptors in human MM.