An antibody to Notch3 reverses the skeletal phenotype of lateral meningocele syndrome in male mice.

Lateral meningocele syndrome (LMS), a genetic disorder characterized by meningoceles and skeletal abnormalities, is associated with NOTCH3 mutations. We created a mouse model of LMS (Notch3tm1.1Ecan ) by introducing a tandem termination codon in the Notch3 locus upstream of the proline (P), glutamic acid (E), serine (S) and threonine (T) domain. Microcomputed tomography demonstrated that Notch3tm1.1Ecan mice exhibit osteopenia. The cancellous bone osteopenia was no longer observed after the intraperitoneal administration of antibodies directed to the negative regulatory region (NRR) of Notch3. The anti-Notch3 NRR antibody suppressed the expression of Hes1, Hey1, and Hey2 (Notch target genes), and decreased Tnfsf11 (receptor activator of NF Kappa B ligand) messenger RNA in Notch3tm1.1Ecan osteoblast (OB) cultures. Bone marrow-derived macrophages (BMMs) from Notch3tm1.1Ecan mutants exhibited enhanced osteoclastogenesis in culture, and this was increased in cocultures with Notch3tm1.1Ecan OB. Osteoclastogenesis was suppressed by anti-Notch3 NRR antibodies in Notch3tm1.1Ecan OB/BMM cocultures. In conclusion, the cancellous bone osteopenia of Notch3tm1.1Ecan mutants is reversed by anti-Notch3 NRR antibodies.

Mammalian NOTCH Receptor Activation and Signaling Protocols.

The NOTCH signaling pathway is one of the highly conserved key pathways involved in most cell differentiation and proliferation processes during both developmental and adult stages in most animals. The NOTCH signaling pathway appears to be very simple but the existence of several receptors and ligands, their posttranslational modifications, their activation in the cell surface and its migration to the cell nucleus, as well as their interaction with multiple signaling pathways in the cytoplasm and the nucleus of cells, make the study of its function very complex.To determine the activation of NOTCH signaling in animal cells, several complementary approaches can be performed. One of them is the analysis of the transcription of NOTCH receptor target genes HES/HEY by qRT-PCR and Western blot. This approach would give us an idea of the global NOTCH activation and signaling. We can also analyze the NOTCH transcriptional activity by luciferase assays to determine the global or specific activation of NOTCH receptors under a given treatment or in response to the modification of gene expression. On the other hand, we can determine the specific activation of each NOTCH receptor by Western blot with antibodies that recognize the active forms of each NOTCH receptor. For this assay will be very important to collect the cells to be analyzed under the appropriate conditions. Finally, we can detect the intracellular domain of each NOTCH receptor into the cell nucleus by confocal microscopy using the appropriate antibodies that recognize the intracellular domain of the receptors.

Top Notch Targeting Strategies in Cancer: A Detailed Overview of Recent Insights and Current Perspectives.

Evolutionarily conserved Notch plays a critical role in embryonic development and cellular self-renewal. It has both tumour suppressor and oncogenic activity, the latter of which is widely described. Notch-activating mutations are associated with haematological malignancies and several solid tumours including breast, lung and adenoid cystic carcinoma. Moreover, upregulation of Notch receptors and ligands and aberrant Notch signalling is frequently observed in cancer. It is involved in cancer hallmarks including proliferation, survival, migration, angiogenesis, cancer stem cell renewal, metastasis and drug resistance. It is a key component of cell-to-cell interactions between cancer cells and cells of the tumour microenvironment, such as endothelial cells, immune cells and fibroblasts. Notch displays diverse crosstalk with many other oncogenic signalling pathways, and may drive acquired resistance to targeted therapies as well as resistance to standard chemo/radiation therapy. The past 10 years have seen the emergence of different classes of drugs therapeutically targeting Notch including receptor/ligand antibodies, gamma secretase inhibitors (GSI) and most recently, the development of Notch transcription complex inhibitors. It is an exciting time for Notch research with over 70 cancer clinical trials registered and the first-ever Phase III trial of a Notch GSI, nirogacestat, currently at the recruitment stage.