Effects of IGHV mutational status on BCR signalling and transcriptomics in CLL cellular models

Chronic lymphocytic leukaemia (CLL) is a B cell disorder with a highly heterogeneous clinical course. The B cell receptor (BCR) is a master regulator of leukemic cells behaviour and is an important therapeutic target. Somatic hypermutation of the immunoglobulin heavy-chain variable region genes (IGHV) influences BCR signalling outcome and represents the most powerful prognostic marker for patients’ stratification, with unmutated patients (IGHV-UM) displaying a clearly inferior clinical outcome. NOTCH1 is the most recurrently mutated gene in CLL and mutations are associated with high-risk cases and therapy refractoriness. The BCR and NOTCH1 were previously shown to be functionally linked, even though the molecular interplays remain to be fully elucidated. The aim of this thesis is to establish a cellular model to study the BCR signaling in the presence of WT or mutated NOTCH1. To do so, we exploited the CLL-like cell line MEC1, reconstituted to carry either an unmutated or a mutated BCR, both in a WT and mutated NOTCH1 background. We showed that short-term stimulation of the BCR triggers a stronger signalling cascade in IGHV-UM than in IGHV-M cells. Similarly, upon long-term BCR engagement, we observed an enhanced transcription of genes involved in survival, proliferation, and inflammation in IGHV-UM clones. These differences were intensified in a NOTCH1m genetic background. When injected in mice, IGHV-UM NOTCH1m MEC1 cells showed the highest growth rate in a murine intravenous injection model. Collectively these results confirm the validity of this model to study BCR and NOTCH1 functional interactions and to test novel drug combinations effective for the most aggressive subset of CLL cells.