APRIL/B Cell Maturation Antigen (BCMA) Signaling Cascades Promote Human Multiple Myeloma Growth and Mediate Immunosuppression in the Bone Marrow Microenvironment Via IL-10, TGF-b, and PD-L1

Expression of B cell maturation antigen (BCMA) and secretion of its ligand a proliferation-inducing ligand (APRIL) are highly elevated in patient multiple myeloma (MM) cells and serum samples, respectively, suggesting that both proteins represent promising targets for novel immunotherapies for MM.  Here, we characterize downstream molecular events following APRIL/BCMA activation in MM cells, and define functional significance of this signaling cascade in MM pathophysiology in addition to myeloma growth and survival.  MM cell lines (H929, MM1S, and RPMI8226 with high, medium, and low BCMA, respectively) were first lentivirally transfected with BCMA shRNA and cDNA to knockdown and overexpress BCMA, respectively.  BCMA downregulation in all MM cells significantly blocks viability and induces caspase3/7 activities, potently reducing colony formation.  Conversely, BCMA overexpression in RPMI8226 (R-BCMA) cells significantly increases growth and colony formation via upregulated phosphorylation of AKT, ERK, and p65, as well as NFkB (p65, p50, p52) DNA binding activity, associated with increased expression of multiple proliferative and anti-apoptotic genes.  Importantly, R-BCMA cells induce earlier onset of tumors when xenografted in mice when compared with parental cells, leading to accelerated tumor growth with increased VEGF and microvessel density (CD31+).  BCMA overexpression further induces osteoclast activation factors (MIP-1α/β, SDF-1), angiogenesis factors (VEGF, CD31, IL-8), adhesion proteins (CD44, ICAM1), as well as immunosuppressive factors including PD-L1, IL-10, TGFβ.  Thirty-nine IL-10 pathway associated genes are consistently elevated by BCMA overexpression in MM cells; conversely, BCMA knockdown blocks expression of these identified genes.  In parallel, osteoclasts and macrophages are key sources for APRIL in the MM BM microenvironment.  APRIL-triggered phosphorylation of AKT and ERK1/2 signaling, as well as NFκB DNA binding activities, are blocked by a blocking anti-APRIL monoclonal antibody hAPRIL01A.  Besides the induction of similar molecular targets defined by BCMA overexpression, APRIL also significantly induces adhesion and migration of MM cells, associated with enhanced angiogenesis and adhesion/chemoattractant factors.  APRIL further induces PD-L1, IL-10, and TGFβ which are blocked by hAPRIL01A.  Importantly, hAPRIL01A suppresses in vivo MM cell growth within implanted human bone chips in SCID mice.  It also blocks promotion of MM cell viability in cocultures with osteoclasts, macrophages, and plasmacytoid dendritic cells.  Finally, lenalidomide enhances cytotoxicity of hAPRIL01A in MM cells cocultured with osteoclasts.  These results define a central role of APRIL/BCMA activation in MM pathophysiology.  Moreover, our study delineates their roles mediating immunosuppression in the MM BM milieu via robust upregulation of PD-L1, IL-10, and TGFβ from MM cells, strongly supporting targeting APRIL and BCMA, alone or together, in novel immunotherapies for MM.