B cell maturation antigen (BCMA), selectively elevated in malignant plasma cells, is an ideal target antigen for immunotherapies for multiple myeloma (MM). Most recently, we reported novel antagonistic anti-BCMA antibody drug conjugates (ADCs) showing potent and specific anti-MM activities via effector cell-dependent and -independent mechanisms in vitro and in vivo (Blood 2014; 123:3128) We here further characterize molecular mechanisms of BCMA activation in MM cells in the bone marrow microenvironment by directly manipulating BCMA receptor levels in MM cells and ligation of a proliferation-inducing ligand (APRIL) to MM cells. Three MM cell lines H929, MM1S, and RPMI8226 with highest, medium, and low BCMA, respectively, were either transfected with lentiviruses of BCMA shRNA or cDNA. First, downregulation of BCMA significantly blocked viability of all 3 MM cells and induced caspase3/7 activities, which led to potent reduction of colony formation in a 3-week methylcellulose culture. Next, MM1R and H929 transfectants with the Doxycyclin (dox)-inducible lentiviral expression vector pTRIPZ shBCMA were generated. Time-dependent BCMA reduction only occurred in dox (1 ug/ml)-containing media. Dox-dependent BCMA inhibition was followed by decreased anti-apoptotic genes (Mcl1, Bcl-2, XIAP, NAIP, NFκB1, NFκB2) and proliferative genes (CCND2, CDK4/6, c-MYC). Conversely, overexpression of BCMA in RPMI8226 by either pCMV6/BCMA vector or pLocBCMA lentiviruses significantly increased NFκB (p65, p50, p52) DNA binding activity. Anti-apoptotic gene and cell proliferation genes were also up-regulated in BCMA-overexpressing MM cells. In addition, osteoclast activation factors MIP-1α/β, SDF-1, angiogenesis factors (VEGF, PECAM-1), adhesion proteins (CD44, ICAM1), as well as immunosuppressive factor TGFβ were augmented in BCMA-overexpressing MM cells. Importantly, opposite effects on these downstream genes were seen in BCMA-knockdown MM cells. Moreover, stimulation of 3 MM cells by APRIL robustly induces NFκB DNA binding activity (p65, p50, and p52, to a lesser extend) and activates PI3K/AKT and ERK1/2 signaling. APRIL also induces pro-survival/anti-apoptotic targets (BCL2A1, NFκB1, NFκB2) and chemotactic/osteoclast activating factors (MIP1α and MIP1β) in a dose-dependent manner. Angiogenesis and adhesion/chemoattractant factors (VEGF, IL-8, CXCL10, and RANTES) were also significantly induced upon APRIL stimulation. In contrast, BCMA-Fc protein that blocks APRIL binding to BCMA, inhibits secretion of these cytokines/chemokines, indicating specific response of engagement of BCMA by APRIL in BCMA-expressing MM cells. APRIL induced adhesion and migration of MM cells whereas BCMA-Fc blocked APRIL-induced responses. Finally, RPMI8226/pLocBCMA cells induce earlier tumor onset and more tumor growth in mouse xenograft model when compared with control RPMI8226 cells. In contrast, pTRIPZ shBCMA H929 cells induce significantly less tumor formation and further prolong survival of mice fed with dox(2 ug/ml)-containing water than those without dox. Together, these results define molecular regulators of active APRIL/BCMA signaling cascade in the MM BM milieu, further supporting targeting APRIL/BCMA in MM.
Anderson:Celgene: Consultancy; Sanofi-Aventis: Consultancy; Onyx: Consultancy; Acetylon: Scientific Founder, Scientific Founder Other; Oncoprep: Scientific Founder Other; Gilead Sciences: Consultancy.
Asterisk with author names denotes non-ASH members.