The role of NOTCH2 in the Extracellular vesicles mediated communication in Multiple Myeloma: a focus on angiogenesis and osteoclastogenesis

Multiple myeloma (MM) is an uncurable hematologic disease, characterized by the proliferation of malignant plasma cells in bone marrow (BM). The poor prognosis is deeply affected by the ability of tumor cells to establish pathological interaction with BM niche, leading to tumorigenic processes crucial for MM progression. One of the actors involved in this pathological communication is the direct cellular interaction mediated by the aberrantly expressed oncogene NOTCH2 by which MM cells trigger the NOTCH pathway activation on BM population boosting their pro-tumorigenic activity, including angiogenesis, osteoclastogenesis and the consequent spread of osteolytic bone lesions. The shaping of BM microenvironment may be also mediated by the ability of MM cells to release extracellular vesicles (MM-EV), which are able to carry key molecular messengers at distant cells of the BM niche. The purpose of our research is to assess the pro-tumorigenic effect of MM-EV and the role played by NOTCH pathway in EV-mediated communication between MM cells and the BM cells, focusing on the role of the NOTCH2 receptor. Our previous findings indicated that the MM-EV may be internalized by endothelial cells and osteoclasts. Moreover, MM-EV carry NOTCH2 and can transfer it between distant cells, increasing tumor angiogenesis and osteoclast differentiation. MM-EV biological outcome was correlated with the levels of NOTCH2 expression in EV-producing MM cells. Here, we get advantage of an inhibitory approach that allows us to silence NOTCH2 expression in MM cells lines, i.e. RPMI8226 and OPM2, and produce MM-derived EV depleted of NOTCH2. In this way, we measured the ability of MM-EV, depleted or not of NOTCH2, to activate NOTCH signaling by using a reporter assay on HeLa cells transfected with a plasmid vector expressing the nanoluciferase under the control of a NOTCH responsive element. Our results confirm the ability of MM-EV to activate Notch signaling only when carrying high levels of NOTCH2. Finally, we demonstrate that MM-EV loaded with NOTCH2 may induce tumor angiogenesis and osteoclastogenesis by activating NOTCH signaling in recipient cells. At this purpose we performed angiogenesis and osteoclastogenesis assay coupling a stimulation with MM-EVs with the pharmacological blockade of NOTCH activation obtained through the administration of the -secretase inhibitor DAPT. As expected, the MM-EV angiogenic and osteoclastogenic potential of MM-EV is strongly reduced in the presence of the DAPT. These results provide the first evidence that NOTCH2 plays a key role in the pro-tumorigenic activity of MM-EV, suggesting that targeting NOTCH pathway may be a promising therapeutic strategy to hamper the pro-tumorigenic role of EV in MM.