GPRC5D Is a Cell Surface Plasma Cell Marker Whose Expression Is High In Myeloma Cells and Reduced Following Coculture With Osteoclasts

Identification of unique cell surface markers on myeloma cells is important for development of targeted therapies and detection of residual disease. GPRC5D is an orphan receptor reportedly expressed at high level in bone marrow (BM) aspirates or biopsies from myeloma patients. Other studies detected GPRC5D in skin and brain but not in other tissues. The aims of the study were to assess GPRC5D gene expression and cell surface localization in myeloma cells, the changes in expression of GPRC5D and other typical myeloma cell markers following coculture of primary myeloma cells with osteoclasts and the consequences of GPRC5D gene silencing on myeloma cell growth. Global gene expression profile (GEP), qRT-PCR and immunohistochemistry revealed exclusive high expression of GPRC5D in normal and myeloma plasma cells but not in B cells, MSCs, osteoclasts or BM mononucleated cells. GPRC5D expression was higher in myeloma plasma cells from newly diagnosed patients (n=698) compared to normal plasma cells (n=26, p<0.0001)). Among molecularly classified groups GPRC5D expression is higher in MS, MF and LB subgroups and lowest in CD2 and MM cell lines. Flow cytometry analysis and immunohistochemistry detected cell surface expression of GPRC5D in myeloma plasma cells while nuclear localization was also detected in certain myeloma cell lines (e.g. H929). Western blots analysis confirmed GPRC5D expression in whole cell lysate and nuclear fraction. We and other demonstrated phonotypical plasticity of myeloma plasma cells capable of altered expression of recognizable plasma cell markers (e.g. CD138, CD45) following coculture with stromal cells (Dezorella et al., 2009) or osteoclasts (Yaccoby, 2005). In coculture of primary CD138-selected myeloma cells with osteoclasts (n=8), CD138 (p<0,004), CD38 (p<0.001) and GPRC5D (p<0.007) were commonly and significantly downregulated, CD45 (p<0.02) was upregulated, and IRF4 expression was unaffected in cocultured myeloma cells compared to the control freshly obtained uncultured myeloma cells assessed by GEP and qRT-PCR. We also observed reduced expression of GPRC5D in MM cells purified from focal lesion compared to interstitial marrow in paired clinical samples (n=176, p<1.21E-09), suggesting that high activity of osteoclasts in osteolytic lesions mediates phenotypical alteration in myeloma cells. Stable knockdown of GPRC5D by 70% in CAG myeloma line using lentiviral particles containing shRNA had no effect on short-term growth of these cells assessed by MTT assay. These data indicate that GPRC5D is a novel cell surface marker for myeloma plasma cells and that its expression is reduced in dedifferentiated myeloma cells.