Abstract
A proliferation-inducing ligand (APRIL), a close member of B-cell-activating factor (BAFF) belonging to the TNF superfamily, is mainly produced by bone marrow (BM) accessory cells to stimulates growth and survival of multiple myeloma (MM) cells. Unlike BAFF, APRIL is dispensable in B cell homeostasis but more critical in plasma cell differentiation and survival. It has higher affinity than BAFF (nanomolar vs micromolar range) to B cell maturation antigen (BCMA) (nanomolar vs micromolar range) which expresses at high levels in all patient MM cells. APRIL also binds to a common plasma cell (PC) marker syndecan-1 (CD138) to induce signaling cascade via TACI, the other APRIL receptor in PC. We here characterize molecular mechanisms regulating APRIL activation in the BM microenvironment and further determine whether a novel anti-APRIL monoclonal antibody hAPRIL.01A inhibits its functional sequelae in MM. First, in vitro osteoclast and macrophage cultures were performed by stimulating CD14+ monocytes from MM patient samples with M-CSF/RANKL and M-CSF, respectively. Osteoclasts and macrophages secret significantly higher levels of APRIL than unstimulated CD14+ monocytes and BM stromal cells (BMSC), as confirmed by ELISA and qRT-PCR. All MM cell lines express cell surface BCMA in significantly higher level than TACI (p < 9.06e-15). H929 MM cells (expressing only BCMA, but not TACI), and other MM cell lines were next stimulated with APRIL, in the presence or absence of hAPRIL.01 Ab followed by immunoblotting and TaqMan® Array assays on harvested protein lysates and mRNA. APRIL stimulation consistently activated NF-kB, PI3K/AKT, and ERK1/2 signaling in MM cells. Importantly, NF-kB-DNA binding activities of p65 and p50 (p52, to a less extent), were significantly upregulated as early as 15 minute and sustain to 4h in all MM cell lines after stimulation. Conversely, hAPRIL.01 Ab completely blocked these signaling cascades, consistent with significantly decreased NF-kB-DNA binding activities in BCMA-knock-downed MM cells by shBCMA lentivirus transfection. APRIL further induced pro-survival proteins (Mcl1, Bcl2, BIRC3, XIAP) and MM cell growth-stimulating regulators (CCDN2, CDK4, CDK6, c-myc), which were completely inhibited by hAPRIL.01 Ab. These results correlated with blockade of hAPRIL.01 Ab in APRIL-promoted viability and colony formation of MM cells, in the presence of osteoclasts or macrophages. APRIL also induces adhesion of MM cells to BMSC, which was blocked by hAPRIL.01 Ab. This concurred with hAPRIL.01 Ab-reduced adhesion molecules (CD44, ICAM-1) induced by APRIL. APRIL-increased VEGF-A and PECAM-1 in MM cells was also significantly reduced by this mAb. APRIL-upregulated chemotactic/osteoclast-activating factors (MIP-1α, MIP1β, SDF-1) were also inhibited by this Ab. Other angiogenesis and adhesion/chemoattractant factors, i.e., IL-8, CXCL10, RANTES and MDC (ccl22), were changed in a similar fashion, indicating specific blocking of hAPRIL.01 Ab to APRIL-induced downstream target genes. This mAb further inhibited APRIL-increased viability of plasmacytoid dendritic cells (pDC) and diminished MM cell viability protected by pDC in 3-d cocultures. Finally, hAPRIL.01 specifically overcame APRIL-, but not IL-6, induced protection in lenalidomide- or dexamethasone-treated MM1S and H929 MM cells. These studies confirm a constitutive APRIL activation via BCMA and TACI in promoting malignancies of myeloma cells, supporting a novel therapeutics of hAPRIL.01 Ab to target MM in the BM microenvironment.
Disclosures
van Eenennaam: BioNovion: Employment. Elsas:BioNovion: Employment. Anderson:Celgene: Consultancy; Onyx: Consultancy; Gilead Sciences: Consultancy; Sanofi-Aventis US: Consultancy; Acetylon: Scientific Founder Other; Oncoprep: Scientific Founder Other.
Sheng Mai San protects H9C2 cells against hyperglycemia-induced apoptosis
