Targeting the Notch pathway in multiple myeloma through small molecules uncoupling Notch-Jag interaction

Multiple myeloma (MM) is still an incurable plasma cell malignancy, mainly due to the localization of MM cells in the bone marrow (BM), where they establish complex interactions with the local milieu, resulting in severe osteolysis, drug resistance (DR) and consequent relapse. Notch signaling is involved in MM cell migration, proliferation, survival and DR. In MM Notch is dysregulated due to the aberrant expression of Notch1-2 receptors and Jag1-2 ligands. The relevance of this dysregulation in MM is evident since through the interaction of Notch receptors and ligands, MM cells shape the nearby niche, promoting key features of MM progression such as osteolysis and DR induced by BM stromal cells (BMSCs). The evidence of our group and literature data showed that all these effects can be interfered by knocking down Jag1-2 in MM cells. This provides a strong rationale for disrupting Notch-Jagged interaction in MM cells and the nearby BMSCs in order to set up a novel therapeutic strategy for relapsing myeloma and advanced bone disease. We have identified in silico 100 small candidate molecules able to disrupt the Notch-Jag complex by screening a large combinatorial database of commercial compounds. The effect of two candidate compounds on Notch transcriptional activity was preliminary analyzed. Notch signal activation was measured in HEK293T, using a Notch responsive Luciferase reporter assay. The compounds efficacy in inhibiting Notch activity was comparable to common anti-Notch agents, i.e. γ-secretase inhibitors (GSIs). Moreover, getting advantage of co-culture systems and Notch responsive luciferase assay, we verified that the compounds in analysis were effective in inhibiting the ability of MM-derived Jag ligands to activate, by heterotypic interaction, Notch signaling in BMSCs. Finally, using different cell lines, we verified that the treatment with compounds induced a dose-dependent cell growth inhibition comparable to GSIs. In conclusion, the proposed approach, aiming at disrupting the Notch-Jag complex using small molecules, promises to be effective to overcome the effect of Notch activation in MM cells as well as in the surrounding stromal cells. We aim to expand the number of candidate compounds in order to successfully identify a lead clinical drug candidate.