Inhibition of endothelial and plasma cells NOTCH mediated comunication reduces angiogenesis in multiple myeloma patients.

Backgound: Multiple Myeloma (MM) is a hematopoietic malignant disease preceded by a Monoclonal Gammophaties of Undetermined Significance (MGUS) stage. Direct interaction of plasmacells (PCs) with endothelial cells (ECs) facilitates the tumor progression stimulating bone marrow angiogenesis. Mammals express four trans-membrane Notch receptors (Notch1-2-3-4), which bind five different ligands (DLL1-3-4 and Jagged1-2). Notch receptors regulate gene expression favoring proliferation, survival and embryonic development. γ-secretase receptor cleavage, mediated by cell interaction, is responsible of Notch pathway activation and, based on its involvement in angiogenesis, we first evaluate Notch basal expression level in ECs of MGUS and MM patients. Subsequently, we determine the role of Notch pathway in MMECs and tumor PCs direct crosstalk. Materials and Methods: RT-PCR, western blotting and immunofluorescence were performed to assess both gene and protein Notch expression. Pathway modulation was evaluated in normal and coculture conditions using siRNA designated for Notch1 and Notch2 receptors and γ-secretase (MK-0752) treatment through functional in vitro assays (adhesion, spontaneous migration, chemotaxis, capillarogenesis in vitro on Matrigel®). Results: Notch1 and Notch2 mRNA expression levels were decreased of 30% and 50% in MMECs than MGECs respectively. Conversely, the relatively protein cleaved form was higher in MMECs than MGECs (50% for Notch1 and 70% for Notch2). Investigating on Notch activation, we found that its targeted genes Hey1 and Hes1 were highly expressed in MMECs compared to MGECs. Regarding functional assays, Notch1 and Notch2 inhibition reduced chemotaxis (40% and 35%), adhesion (10% and 50%), spontaneous migration and in vitro angiogenesis on Matrigel® in MMECs. In co-culture experiments, Hey1 expression was no significantly different between direct/indirect crosstalk. On the other hand, direct and indirect culture determined an increase of Hes1 expression both at 24h and 48h. Adhesion assay revealed that MK-0752 treatment decrease cells adherence capability of almost 20%. Moreover, chemotaxis reduction (20%), decreased spontaneous migration after wound, in vitro reduced angiogenesis on Matrigel® and inhibition of pro-angiogenic cytokines release were also noticed after MK-0752 treatment. Conclusions: Due to the angiogenic modulation orchestrated by Notch we identify this pathway as an innovative target for thedevelopment of new drugs in MM therapy. Indeed, both the higher gene expression levels of Hey1 and Hes1 in MMECs and the higher activation of Notch1 and Notch2 receptors than MGECs confirm our hypothesis. In co-culture experiments we demonstrate an active role of PCs in enhancing ECs angiogenic abilities by stimulation of Notch pathway. On the other hand, siRNA silencing and treatments with γ-secretase negatively affect those acquired capabilities, suggesting the development of Notch inhibitors based therapies.