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Impact of tumour microenvironmental factors on B-cell signalling, migration and adhesion in chronic lymphocytic leukaemia

Impact of tumour microenvironmental factors on B-cell signalling, migration and adhesion in chronic lymphocytic leukaemia
Impact of tumour microenvironmental factors on B-cell signalling, migration and adhesion in chronic lymphocytic leukaemia
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.
University of Southampton
Dobson, Rachel Colette
c2ef3195-3497-4d01-b75e-e0f2dd668ec3
Dobson, Rachel Colette
c2ef3195-3497-4d01-b75e-e0f2dd668ec3
Steele, Andrew
4349f6aa-2e3a-49a8-be73-7716056ae089
Cragg, Mark
ec97f80e-f3c8-49b7-a960-20dff648b78c
Strefford, Jon
3782b392-f080-42bf-bdca-8aa5d6ca532f

Dobson, Rachel Colette (2018) Impact of tumour microenvironmental factors on B-cell signalling, migration and adhesion in chronic lymphocytic leukaemia. University of Southampton, Doctoral Thesis, 257pp.

Record type: Thesis (Doctoral)

Abstract

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.

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Rachel Dobson - Impact of tumour microenvironmental factors on B-cell signalling, migration and adhesion in chronic lymph~1 - Version of Record
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Published date: April 2018

Identifiers

Local EPrints ID: 435488
URI: http://eprints.soton.ac.uk/id/eprint/435488
PURE UUID: 190822e4-aba8-4089-8862-70e7ff042aa3
ORCID for Andrew Steele: ORCID iD orcid.org/0000-0003-0667-1596
ORCID for Mark Cragg: ORCID iD orcid.org/0000-0003-2077-089X
ORCID for Jon Strefford: ORCID iD orcid.org/0000-0002-0972-2881

Catalogue record

Date deposited: 07 Nov 2019 17:30
Last modified: 08 Nov 2019 01:37

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Contributors

Author: Rachel Colette Dobson
Thesis advisor: Andrew Steele ORCID iD
Thesis advisor: Mark Cragg ORCID iD
Thesis advisor: Jon Strefford ORCID iD

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