Dissecting the structural requirements for Notch/ligand interactions with mutations derived from cancer genome sequencing

Abstract

Notch is a cell surface receptor with critical roles in development and cellular differentiation, and its altered activity is frequently associated with cancer. Despite extensive sequencing efforts identifying numerous cancer-associated mutations in Notch, the functional consequences of many of these mutations remain poorly understood. Notch activation is mediated by its interaction with cell surface ligands, Delta and Serrate/Jagged, triggering proteolytic cleavage events that release the Notch intracellular domain (NICD). The NICD translocates to the nucleus to regulate transcription, and Notch can also be activated through ligand-independent mechanisms following endocytosis. This thesis exploits the high sequence conservation between human and Drosophila Notch to dissect how cancer-associated mutations alter receptor functionality, using an approach comprising in vitro and in vivo analyses. This work focuses on mutations within the ligand-binding region that are associated with cancers where Notch acts as a tumour suppressor. Through cell culture assays, I categorised Notch mutants based on their ligand-binding properties, signalling efficiencies, and ligand-independent activities. Using CRISPR/Cas9, these mutations were introduced into the Drosophila genome, enabling a comprehensive study of their phenotypic consequences. My analyses revealed diverse mutant classes, including those that completely eliminate signalling or retain partial functionality. For example, I identified mutants that discriminated between different Notch ligands, removed ligand-dependent signalling while retaining ligand-independent activity, and one mutant that retained ligand-dependent activation but removed ligand-independent activity. I also found that different cell-based assays could distinguish between levels of ligand associations required for cell adhesion and cell signalling. In vivo genetic interaction studies further refined our understanding of mutant classifications, revealing Notch mutant-specific interactions with a panel of genetic modifiers and uncovering phenotypes that deviated significantly from null-like behaviour. Additionally, I demonstrated that several mutations compromised cis-inhibitory interactions, with heterogeneous impacts on this regulatory mechanism across different developmental contexts. These findings provide valuable insights into the regulatory dynamics of Notch signalling, paving the way for advancing our understanding of the pathway’s role in development and disease.