The New Immunoreceptor SLAMF3 Promotes Aggressive Biological and Clinical Characteristics in Multiple Myeloma

Introduction: The signaling lymphocyte activation molecule family member 3 (SLAMF3), also known as Ly9 or CD229, is a member of the immunoglobulin superfamily expressed on T, B, and natural killer cells and modulates the activation and cytotoxicity of these cells via self-ligand binding. We reported that SLAMF3 was highly and constitutively expressed on plasma cells from patients with multiple myeloma (MM) regardless of disease stage [Yamada et al., ASH 2014 Abstract #2063], although its role in MM pathogenesis remains unclear. We thus investigated the biologic functions and single-nucleotide polymorphism (SNP) rs509749 of SLAMF3 in MM and whether serum levels of its soluble form in MM patients are a prognostic factor.

Materials and Methods: 1) Peripheral blood and bone marrow samples were obtained from 96 newly diagnosed (17 asymptomatic and 79 symptomatic) MM patients and 16 patients with monoclonal gammopathy of undetermined significance (MGUS). 2) The expression of cell-surface antigen, drug sensitivity to anti-MM agents (melphalan and bortezomib), and proliferative potential of MM cells were analyzed using flow cytometry or MTT assay. SLAMF3 knockdown MM cell lines were obtained using the lentiviral shRNA system. Full-length SLAMF3 and cytoplasmic domain-truncated SLAMF3 (ΔSLAMF3)-overexpressing KMS34 cells, which lack SLAMF3 expression, were established through corresponding vectors. The adaptor protein of SLAMF3 and signal transduction proteins were identified by western blotting and immunoprecipitation. Gene expression profiling was performed using an oligonucleotide microarray, followed by gene set enrichment analysis (GSEA). SNP genotyping was analyzed by allele-specific real-time PCR. Soluble SLAMF3 (sSLAMF3) levels were measured using ELISA.

Results: 1) SLAMF3-positive KMS18 and U266 cell lines contained major SLAMF3^high and minor SLAMF3^low cell fractions. The proliferative potential and percentages of anti-MM agent-induced apoptosis in SLAMF3^high cells were significantly higher and lower than in SLAMF3^low cells, respectively. The high malignant potential in MM cells was abolished by SLAMF3 knockdown and inhibited by anti-SLAMF3 antibody. Furthermore, SLAMF3 overexpression in KMS34 cells promoted the proliferative and antiapoptotic potential in comparison with ΔSLAMF3 cells. Almost all MM cell lines expressed adaptor proteins SHP2 and GRB2, but not SAP or EAT2. SLAMF3 interacted directly with SHP2 and GRB2, and SHP2 also interacted with GRB2. SHP2 inhibitor-treated or SHP2/GRB2-knockdown cells had characteristics similar to SLAMF3-knockdown cells. Furthermore, the phosphorylated ERK protein was decreased in SLAMF3 knockdown, ΔSLAMF3, and SHP2 inhibitor-treated cells. GSEA also showed the MAPK/ERK signal pathway to be significantly downregulated in ΔSLAMF3 cells compared with SLAMF3 cells. 2) The frequency of GG genotypes of SNP rs509749 in MM patients was 63.6% (n=28), of AG 29.5% (n=13), and of AA 6.8% (n=3), while those 3 genotypes were present nearly equally in healthy controls. Patients with GG genotypes had significantly more advanced stage and tended to have shorter overall survival times than patients with AG genotypes. Consistent with the results, SLAMF3.KMS34 cells with the G allele of rs509749 had higher proliferative capacity and were more resistant to anti-MM agents than those with the A allele. 3) sSLAMF3 levels were significantly higher in symptomatic MM than in asymptomatic MM and MGUS and markedly increased in advanced MM. MM patients with high levels (≥3.3 ng/ml, n=62) of sSLAMF3 had aggressive clinical characteristics and shorter progression-free survival times than those with low levels (<3.3 ng/ml, n=32; P=0.032). sSLAMF3 was produced by SLAMF3-expressing MM cells via cleavage by autocrine secretion of MMP-9.

Conclusions: This study revealed that SLAMF3 molecules consistently expressed on MM cells transmit MAPK/ERK signals mediated via the complex of SHP2 and GRB2 by self-ligand interaction between MM cells and induce a high malignant potential in MM. Furthermore, high levels of serum sSLAMF3 may reflect MM disease progression and be a useful prognostic factor. Thus, SLAMF3 may be a new therapeutic target for immunotherapy and novel agents such as small-molecule inhibitors.