The signaling lymphocytic activation molecule family (SLAMF7; also known as CS1 or CD319) is highly expressed on plasma cells from multiple myeloma (MM) as well as natural killer (NK) cells and is a well-known therapeutic target of elotuzumab. The objective of this study was to evaluate the clinical significance of serum soluble SLAMF7 (sSLAMF7) levels in patients with MM (n=103) and furthermore the impact of sSLMF7 on the antitumor activity of anti-SLAMF7 antibody. Thirty-one percent of MM patients, but not patients with monoclonal gammopathy of undetermined significance and healthy controls, had detectable levels of serum sSLAMF7, which were significantly increased in advanced MM patients. Further, MM in sSLAMF7-postive patients exhibited aggressive clinical characteristics with shorter progression-free survival times in comparison with sSLAMF7-negative patients. In responders to MM therapy, the levels of sSLAMF7 were undetectable or decreased compared with those before treatment. In addition, the anti-SLAMF7 antibody-mediated antibody-dependent cellular cytotoxicity of NK cells against MM cell lines was inhibited by recombinant SLAMF7 protein. Thus, our findings suggest that high concentrations of sSLAMF7, which could transiently suppress the therapeutic effects of elotuzumab, may be a useful indicator of disease progression in MM patients.

The signaling lymphocytic activation molecule family 3 (SLAMF3) is a member of the immunoglobulin superfamily expressed on T, B, and natural killer cells and modulates the activation and cytotoxicity of these cells. SLAMF3 is also expressed on plasma cells from patients with multiple myeloma (MM), although its role in MM pathogenesis remains unclear. This study found that SLAMF3 is highly and constitutively expressed on MM cells regardless of disease stage and that SLAMF3 knockdown/knockout suppresses proliferative potential and increases drug-induced apoptosis with decreased levels of phosphorylated ERK protein in MM cells. SLAMF3-overexpressing MM cells promote aggressive myeloma behavior in comparison with cytoplasmic domain-truncated SLAMF3 (DSLAMF3) cells. SLAMF3 interacts directly with adaptor proteins SH2 domaincontaining phosphatase 2 (SHP2) and growth factor receptor bound 2 (GRB2), which also interact with each other. SLAMF3 knockdown, knockout, DSLAMF3, and SHP2 inhibitor-treated MM cells decreased phosphorylated ERK protein levels. Finally, serum soluble SLAMF3 (sSLAMF3) levels were markedly increased in advanced MM. Patients with high levels of sSLAMF3 progressed to the advanced stage significantly more often and had shorter progression-free survival times than those with low levels. 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.Implications: SLAMF3 may be a new therapeutic target for immunotherapy and novel agents such as small-molecule inhibitors.

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<p>Table S1. Primer sequences Table S2. Patient characteristics in cell - surface SLAMF3 and CD138 expression analysis. Table S3. One hundred and sixty-eight gene sets that were significantly upregulated in SLAMF3-KMS34 cells compared with Î"SLAMF3 cells (NOM P-value <0.05, FDR q-value <0.20). Table S4. Core enrichment genes in MAPK/ERK signal-related gene sets which were significantly enriched in SLAMF3.KMS34 cells compared with Î"SLAMF3 cells. Table S5. Characteristics of MGUS and newly diagnosed MM patients. Figure S1. SLAMF3 expression in MM patients. Figure S2. Expression of cell markers CD38, CD138, CD34, CD45, CD19, and CD27 in SLAMF3high and SLAMF3low cell fractions of U266 cells. Figure S3. SLAMF3, SHP2, and GRB2 mRNA in MM cell lines and patient samples. Figure S4. Melphalan sensitivity and BrdU incorporation in MM cells treated with SHP2- and GRB2-specific siRNA and NSC87877. Figure S5. Enrichment plots for the gene sets in Supplementary Table S3 which were associated with survival and cell proliferation. Figure S6. SLAMF3 expression on MM cells induced gene expression associated with survival and cell proliferation. Figure S7. Soluble SLAMF3 (sSLAMF3) in MM patients and cell lines. Figure S8. Correlation of serum sSLAMF3 levels with the percentage of BM plasma and SLAMF3-positive cells, and serum IL-6 concentrations. Figure S9. Receiver operating characteristics (ROC) curve for predicting the R-ISS score (I vs. II/ III) for patients with MM. Figure S10. Effect of soluble SLAMF3 on MM cells. Figure S11. Receiver operating characteristics (ROC) curve (ROC) curve Receiver operating characteristics (ROC) curve for predicting the R-ISS score (I vs. II/ III) for patients with MM. Figure S12. Effect of soluble SLAMF3 on MM cells. Figure S13. Proposed mechanism of disease progression via SLAMF3 signaling in MM. Figure S14. Expression of cell markers CD38, CD138, CD34, CD45, CD19, CD27 and c -kit in SLAMF3high and SLAMF3low cell fractions of U266 cells. Figure S15. Characteristics of SLAMF3high and SLAMF3low U266 cells.</p>

<p>Table S1. Primer sequences Table S2. Patient characteristics in cell - surface SLAMF3 and CD138 expression analysis. Table S3. One hundred and sixty-eight gene sets that were significantly upregulated in SLAMF3-KMS34 cells compared with Î"SLAMF3 cells (NOM P-value <0.05, FDR q-value <0.20). Table S4. Core enrichment genes in MAPK/ERK signal-related gene sets which were significantly enriched in SLAMF3.KMS34 cells compared with Î"SLAMF3 cells. Table S5. Characteristics of MGUS and newly diagnosed MM patients. Figure S1. SLAMF3 expression in MM patients. Figure S2. Expression of cell markers CD38, CD138, CD34, CD45, CD19, and CD27 in SLAMF3high and SLAMF3low cell fractions of U266 cells. Figure S3. SLAMF3, SHP2, and GRB2 mRNA in MM cell lines and patient samples. Figure S4. Melphalan sensitivity and BrdU incorporation in MM cells treated with SHP2- and GRB2-specific siRNA and NSC87877. Figure S5. Enrichment plots for the gene sets in Supplementary Table S3 which were associated with survival and cell proliferation. Figure S6. SLAMF3 expression on MM cells induced gene expression associated with survival and cell proliferation. Figure S7. Soluble SLAMF3 (sSLAMF3) in MM patients and cell lines. Figure S8. Correlation of serum sSLAMF3 levels with the percentage of BM plasma and SLAMF3-positive cells, and serum IL-6 concentrations. Figure S9. Receiver operating characteristics (ROC) curve for predicting the R-ISS score (I vs. II/ III) for patients with MM. Figure S10. Effect of soluble SLAMF3 on MM cells. Figure S11. Receiver operating characteristics (ROC) curve (ROC) curve Receiver operating characteristics (ROC) curve for predicting the R-ISS score (I vs. II/ III) for patients with MM. Figure S12. Effect of soluble SLAMF3 on MM cells. Figure S13. Proposed mechanism of disease progression via SLAMF3 signaling in MM. Figure S14. Expression of cell markers CD38, CD138, CD34, CD45, CD19, CD27 and c -kit in SLAMF3high and SLAMF3low cell fractions of U266 cells. Figure S15. Characteristics of SLAMF3high and SLAMF3low U266 cells.</p>