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The Role of the NFAT Signalling Pathway in Diffuse Large B-Cell Lymphoma

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The Role of the NFAT Signalling Pathway in Diffuse Large B-Cell Lymphoma The role of the NFAT signalling pathway in Diffuse Large B-cell Lymphoma Holly White Doctor of Philosophy Thesis September 2015 Supervisors: Dr Chris Bacon, Professor Neil Perkins & Dr Vikki Rand Northern Institute for Cancer Research Word count: 66,024 1 Abstract Diffuse Large B-Cell Lymphomas (DLBCL) are common, aggressive malignancies of mature B-lymphocytes that represent ~40% of lymphomas. Despite the widespread use of combined immunochemotherapy, approximately 50% of patients with DLBCL die from their disease. The two main DLBCL subgroups resemble activated B cells (ABC) or germinal centre B cells (GCB), where patients with ABC-DLBCL have significantly worse outcome. There is urgent need for novel therapeutic strategies in the treatment of DLBCL, which requires a better understanding of the molecular pathways upon which tumours depend. Accumulating evidence suggests that the signalling networks promoting and sustaining DLBCL derive from dysregulation of the normal pathways controlling B-lymphocyte activation and differentiation. There is increasing evidence indicating important roles for the NFAT family of transcription factors in DLBCL. Constitutively-active nuclear NFAT2 has been demonstrated in approximately 40% of primary DLBCL samples and NFAT has been shown to regulate a small number of genes associated with DLBCL growth/survival. This project investigated the role of NFAT in DLBCL. Nuclear localisation and activation of NFAT family members were characterised in a panel of DLBCL cell lines and chemical inhibition of calcineurin/NFAT, using Cyclosporin A (CsA), indicated dependency on the calcineurin/NFAT pathway for survival. Gene expression microarray analysis performed in DLBCL cell lines treated with CsA revealed potential NFAT target genes involved in the tumour microenvironment and anergy. These data revealed that the cytokine TNFα was downregulated by CsA in ABC, but not GCB cell lines. TNFα expression has recently been reported a significant prognostic factor for OS and PFS in DLBCL and evidence suggests dependency of some DLBCL on autocrine TNFα signalling for survival. Biologically active TNFα was produced by DLBCL cell lines, however inhibition of TNFα signalling using Infliximab and Etanercept had no effect on cell viability, suggesting that TNFα may be functionally important in DLBCL by other mechanisms. i PhD Thesis Holly White Acknowledgements It is a pleasure to thank those who have made this research project possible. Thank you to my supervisors Dr Chris Bacon, Professor Neil Perkins and Dr Vikki Rand for their support and guidance throughout my PhD. I am especially grateful to Chris, who during times when I doubted myself, gave me the confidence to push myself further and gain a great sense of achievement. I would also like to thank Cancer Research UK for giving me the opportunity to complete my PhD by funding this project. I am also thankful to Dr Anja Krippner who kindly provided the anti-TNFα agents for the TNFα investigation. I would also like to express my gratitude to my colleagues at the Northern Institute for Cancer Research, particularly within the lymphoma research group. I fully appreciate the technical support and advice given during the last three years. Many thanks also go to researchers in the Neil Perkins laboratory, especially to Dr Alex Sfikas for helping me with the NF-κB luciferase assays and to Dr Jill Hunter for her enthusiasm and guidance during my studies. I am also very thankful for the friendships I have made during my PhD, especially with Laura Ogle, Sarah Wilkinson and Paul Gibson, who always brightened my days. Finally, I would like to thank my boyfriend Iain for being my shoulder to cry on, and my family, who gave me emotional support through all the ups and downs during the past three years. ii PhD Thesis Holly White Table of Contents Abstract .............................................................................................................................. i Acknowledgements ........................................................................................................... ii List of Figures ................................................................................................................... x List of Tables.................................................................................................................. xvi List of Abbreviations.................................................................................................... xviii 1. Introduction ................................................................................................................ 2 1.1. Lymphoma .......................................................................................................... 2 1.2. B-Cell Non-Hodgkin Lymphoma ....................................................................... 3 1.3. Diagnosis, prognosis and treatment of DLBCL ................................................. 4 1.4. Sub-classification of DLBCL by cell of origin (COO) ...................................... 5 1.5. Pathogenesis of DLBCL ..................................................................................... 7 1.5.1. Genetic abnormalities in DLBCL ............................................................... 7 1.5.2. Deregulation of BCL2 activity .................................................................... 8 1.5.3. Deregulation of BCL6 activity .................................................................... 9 1.5.4. Disruption of terminal differentiation ....................................................... 10 1.5.5. Alterations in epigenetic modifiers ........................................................... 11 1.6. B-Cell Receptor signalling ............................................................................... 13 1.6.1. Normal B-Cell receptor signalling ............................................................ 13 1.6.2. B-Cell receptor signalling in DLBCL ....................................................... 14 1.6.3. Chronic active BCR signalling ................................................................. 14 1.7. The NF-κB signalling pathway ........................................................................ 15 1.7.1. Biology of NF-κB/primary structure of NF-κB ........................................ 15 1.7.2. Activation of the NF-κB signalling pathway ............................................ 16 1.7.3. NF-κB signalling in DLBCL ..................................................................... 18 1.7.4. CD79A/B mutations .................................................................................. 18 1.7.5. Alterations in positive regulators of NF-κB .............................................. 18 1.7.6. Alterations in negative regulators of NF-κB ............................................. 20 1.7.7. Targeting NF-κB in DLBCL ..................................................................... 20 1.8. Tonic BCR signalling and the PI3K/AKT signalling pathway ........................ 22 1.9. Pathway lesions downstream of TLR signalling .............................................. 25 1.10. Lymphoma summary .................................................................................... 26 1.11. Nuclear factor of activated T-cell (NFAT) family of proteins ..................... 28 iii PhD Thesis Holly White 1.12. Biology of NFAT/primary structure of NFAT ............................................. 29 1.13. NFAT isoforms ............................................................................................. 30 1.14. NFAT2 autoregulation .................................................................................. 33 1.15. The NFAT signalling pathway ..................................................................... 34 1.16. Regulation of NFAT ..................................................................................... 36 1.16.1. Kinase regulation of NFAT ................................................................... 36 1.16.2. Regulation of NFAT by calcineurin ...................................................... 36 1.16.3. Post-translational modifications ............................................................ 37 1.17. NFAT transcriptional partners ...................................................................... 37 1.18. Role of NFAT in Cancer ............................................................................... 38 1.18.1. Study of NFAT using mouse models .................................................... 41 1.18.2. Roles of NFAT in cancer development and progression ...................... 42 1.18.3. Role of NFAT in the malignant transformation and cell proliferation .. 42 1.18.4. Role of NFAT in cell invasion and metastasis ...................................... 43 1.18.5. Role of NFAT in angiogenesis and lymphangiogenesis ....................... 44 1.18.6. Role of NFAT in the tumour microenvironment................................... 45 1.19. Targeting the NFAT pathway in cancer therapy .......................................... 48 1.20. Role of NFAT in haematological malignancies ........................................... 49 1.21. Evidence for cooperative roles of NFAT and NF-κB in DLBCL ................ 50 1.22. Importance of the BCR and NFAT signalling .............................................. 51 1.23. NFAT summary ............................................................................................ 52 1.24. Aims .............................................................................................................
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