A new therapeutic strategy in multiple myeloma based on small molecules directed to notch pathway

BACKGROUND: Multiple myeloma (MM) is the second most common hematological malignancy. Although clinical advances, it is still incurable disease, sustained by a tight interaction of malignant plasma cells with the bone marrow (BM) microenvironment that promotes tumor growth, immunosuppression, drug resistance, neoangiogenesis and bone destruction. The oncogenic Notch signaling plays a crucial role in MM. In particular, aberrant Notch2 receptor activation and Jag1 and 2 ligands overexpression stimulate MM cells to establish pathological interactions with BM that trigger MM progression. Our previous data showed that these effects can be interfered by knocking down of Jag1 and 2 expression. Indirect approaches to inhibit Notch signaling are mainly based on inhibition of \uf067-Secretase that catalyzes Notch activation along with other several \uf067-Secretase substrates. Moreover, inhibition of all four Notch receptors is associated with a gut toxicity. This evidence prompted us to develop a therapeutic tool to selectively inhibit Notch2 signaling triggered by Jag1 and 2. METHODS: We applied in silico protein-protein docking and virtual high-throughput screening (HTS) of a chemoteque to select druglike small molecules. The biological activity was validated by a Notch responsive reporter assay and co-culture assay that allow to measure Notch2 transcriptional activity induced either by Jag or Dll ligands. Based on previous setup of integrated in silico pipeline, we applied a strategy to exclusively uncouple Notch2::Jag1/2, leaving unaltered the interaction with Dll. 100 top-scoring compounds directed exclusively to Notch2::Jag2 surface were selected by HTS of the chemoteque. 2 of 100 compounds were validated in vitro by the Notch responsive reporter assay on HEK293T cells and showed a significant reduce in Notch transcriptional activity. The co-culture assay of Hela cells overexpressing Notch2 with NIH3T3 overexpressing Jag1 or Dll4 ligands identified one promising compound that specifically inhibited Notch2::Jag1 and not Notch2::Dll4 interactions. Overall, our results showed that the identified compounds can selectively antagonize Notch activation providing a rationale for an effective and safe anti-Notch therapy in MM and other Notch-dependent tumors