Jagged ligands as new targets in multiple myeloma associated angiogenesis

Multiple myeloma (MM) is an incurable hematologic disease. Its progression and aggressiveness stem from its peculiar localization in the bone marrow (BM) and the ability to promote angiogenesis, inducing in the BM the release of angiogenic factors, such as VEGF thereby boosting the number of vessels which represents a source of nutrients, oxygen and a way of metastatic dissemination. The family of Notch receptors regulates the aberrant interaction between MM cells and the BM population. MM cells overexpress the Notch ligands Jagged1 and 2 leading to the activation of Notch signaling both in MM cells and the surrounding BM population such as endothelial cells (ECs) and BM stromal cells (BMSCs). In this work, we investigated the role Jagged1 and 2 in MM-associated angiogenesis and elucidated different mechanisms involved. In order to assess the role of Jagged1 and 2, we knocked down their expression in two MM cell lines (OPM2 and RPMI8226) by using specific siRNA (MMJ1/2KD) and evaluated MM and MMJ1/2KD cell ability to directly promote angiogenesis by measuring EC adhesion, motility and tube-formation, or to indirectly increase the pro-angiogenic ability of BMSCs assessing their release of angiogenetic factors. The MM and MMJ1/2KD potential to induce the formation of new vessels was confirmed in vivo by using a zebrafish embryo model of MM and validated by analyzing the BM biopsies (BMBs) of MM patients. The results obtained indicated that MM-derived Jagged ligands played a role in inducing the angiogenic switch. Indeed, the injection of MM cells in the MM zebrafish model induced the formation of new vessels, which was reduced if MMJ1/2KD were injected. The in vitro experiments allowed us to explore the mechanisms involved. First, MM cells increase the angiogenic potential of ECs inducing the activation of Notch signaling or the release of VEGF. More importantly, Jagged ligands induce the angiogenic process by stimulating the Notch pathway-mediated VEGF secretion in BMSCs. Interestingly, the immunohistochemical analysis of MM patients’ BMBs confirmed these results showing that MM infiltration levels in the neoplastic area correlated with Jagged ligands expression, which is positively associated with Notch signaling activation in both MM cells and non-tumor stromal cells. Additionally, Notch activation correlated with the expression of VEGF-A in the BM microenvironment. In this research, we obtained in vitro, in vivo and ex vivo results confirming the role of Jagged1 and 2 in MM-associated angiogenesis, providing a rational for a Jagged-directed therapy in MM.