Notch targeting prevents multiple myeloma associated osteoclastogenesis

Introduction: Multiple myeloma is an incurable hematological tumor stemming from malignant plasma cells. MM cells accumulate in the bone marrow (BM) and establish complex interactions with normal BM stroma, which promotes tumor survival, bone disease and drug resistance. Recent evidence indicate that Notch pathway is deregulated in MM and plays a role in the pathogenesis of this tumor by modulating tumor cell biology and the crosstalk between MM cells and the BM niche. Activation of the Notch signaling is mainly due to the the aberrant expression of two of the Notch ligands, Jag1 and Jag2, that causes an increase in the ability of MM cells to trigger Notch activation in neighboring tumor cells and cells of the BM niche. In this work, we investigated the contribution of Jag1/2 to the osteoclastogenic potential of MM. Methods The γ-secretase inhibitor DAPT was used at a final concentration of 50μM. Osteoclasts (OCL) differentiation of Raw264.7 cells was induced by treatment with 50ng/ml mRANKL or co-culturing with MM cells or their conditioned medium. After 5-7days cells were stained using the TRAP Kit and counted. For bone resorption assay, Raw264.7 cells were cultured on Osteo Assay Surface plates under differentiation conditions for 7-10 days. Images of the resorbed areas were captured and the % of resorbed area was measured using the Wimasis image analysis software. Select RNAiTM siRNA system (Invitrogen) was used according to the manufacturer instructions for the selective inhibition of Jag1 and Jag2. Transfection was performed by electroporation using two plasmids coding the intracellular Notch1 and Notch2.Total RNA was isolated using TRI-Reagent. cDNA was prepared through MMLV-RT, then quantitative PCR was performed by Maxima SYBR Green qPCR Master Mix. ELISA Assay was performed using biotin-conjugated goat anti-human RANKL (Merck-Millipore) and Streptavidin-HRP-labeled secondary antibody. Flow citometry was performed using an anti-human RANKL antibody (Abcam) and a Alexa488-conjugated secondary antibody. Jag1 recombinant peptide was used at 0.5μg/ml. anti-RANKL neutralizing antibody was used at 0.1μg/ml. Results Our work demonstrates that Notch signaling drives MM-induced osteoclastogenesis and osteolysis. The underlying molecular mechanisms is based on Jag ligands dysregulation that allows MM cells to promote OCLs differentiation through the release of RANKL or by direct contact with OCL precursors which direct respectively the activation of the osteoclastogenic NF-kB and Notch pathways. Moreover, Jag ligands are able to mediate the interactions of MM cells with the BM niche which further promotes MM cell osteoclastogenic ability. Conclusions Our results suggest that Jag1/2 might present new promising therapeutic targets to reduce MM-associated bone disease.