Anaplastic Large Cell Lymphoma (ALCL) is a T cell Non-Hodgkin Lymphoma, mainly presenting in paediatric and young adult patients. The majority of cases express a chimeric fusion protein resulting in hyperactivation of Anaplastic Lymphoma Kinase (ALK) as the consequence of a chromosomal translocation. Patients diagnosed with ALCL are still treated with toxic multi-agent chemotherapy and as many as 25-50% of patients relapse. It is clear that continued adaption of current therapies will likely not improve these statistics and for progress to be made, integration of biology with the design and implementation of future clinical trials is required.

To understand disease pathology and to uncover novel targets for therapy, Whole-Exome Sequencing (WES) of ALK+ ALCL was performed, as well as Gene-Set Enrichment Analysis. This revealed that the Notch pathway was the most enriched in mutations. In particular, variant T349P of Notch1, which confers a growth advantage to cells in which it is expressed, was detected in 12% of ALK+ and ALK- ALCL patient samples. Furthermore, we demonstrate that the nucleophosphomin 1-Anaplastic lymphoma kinase (NPM-ALK) chimeric protein promotes Notch1 expression through binding of Signal Transducer and Activator of Transcription 3 (STAT3) upstream of notch1. Moreover, inhibition of Notch1 with γ-secretase inhibitors (GSI) or silencing by shRNA leads to apoptosis; co-treatment in vitro with the ALK inhibitor crizotinib led to additive/synergistic anti-tumour activity suggesting this may be an appropriate combination therapy for future use in the circumvention of ALK inhibitor resistance. Indeed, crizotinib-resistant and sensitive ALCL cells were equally sensitive to GSIs. In conclusion, we show a variant in the extracellular domain of Notch1 that provides a growth advantage to cells and confirm the suitability of the Notch pathway as a second-line druggable target in ALK+ ALCL.