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Title: Impact of tumour microenvironmental factors on B-cell signalling, migration and adhesion in chronic lymphocytic leukaemia
Author: Dobson, Rachel Colette
ISNI:       0000 0004 8501 8268
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2018
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A key characteristic of chronic lymphocytic leukaemia (CLL) is the accumulation of malignant Bcells in the patients' lymph nodes, where the microenvironment promotes CLL cell survival and proliferation, in part via B-cell receptor (BCR) signalling and interleukin-4 (IL-4) signalling. BCR signalling is pivotal for disease progression and development. CLL prognosis is heterogeneous, with patients with unmutated (U-CLL) immunoglobulin heavy chain variable (IGHV) being associated with worse prognosis than those with mutated (M-CLL) IGHV. Recently, BCR kinase inhibitors have revolutionised CLL treatment. Nevertheless, some patients fail to respond, or develop resistance by some known and unknown reasons. IL-4 induced pSTAT6(Y641) (phosphorylated signal transducer and activator of transcription 6) signalling enhances BCR expression and signalling in murine splenic B-cells. Therefore, IL-4 may drive BCR signalling and resistance against BCR kinase inhibitors in CLL. In this thesis, I investigated the hypothesis that IL-4 induced pSTAT6(Y641) signalling promotes functional effects associated with disease pathogenesis. I demonstrated, via immunoblotting, that whilst IL-4 induced JAK/STAT6 signalling is not clearly associated with prognostic factors, the cytokine treatment significantly increased expression of the suppressor of cytokine signalling protein 3 more prominently in U-CLL samples. I conclude that IL4 induced signalling can differentially regulate target proteins between prognostic subsets. Using immunophenotyping, I demonstrated that IL-4 induces sIgM expression on CLL cells, with more prominent effects in the U-CLL subset. Intracellular calcium flux analysis demonstrated that IL-4 enhances anti-IgM induced signalling and immunoblotting showed that IL-4 increases expression of proteins known to promote BCR signalling. The survival factors CD40 ligand and Bcell activating factor had no clear effects on BCR expression or signalling. I also demonstrated that IL-4 can reduce the ability for BCR kinase inhibitors to induce apoptosis and block anti-IgM induced signalling. I propose that co-treating CLL patients with BCR kinase inhibitors in combination with inhibitors, such as JAK3 inhibitors, targeting IL-4 signalling could have therapeutic potential. I further showed, with flow cytometry, that IL-4 reduces chemokine receptor expression and enhances the expression of adhesion molecule CD44 on CLL cells. Adhesion assays showed that IL4 can enhance anti-IgM induced CLL cell adhesion to the extracellular matrix component fibronectin. Collectively, these findings suggest that IL-4 may promote CLL cell localisation and retention in the lymph node. Together these findings mostly support the hypothesis and suggest that IL-4 induced pSTAT6(Y641) signalling may promote CLL pathogenesis in vitro. This thesis advances the field by further suggesting that IL-4 may promote disease pathogenesis and showing how small inhibitors targeting the cytokine signalling could provide a potential therapeutic target for CLL.
Supervisor: Steele, Andrew ; Cragg, Mark ; Strefford, Jon Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available