Notch-Jag axis in the interplay between multiple myeloma and endothelium

Multiple myeloma(MM) is an incurable plasma cells malignancy. MM is characterized by aberrant activation of Notch signaling pathway due to Jag1 and 2 ligands overexpression. Notch activation occurs through homotypic interaction among MM cells and through heterotypic interaction of MM with surrounding bone marrow(BM) cells. In BM microenvironment, stromal cells support MM through Jag-Notch interaction and endothelial cells (ECs) are involved in MM progression and dissemination, indeed MM patients display high level of angiogenesis. The aim of this project is to study if MM cells influences BM-ECs behavior through Jag-mediated activation of Notch signaling. Jag1 and 2 were silenced in MM cell line using lentiviral vector (RPMI8226shJAG1/2) were cultured with human pulmonary aortic endothelial cells (HPAECs) used as model of ECs. Angiogenesis was assessed by Matrigel assay at 24 h. To assess the exclusive effect of Jag ligands on angiogenesis, HPAEC were directly stimulated with soluble Jag1. Wound healing assay was set up to assess variation in the migration ability of HPAEC using conditioned media from RPMI8226SCR and RPMI8226shJAG1/2 cells. Moreover, a 3D organoid was set up to mimic BM niche to study the interplay between MM cells and ECs. Matrigel assays indicate that an active Jag-Notch axis directed by MM cells stimulates angiogenesis. Indeed, the absence of Jag ligands in MM cells reduces their angiogenic potential. Moreover, Jag1 alone is sufficient to promote EC angiogenic capability. Also ECs motility is modulated through Notch signaling, more specifically CM from silenced MM cells have a reduced ability to activate cell motility suggesting that MM cells release Notch dependent soluble factors that stimulate migration. Assays with 3D organoids show that MM cells support ECs survival. These data suggest that the Jag-Notch axis is necessary for MM-associated angiogenesis.