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The BCL6-IRF4-BLIMP1 Transcription Factor Axis As a Therapeutic Target in T- Cell Lymphoma

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The BCL6-IRF4-BLIMP1 Transcription Factor Axis As a Therapeutic Target in T- Cell Lymphoma The BCL6-IRF4-BLIMP1 Transcription Factor Axis as a Therapeutic Target in T- cell Lymphoma PAUL DAVID GIBSON Thesis submitted for the degree of Doctor of Philosophy Northern Institute for Cancer Research, Newcastle University September 2015 i i Abstract Peripheral T-cell lymphomas (PTCL) are a heterogeneous group of clinically aggressive malignancies derived from mature (post-thymic) T-cells or Natural Killer cells, which comprise approximately 10-15% of all non-Hodgkin lymphomas. In contrast to aggressive B-cell malignancies, which are often curable and for which advances in understanding disease biology have resulted in new targeted treatment approaches, the treatment of PTCL remains inadequate. Apart from those with ALK-positive anaplastic large cell lymphoma (ALCL), patients presenting with PTCL have a poor outcome with only approximately 25% cured of their disease. The pathogenesis of T-cell lymphoma is poorly understood and few new targeted therapies are emerging. The transcription factors BCL6, IRF4 and BLIMP1 function in a regulatory network to direct mature B-cell differentiation. They are genetically altered and dysregulated in B-cell malignancy, and BCL6 and IRF4 represent potential therapeutic targets. These transcription factors also interact to regulate T-cell differentiation and emerging data indicated genetic alteration in some PTCL. This project investigated the importance of BCL6, IRF4, and BLIMP1 in the regulation of PTCL cell line proliferation and survival using ALCL cell lines in vitro as a model. Lentiviral-mediated knockdown of BCL6 and IRF4, and overexpression of BLIMP1, each resulted in reduced proliferation / survival of some, but not all, ALCL cell lines tested, and no clear pattern of response emerged. These effects were associated with small changes in cell cycle progression and induction of apoptosis. Modulation of each of the transcription factors had small effects on the expression of the others, again with variable patterns between cell lines. IRF4 knockdown revealed a positive interaction with c-MYC ad BLIMPα i / ALK+ ALCL cell lines. Intriguingly, ALK inhibition with crizotinib revealed different patterns of NPM-ALK mediated dysregulation of the transcription factors across the cell lines. These data support a positive role for BCL6 and IRF4 in the maintenance of ALCL, and an inhibitory / tumour suppressor role for BLIMP1, but show variability in dependencies between cell lines which could reflect clinically important disease heterogeneity which must be considered when targeting this transcription factor axis therapeutically. ii The ak of a peso is defied ho the ope i the fae of adesit iii Acknowledgements I must thank my supervisory team for giving me the opportunity to undertake my PhD, Dr. Chris Bacon, Dr. Vikki Rand, Dr. James Allan, and Dr. Luke Gaughan. I cannot express the gratitude for the teaching I have received, helping me to not only produce this thesis, but to further myself in ways I did not think possible. Special thanks go to Chris, who throughout the last three years has taught me not only to be independent but provided invaluable advice in every aspect of my life and has always encouraged me to push myself further. I am also thankful to Bright Red who provided me with the opportunity to complete my PhD by funding my studentship. I am also thankful to Dr. Paul Sinclair who provided the BLIMP1-SIEW, and SIN- SIEW overexpression vectors for my BLIMP1 investigations. I am grateful to all the members of the lymphoma research group who have given me technical help and advice throughout my project and offered a fresh pespetie o a thigs I thought hopeless. Not least is the suppot I’e eeied from Sarah and Holly who have always helped to brighten my day even after a string of failed experiments. I am also thankful to all members of the NICR who have been enormously friendly and always willing to help. Finally, I am thankful to my loving wife Loren. She has heard it all, through good ad ad esults, ag ats aout h thigs do’t ok, to just listeig to e ramble on about my project. She has been my inspiration and encouragement to complete the PhD from the start. iv v Contents Abstract ............................................................................................................................. ii Acknowledgements .......................................................................................................... iv List of figures .................................................................................................................... xi List of tables ................................................................................................................... xiv Abbreviations .................................................................................................................. xv 1. Introduction ................................................................................................................ 22 1.1 The immune system .............................................................................................. 22 1.1.1 B-cell development ............................................................................................. 22 1.1.2 T-cell development ............................................................................................. 26 1.2 Lymphoma ............................................................................................................. 31 1.3 B-cell Non-Hodgkin lymphoma.............................................................................. 31 1.4 Peripheral T-cell lymphoma .................................................................................. 34 1.4.1 Peripheral T-cell Lymphoma – Not otherwise Specified ................................ 36 1.4.2 Angioimmunoblastic T-cell lymphoma ........................................................... 38 1.4.3 Anaplastic Large Cell Lymphoma .................................................................... 39 1.4.3.1 Genetics of ALK+ ALCL .............................................................................. 41 1.4.3.2 Genetics of ALK- ALCL .............................................................................. 45 1.5 B-cell Lymphoma 6 (BCL6) ..................................................................................... 47 1.5.1 BCL6 Structure ................................................................................................ 47 1.5.2 BCL6 in B-cells ................................................................................................. 49 1.5.3 BCL6 in B-cell lymphoma ................................................................................ 51 1.5.4 BCL6 in T-cells ................................................................................................. 54 1.5.5 BCL6 in T-cell lymphoma ................................................................................. 57 1.6 Interferon Regulatory Factor 4 (IRF4) ................................................................... 58 1.6.1 IRF4 structure.................................................................................................. 58 1.6.2 IRF4 in B-cells .................................................................................................. 59 1.6.3 IRF4 in B-cell lymphoma ................................................................................. 62 1.6.4 IRF4 in T-cells .................................................................................................. 63 1.6.5 IRF4 in T-cell lymphoma .................................................................................. 69 1.7 B-lymphocyte Induced Maturation Protein 1 (BLIMP1) ........................................ 71 1.7.1 BLIMP1 structure ............................................................................................ 71 1.7.2 BLIMP1 in B-cells ............................................................................................. 72 vi 1.7.3 BLIMP1 in B-cell lymphoma ............................................................................ 73 1.7.4 BLIMP1 in T-cells ............................................................................................. 74 1.7.5 BLIMP1 in T-cell lymphoma ............................................................................ 76 1.8 Aims ....................................................................................................................... 78 2. Materials and Methods ............................................................................................... 82 2.1 Mammalian cell culture ......................................................................................... 82 2.1.1 Cell culture conditions .................................................................................... 82 2.1.2 Thawing of cryopreserved cells ...................................................................... 83 2.1.3 Counting cells using a Haemocytometer and Trypan Blue exclusion ............. 83 2.1.4 Freezing cells ................................................................................................... 83 2.1.5 Mycoplasma testing ........................................................................................ 85 2.2 Drugs/Inhibitors .................................................................................................... 85 2.2.1 BCL6 inhibitor, 79-6 ........................................................................................ 85 2.2.2 ALK inhibitor, Crizotinib .................................................................................
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