Anti-β²-Microglobulin Monoclonal Antibodies Recruit MHC Class I to and Exclude Growth and Survival Cytokine Receptors from Lipid Rafts and Induce Apoptosis in Myeloma Cells.

Multiple myeloma (MM) is a B-cell malignancy dependent on the bone marrow microenvironment, where a number of cytokines contribute to myeloma cell growth, survival, migration and resistance to conventional chemotherapy. We have recently shown that β₂M-specific mAbs have remarkably strong antimyeloma activities by binding to β₂M and recruiting MHC class I to the lipid rafts, in turn activating JNK and inhibiting ERK and PI3K/Akt pathways, whereas, neither interleukin-6 (IL-6) nor insulin-like growth factor-I (IGF-I) affect mAb-tumoricidal effects. This study was undertaken to elucidate the mechanisms underlying anti-β₂M mAb-induced PI3K/Akt and ERK inhibition and the inability of IL-6 and IGF-I to protect myeloma cells from mAb-induced apoptosis. In our experiments, β₂M-specific mAbs were added to cultures of myeloma cells, including tumor cell lines and freshly isolated primary tumor cells from patients with MM, with or without addition of IL-6 or IGF-I. Cell apoptosis was examined 48 hours later by Annexin-V staining assay. The results confirmed that the addition of IL-6 or IGF-I significantly undermined dexamethasone-induced cell death, but did not abrogate mAb-induced apoptosis in myeloma cells. As lipid rafts are considered to function in part as a platform for signaling from the receptors, and ERK and PI3K/Akt are downstream of IL-6 and IGF-I receptor activation, we hypothesized that MHC class I relocation to lipid rafts by mAbs may disrupt IL-6 and IGF-I receptor signaling. For this purpose, myeloma cells were incubated with cytokines, with or without mAbs. After 30 minutes of treatment, lipid rafts were prepared and separated using a discontinuous sucrose gradient ultracentrifugation followed by immunoblotting with specific antibodies. The results showed that by recruiting MHC class I into lipid rafts, anti-β₂M mAbs excluded IL-6 and IGF-I receptors and their substrates from the rafts. Likewise, upon immunoprecipitation, IL-6 and IGF-I receptors in cytokine-treated cells were physically associated with caveolin-1, a lipid raft-associated protein. Such an association was missing or less prominent in cytokine- and β₂M-specific mAb-treated myeloma cells. Treatment with methyl-β-cyclodextrin (MCD), an agent that disrupts the structure of lipid rafts in the cell membrane, could abrogate both IL-6-mediated protection against apoptosis induced by dexamethasone and β₂M-specific mAb-induced cell death of myeloma cells. Using Western blotting assay, we further found that β₂M-specific mAbs were not only redistributed the receptors in cell membrane, but also abrogated cytokine-mediated JAK/STAT3, PI3K/Akt, and Ras/Raf/ERK pathway signaling, which are otherwise constitutively activated in myeloma cells. Thus, this study further defines the tumoricidal mechanism of the β₂M-specific mAbs and provides strong evidence to support the potential of these mAbs as therapeutic agents for myeloma.