Impaired Osteogenic Differentiation of Mesenchymal Stem Cells Derived From Multiple Myeloma Patients Is Associated with a Failure In the Deactivation of the NOTCH Pathway

Abstract 1894

Mesenchymal stem cells (MSCs) give rise to bone marrow (BM) stromal cells and play an essential role in the formation and function of the MM microenvironment. Some recent studies revealed that MSCs from myeloma patients (MM-hMSCs) show an enhanced spontaneous and myeloma cell-induced production of cytokines and a distinctive gene expression profile, when compared to MSCs from normal donors (ND-hMSCs). However, regarding the osteogenic differentiation ability of MM-hMSCs conflicting observations were reported. In this study, we observed that MM-hMSCs, especially for those from MM patients with bone lesions, exhibited in the presence of osteogenic differentiation (OD) medium, significantly decreased alkaline phosphatase (ALP) activity, reduced expression of specific osteogenic markers (OPN, BMP2, OTX and BSP) and impaired matrix mineralization, compared to ND-hMSCs. However, MGUS-hMSCs, did not show a significantly impaired osteogenesis ability. Primary CFU-ALP assay from BM samples of diseased mice in the 5T33MM model also confirmed that the osteogenic differentiation ability of MSCs was impaired. Previous reports indicated that MM cells can suppress MSCs osteogenesis by HGF and DKK1 as observed in vitro (Giuliani et al, Cancer Res. 2007; Standal et al, Blood. 2007). Since MM-hMSCs have been cultured in vitro for several weeks and without any stimulation of MM cells, we believe that the impaired osteogenic differentiation of MM-hMSCs was due to an intrinsic abnormality. Several reports suggested that NOTCH signalling can maintain bone marrow mesenchymal progenitors in a more undifferentiated state by suppressing osteoblast differentiation (Hilton et al, Nat Med. 2008; Zanotti et al, Endocrinology. 2008). Therefore, we postulate that impaired osteogenic ability of MM-hMSCs might be (at least partly) related to abnormal NOTCH activity during osteogenesis. We found by quantitative real time PCR that NOTCH1, NOTCH2, Dll-1, Jagged-1, and NOTCH pathway downstream genes hes1, hey1, hey2, heyL were considerably decreased in ND-hMSCs after shifting them from normal culture medium to OD medium, indicating that NOTCH signalling was gradually suppressed during MSC osteogenesis. However, it was observed that the expression of NOTCH1, Jagged-1, Hes1 and Hes5 in MM-hMSCs did not decrease to the level of ND-hMSC with statistical difference. This implicates that the NOTCH signaling pathway remains in MM-hMSCs over-activated even in the presence of osteogenesis inducing signals. When the NOTCH signalling inhibitor DAPT was added to MM-hMSCs in OD medium, we found that hes1 expression was suppressed while, RUNX2 expression, a key transcription factor for osteoblastogenesis, as well as ALP activity, osteogenic genes expression and mineralization deposition were all increased. In conclusion our data indicate that MM-hMSCs exhibit in vitro lower osteogenic differentiation ability compared to ND-hMSCs, and that this impairement is associated with an inappropriate NOTCH pathway deactivation during the osteogenesis process. Targeting hMSCs in vivo by NOTCH inhibitors might have therapeutical potential to control bone disease in MM patients.