Activation of B-Cell Maturation Antigen (BCMA) on Human Multiple Myeloma Cells by a Proliferation-Inducing Ligand (APRIL) Promotes Myeloma Cell Function in the Bone Marrow Microenvironment.
Authors: Leiba Merav , Xu Jiangchun , Song Weihua , Li Xian-Feng , Burger Peter , Kordich Jennifer , Cassiano Linda , Podar Klaus , Chauhan Dharminder , Hideshima Teru , Munshi Nikhil C. , Richardson Paul , Anderson Kenneth C. , Tai Yu-Tzu 
Published: April 2020

Abstract
A proliferation-inducing ligand (APRIL), a close member of B-cell-activating factor (BAFF) belonging to the TNF superfamily that stimulates growth and survival of multiple myeloma (MM) cells, is mainly produced by MM patient bone marrow stromal cells (BMSCs) and osteoclasts in the BM microenvironment. Like BAFF, APRIL binds two receptors, B-cell maturation antigen (BCMA) and transmembrane activator and calcium modulator (TACI). Since BCMA has significantly higher affinity for APRIL than for BAFF (nM vs. mM range), APRIL/BCMA signaling may be more important than BAFF/BCMA in MM. Specifically, BCMA but not TACI is unequivocally expressed at mRNA level in the majority of CD138-expressing MM cell lines and patient MM cells. To date, it is unclear how APRIL signaling through BCMA contributes to BM microenvironment in support of MM cells. We here studied the functional sequelae of APRIL/BCMA pathway in MM cells, and further identified molecular mechanisms regulating these processes. Using flow cytometric analysis, cell surface BCMA expression was confirmed in the majority of MM cell lines. Microarray gene expression analysis showed significant expression of BCMA mRNA in CD138+ patient MM cells (n=105). Higher BCMA mRNA levels were observed in patient MM cells than in normal plasma cells (n=27) (p&lt; 0.03). Importantly, CD138+ patient MM cells express BCMA protein on the cell surface (n=15), as confirmed by CD38+BCMA+ dual immunofluorescence staining followed by flow cytometric analysis and immunohistochemical (IHC) study. Next, H929 MM cell line that expresses only BCMA, but not TACI, and Daudi B cells transduced with huBCMA (huBCMA-Daudi) were stimulated with APRIL. These cells were subjected to mRNA extraction at different time points followed by microarray analysis to identify downstream transcriptional targets. Stimulation of these two cell lines by APRIL activated the NF-kB signaling in a dose-dependent manner. More than 6-fold increase in NF-kB activity was induced by APRIL (100 ng/ml) using a p65 enzyme-linked DNA/protein interaction assay. IL-6 and PI3K/AKT signaling cascades were concurrently induced. Additionally, APRIL upregulated chemotactic/osteoclast activating factors, i.e., MIP-1a and MIP1b, in a dose-dependent manner, which was further validated by specific ELISA. Multi-Analyte Profiles also confirmed that angiogenesis and adhesion/chemoattractant factors, i.e., IL-8, CXCL10, RANTES, MDC (ccl22), were significantly induced upon APRIL stimulation. Conversely, a soluble BCMA-Fc protein inhibited APRIL binding to BCMA and blocked secretion of APRIL-induced chemokines, indicating specific functional interaction of BCMA with APRIL. Importantly, APRIL similarly induced these signaling cascades in CD138+ patient MM cells. Taken together, our studies are the first to demonstrate cell surface BCMA on the majority of CD138+ patient MM cells. Furthermore, APRIL induced osteoclasts-, migration-, and angiogenesis-associated factors in MM cells. These studies therefore establish a role of BCMA activation in the BM microenvironment that provides a niche for MM disease progression, supporting novel therapeutics specifically targeting APRIL/BCMA pathway in MM.