Hyperactivated Notch signalling has been implicated in breast cancer, but how elevated levels of Notch signalling contribute to mammary dysplasia and tumorigenesis is not fully understood. In this study, we express an activated form of Notch1 in the mouse mammary luminal lineage and analyse the consequences for tumour formation and the transcriptomic landscape in the luminal lineage. Simultaneous conditional activation of a Notch1 intracellular domain (Notch1 ICD) and EGFP in the luminal lineage was achieved by removal of a stop cassette by CRE-recombinase expression from the whey acidic protein (WAP) promoter. Mice in which Notch1 ICD was activated in the luminal lineage (WAP-CRE;R26-N1ICD mice) exhibit ductal hyperplasia after lactation with an increase in branching frequency and in the number of side-branch ends in the ductal tree. A subset of the mice developed mammary tumours and the majority of the tumour cells expressed EGFP (as a proxy for Notch1 ICD), indicating that the tumours originate from the Notch1 ICD-expressing cells. Single-cell transcriptome analysis of the EGFP-positive mammary cells identified six subtypes of luminal cells. The same six subtypes were found in control mice (WAP-CRE;R26-tdTomato mice expressing the tdTomato reporter from WAP-CRE-mediated activation), but the proportion of cells in the various subtypes differed between the WAP-CRE;R26-N1ICD and control WAP-CRE;R26-tdTomato mice. In conclusion, we show that Notch1 ICD expression in the luminal lineage produces a ductal hyperplasia and branching phenotype accompanied by altered luminal cell subtype partitioning.

In T-cell acute lymphoblastic leukemia (T-ALL) NOTCH 1 receptors are frequently mutated. This leads to aberrantly high Notch signaling, but how this translates into deregulated cell cycle control and the transformed cell type is poorly understood. In this report, we analyze downstream responses resulting from the high level of NOTCH 1 signaling in T-ALL. Notch activity, measured immediately downstream of the NOTCH 1 receptor, is high, but expression of the canonical downstream Notch response genes HES 1 and HEY 2 is low both in primary cells from T-ALL patients and in T-ALL cell lines. This suggests that other immediate Notch downstream genes are activated, and we found that Notch signaling controls the levels of expression of the E3 ubiquitin ligase SKP2 and its target protein p27Kip1. We show that in T-ALL cell lines, recruitment of NOTCH 1 intracellular domain (ICD) to the SKP2 promoter was accompanied by high SKP2 and low p27Kip1 protein levels. In contrast, pharmacologically blocking Notch signaling reversed this situation and led to loss of NOTCH 1 ICD occupancy of the SKP2 promoter, decreased SKP2 and increased p27Kip1 expression. T-ALL cells show a rapid G1-S cell cycle transition, while blocked Notch signaling resulted in G0/G1 cell cycle arrest, also observed by transfection of p27Kip1 or, to a smaller extent, a dominant negative SKP2 allele. Collectively, our data suggest that the aberrantly high Notch signaling in T-ALL maintains SKP2 at a high level and reduces p27Kip1, leading to more rapid cell cycle progression.