Targeting extracellular vesicles in multiple myeloma: a new role for the Notch pathway

Multiple myeloma (MM) is the second most frequently diagnosed hematological malignancy and today is still incurable, mainly due to the interaction between the bone marrow (BM) niche. Indeed, MM cells accumulate in the BM and establish complex interactions with the surrounding normal cells, forcing them to assume a pro-tumor behavior. In this process, a key role is played by Notch2 and two Notch ligands Jagged1 and 2, whose deregulated expression causes an aberrant activation of the Notch pathway both in MM cells and in the BM niche cells mediating a pathological communication. In this complex picture, new players have recently come to light: the extracellular vesicles (EVs). EVs include a heterogeneous group of cell-derived membranous structures classified into exosomes and microvesicles, which are key mediators in the communication between tumor and stroma. In this work, we aimed to elucidate the possible role of the Notch pathway in EV-mediated communication between MM cells and non-tumor cells of the BM niche, by investigating the pro-tumorigenic activity of EVs from the MM cell line RPMI8226 and the changes occurring in EVs from RPMI8226 constitutively inhibited for Jagged1/2 (MMJ1/2KD) and Notch2 (MMN2KD). The ability of MM-EVs to interact with normal cells and activate the Notch pathway in the recipient cells was evaluated in vivo, by injecting MM-EVs in the duct of Cuvier of 2 days post fertilization Notch-reporter Tg(T2KTp1bglob:hmgb1-mCherry)jh transgenic zebrafish embryos. Results showed that MM-EVs are uptaken by embryo’s cells and induce Notch activation in the intersegmental vessels, caudal artery and in the area of the caudal hematopoietic tissue, the main hematopoietic organ, analogous to the human BM. By contrast, this effect is reduced when EVs were produced by MMJ1/2KD and MMN2KD cells. We also demonstrated that MM-EVs promote three key processes involved in myeloma progression: osteoclastogenesis, angiogenesis and drug resistance. Interestingly, Jagged1/2 or Notch2 knock down reduces MM-EVs pro-tumor activity. Indeed, MMJ1/2KD-EVs and MMN2KD-EV lost the pro-osteoclastogenic effect on the monocyte cell line Raw264.7 and the ability to induce human endothelial cells to organize tubular structures, a key step of angiogenesis. Finally, the level of pro-tumor cytokines released by the bone marrow stromal cells (BMSCs) HS5 was also reduced when they were treated with MMJ1/2KD-EVs and MMN2KD-EVs along with their ability to promote MM cells resistance to bortezomib and melphalan. In this work, we provide the first evidence that targeting the Notch pathway may represent a suitable strategy to hamper MM-EVs-mediated communication with the BM niche and the nasty consequence on the progression of this disease