Analysis of cancer mutations introduced into the <i>Drosophila melanogaster</i> Notch negative regulatory region uncovers a diversity of regulatory outcomes.

Abstract

Activating mutations of Notch are drivers of the blood cell cancer, T-ALL, and some solid tumours. The negative regulatory region (NRR) of the extracellular domain (ECD) and the PEST region of the intracellular domain (ICD) are mutation hot spots which can act synergistically in T-ALL. The NRR, comprised of a heterodimerisation domain (HD) and three Lin12/Notch repeats (LNR A-C), masks the S2 cleavage site, normally only exposed following ligand binding and cleaved as the first step that ultimately leads to ICD release. <i>Drosophila</i> mutants have played a key role in analysing Notch structure/function, but there have been few mutational studies of the NRR. Here, we expressed, in S2 cells, over 20 cancer mutations located in the HD, LNR and LNR/HD interface, introduced into <i>Drosophila</i> Notch. Mutations in the HD domain core did not activate, likely due to absence in <i>Drosophila</i> of an S1 cleavage within the HD required for mammalian Notch activity. In contrast, mutations in the LNR/HD interface behaved similarly to T-ALL, activating constitutively with no further ligand induction and were synergistic with PEST deletion. Mutations of surface-exposed residues of LNR-C also activated constitutively but remained inducible both by ligand and by an intracellular endocytic regulator, Deltex, and were not synergistic with PEST deletions. These mutations caused elevated Notch levels and decreased turnover, suggesting a novel regulatory mechanism. Our results, therefore, uncover a variety of outcomes arising from perturbations of the NRR and will facilitate the establishment of <i>Drosophila</i> cancer models and the development of mutant-specific approaches to effective therapies.