Notch signaling dysregulation promotes multiple myeloma-associated bone disease

Introduction The Notch pathway is dysregulated in multiple myeloma (MM), due to the hyperexpression of Notch receptors and Jag1/2 ligands. This alteration affects MM cell biology and improves the ability of MM cell to shape the bone marrow (BM) niche inducing a supportive behavior that promotes tumor progression.\u2028 BM niche alterations include an increased osteolytic activity results in bone disease which affects patients quality of\u2028 life and promotes tumor growth and survival, contributing to the fatal outcome of MM. Notch has been reported as a key regulator of bone tissue remodeling. The aim of this work was to study the role of Notch signaling in MM-associated bone disease and, specifically, to verify the effect of Notch signaling activated by the two dysregulated ligands, Jag1 and 2. \u2028Methods The experiments took advantage of cell lines as well as primary cells including MM cells, osteoclasts (OCLs) and BM stromal cells (BMSCs). OCL differentiation was induced for 5-7days cells by: i) adding 50ng/ml RANKL, ii) co-culturing with MM cells or iii) addition of MM cell lines conditioned media (CM). OCLs were enumerated by TRAP staining. Notch signaling was inhibited by the g-secretase inhibitor, 25-50 mM DAPT, or by Jag1-2 silencing performed using the RNAiTM siRNA system (Invitrogen). Quantitative PCR reactions were carried with MaximaTM SYBR Green qPCR Master Mix.\u2028 RANKL was quantified by ELISA and flow cytometry. \u2028Results Our findings indicate that Notch activity induces the autonomous release of the osteoclastogenic factor RANKL by MM cells, inducing OCLs differentiation and osteolytic activity.\u2028 In addition, MM-derived Jag1-2 directly activate the pro-osteoclastogenic Notch signaling in the neighboring OCL progenitors, thereby boosting their differentiation.\u2028 Finally, MM cell-derived Jag1-2 activate Notch signaling in BM stromal cells (BMSCs), which, in turn, enhance the osteoclastogenic potential of MM cells, by boosting their secretion of RANKL.\u2028 Conclusion: This study provides the first evidence that Jag1 and \u20282 ligands aberrantly expressed by MM cells shape the BM niche and foster MM-induced OCLs differentiation and bone resorption activity. Our results suggest that inhibiting the engagement between the Jag1-2 ligands expressed by MM cells and Notch receptors may prevent MM-associated bone disease. This evidence strengthen the rational of a molecular therapy in MM myeloma directed to Notch ligands, Jag1 and 2