EXTRACELLULAR VESICLES-MEDIATED COMMUNICATION IN REMODELING MULTIPLE MYELOMA MICROENVIRONMENT: A NEW ROLE FOR THE NOTCH PATHWAY

Multiple myeloma (MM) is a still incurable hematological neoplasm mainly due to the localization of malignant plasma cells (PC) in the bone marrow (BM) where they can create pathological interaction with the surrounding resident cell populations. Indeed, upon localization within the BM, myeloma cells “educate” the resident cells to support the progression from monoclonal gammopathy of undetermined significance (MGUS) to active MM. The pathological crosstalk is also mediated by aberrant Notch signaling pathway due to the overexpression of Notch pathway members, such as Notch2 and the ligands Jagged1 and Jagged2. MM cells can enhance Notch signaling activation in the BM cells, leading to pro-tumorigenic processes such as drug resistance, osteoclastogenesis and angiogenesis. In this complex microenvironment, extracellular vesicles (EVs) shed from MM cells (MM-EVs) are reported to be involved the BM reprogramming thanks to their ability to carry molecular messengers both at long and short distance. Starting from this evidence, the aims of this thesis are: 1) to investigate the role of Notch pathway in MM microenvironment, focusing on the incompletely explored role of Jagged ligands in MM angiogenesis; 2) to evaluate the role of Notch in the ability of MM-EVs to induce osteoclastogenesis and angiogenesis, focusing on Notch2 receptor. To assess the first point, I used an inhibitory approach to study the role of Jagged ligands on MM cells and their outcome on MM-induced angiogenesis was assessed in vitro. To investigate the second aim, I used in vitro, in vivo and ex vivo models to assess the ability of EVs to carry and transfer the Notch2 to recipient cells, activating the Notch signaling and leading to their pro-tumorigenic effect. The role of Notch2 was assessed by RNA interference using lentiviral inducible vectors. The results obtained in the first part of this thesis indicate that Jagged ligands on MM cells can induce the angiogenic activity of endothelial cells promoting MM cell release of vascular endothelial growth factor (VEGF) and by directly activating the endothelial Notch pathway. Moreover, Jagged-mediated Notch pathway activation in BMSCs boosts their support to the angiogenic process. The second part of my PhD work focused on MM-EVs mediated communication showing that Notch2 is transferred to distant cells via MM-EVs and is able to increase Notch signaling in recipient cells also at distant sites. MM-EVs may increase the osteoclastogenic potential and the angiogenic ability in the tumor microenvironment in a Notch2 dependent way. Indeed, interfering with Notch2 expression decreases the amount of Notch2 in MM-EVs and their pro-tumorigenic effect, suggesting that MM-EVs may be the target of a Notch-directed therapeutic approach. I investigated the possibility to translate these results in the clinical practice using currently available Notch inhibitors, such as a γ-secretase inhibitor, i.e. DAPT. I found out that DAPT may hamper the effect of MM-EVs on both angiogenesis and osteoclastogenesis. The effectiveness of this pharmacological approach was confirmed ex vivo demonstrating DAPT ability to reduce the angiogenic effect of EVs from the BM aspirate of MM patients which better recapitulate the complexity of MM microenvironment. Overall, these results contribute to increase the knowledge on the role of Notch pathway in two pro-tumorigenic processes involving the MM microenvironment, such as tumor angiogenesis and osteoclastogenesis, indicating that the pathological Notch pathway activation may be triggered also at long distance via MM-EVs. Thereby, we provide the proof of concept that to hamper the pro-tumorigenic role of EVs could be a promising therapeutic strategy in MM as well as other tumors.