CD200 Is a Potent New Growth Factor in Multiple Myeloma through IGF-1R Interaction

Multiple myeloma (MM) is the second most common hematologic malignancy characterized by the accumulation of clonal plasma cells within the bone marrow.

We previously identified that CD200 is expressed in Multiple Myeloma Cells (MMCs) of 78% of newly MMdiagnosed patients and this expression is associated with a bad prognosis. CD200 is a type I immunoglobulin superfamily transmembrane glycoprotein that is expressed by various cell types including B cells and T cells. In tumors cells, interaction of CD200 with its known receptor CD200R, inhibits T-cell mediated immune response and induces immunosuppressive responses. However, the role of CD200 in tumorigenesis is poorly understood. Recently, a study shown that CD200 can induce neuritogenesis and promote neuronal survival of primary neurons through interaction and activation of the fibroblast growth factor receptor.

Here, we investigated for the first time the role of CD200 in MM cell biology. First, we identified that CD200 protein expression is present only in 3 out of 9 Human Myeloma Cell lines (HMCLs). Then, we confirmed that CD200 protein is strongly expressed in MMCs of 73% of the 35 previously untreated MM patients and that CD200 is never expressed in the normal plasma cell counterparts.

CD200 overexpression using lentiviral vector significantly increased HMCLs cell growth in a serum free culture medium (SYN-H) compared to GFP overexpression (fold increasing: 1.6 and 1.4 at day 4; 2.5 and 2.1 at day 7 for LP1 and XG1 respectively). Furthermore, CD200 overexpression results in significant increasing of the number of colonies for HMCLs CD200^positive compared to HMCLs CD200^negative in clonogenic assays (4.3 fold and 13.1 fold for LP1 and XG1 respectively, p<0.001). We confirmed also the growth advantage linked to CD200 overexpression in coculture model. These effects are significantly inhibited by a monoclonal antibody (MoAb) against CD200 compared to treatment with a no-relevant anti-IgG1. We also confirmed the significant inhibition of proliferation with the anti-CD200 MoAb using clonogenic assays. The anti-CD200 MoAb has no effect on the GFP transduced HMCLs.

To investigate the implication of CD200R in CD200-induced MM cell growth, inducible CD200R depletion was analyzed in CD200^positive and CD200^negative, using lentiviral vectors. After doxycycline addition, the shCD200R reduced CD200R expression by 95-100% but had no significant effects on the proliferation advantage mediated by CD200 demonstrating that the growth advantage supported by CD200 overexpression is independent of CD200R.

Thus, to investigate the possibility of another receptor activated by CD200 interaction, we added different essential MM growth factors to the serum free medium. Addition of fetal calf serum, IGF1 or insulin in SYN-H medium abrogated the growth advantage provided by CD200 overexpression in HMCLs. These data suggest that CD200 could act as a growth factor through interaction and activation of IGF1R. In addition, overexpression of CD200 in a IGF-1R negative XG12 HMCL did not stimulate its cell growth or clonogenic activity. Furthermore, anti-IGF-1R monoclonal antibody completely abrogated the CD200-dependent growth activity at day 4 and at day 7 in LP1CD200^positive cell line compared to non-relevant antibody. The anti-IGF-1R MoAb has no effect on control LP1GFP ^positive cell line cultured in serum free culture medium.

In order to identify a direct interaction between CD200 and IGF-1R in serum free culture conditions, IGF-1R immunoprecipitation in denaturing conditions was performed at day 4 using LP1GFP and LP1CD200 cell lines. CD200 and IGF-1R interaction was identified by mass spectrometry and western-blotting.

According to these data, MM patients with CD200^high and IGF1R^high gene expression have a poor prognosis (OS) compared to patient with only CD200^high or IGF1R^high expression (P=0.03, 52.7 vs 61.9 months in HM series; and P=0.02, 58.9 vs 68.1 months in TT2 series) and compared to patients with CD200^low and IGF1R^low gene expression (P=0.002, 52.7 vs 71.6 months in HM series; and P=0.01, 58.9 vs 70.2 months in TT2 series). Same results are observed for EFS.

Altogether, these data demonstrated a new role of CD200 as a MM growth factor through interaction with IGF1R. Our results suggest that CD200 and IGF1R inhibition by monoclonal antibodies could represent a promising strategy in MM.