Notch-targeted therapeutic strategy in multiple myeloma based on small molecules hampering receptor-ligand interaction

Multiple myeloma (MM) is an incurable hematological cancer characterized by MM cells accumulation in the bone marrow (BM) where they interact and shape the nearby BM milieu, resulting in tumor progression, acquisition of drug resistance and consequent patient's relapse. Despite recent advances, poor clinical response and relapse remain major problems. The oncogenic Notch signaling consists of 4 receptors (Notch1-4) and 5 ligands (Jag1,2 and Dll1,3,4) and plays a crucial role in MM. In particular, aberrant Notch2 activation and Jag1 and 2 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. Currently, indirect approaches to inhibit Notch signaling are mainly based on inhibition of -Secretase that catalyzes Notch activation along with other several -Secretase substrates. Moreover, inhibition of all four Notch receptors is associated with gut toxicity that might be avoided by selectively blocking Notch signaling triggered by only one family of ligands, Jag or Dll ligands. This evidence prompts us to develop a therapeutic tool to selectively inhibit Notch2 signaling triggered by Jag1 and 2. 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. This lays a basis for an effective and safe Notch-directed anti-tumor therapy in MM and other Notch-dependent tumors.