This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ Functional interrogations of Phospholipase C Gamma 1 (PLCG1) mutations in Sézary Syndrome Patel, Varsha Maheshkumar Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). Non Commercial: You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 11. Oct. 2021 Functional interrogations of Phospholipase C Gamma 1 (PLCG1) mutations in Sézary Syndrome Varsha Maheshkumar Patel Skin Tumour Unit, St John’s Institute of Dermatology, School of Basic and Medical Biosciences, King’s College London. PhD thesis submitted to King’s College London for the degree of Doctor of Philosophy. 2019 1 Signed declaration I declare that the work presented in this thesis is my own unless clearly stated otherwise. Varsha M. Patel 2 Abstract Sézary Syndrome (SS) is an aggressive leukaemic variant of cutaneous T-cell lymphoma (CTCL). After the tumour suppressor gene TP53, PLCG1 is the most frequently mutated gene in CTCL. PLCγ1 (encoded by PLCG1) is fundamental in T- cell receptor (TCR) signalling as it hydrolyses a plasma membrane component to trigger pathways that induce NFκB, NFAT and AP-1 transcriptional activity. This thesis aimed to functionally interrogate nine PLCγ1 mutations (p.R48W, p.S312L, p.D342N, p.S345F, p.S520F, p.R1158H, p.E1163K, p.D1165H and the indel p.VYEEDM1161V) identified in SS. PLCG1 mutations detected in diagnostic samples persisted in multiple tumour compartments several months after diagnosis, suggesting that these are likely driver gene mutations. A comprehensive analysis of whole-exome and targeted gene sequencing studies in addition to database interrogations revealed frequent PLCG1 mutations in 7/10 different types of mature T-cell lymphomas and highlighted five hotspot mutations. In basal conditions, five mutant proteins directly increased PLCγ1 activity by elevating inositol phosphate production and significantly enhanced downstream NFκB and NFAT activity, demonstrating bona fide gain-of-function properties. The hotspot p.R48W protein required stimulation to significantly elevate NFκB activity. Four activating mutations mapped to the PLCγ2 protein surface that likely interacts with the plasma membrane. These four mutant proteins are hypothesised to have increased access to substrate, resulting in augmented TCR signalling. Abrogation of the key PLCγ1 phosphorylation residue did not influence the elevated NFκB, NFAT and AP-1 activity induced by gain-of-function proteins in basal conditions or NFκB activity in stimulated cells, suggesting that the mutant proteins act in a phosphorylation-independent manner. The indel in the C2 domain of PLCγ1 reduced total protein expression but importantly mediated gain-of-function, proposing a novel and critical role for this domain in regulating protein activity. An IKKβ inhibitor was ineffective at reducing PLCγ1- induced NFκB activity. In conclusion, PLCG1 mutations frequently occur in mature T-cell lymphomas and persist in multiple tumour tissues throughout the course of disease in SS. Five mutant proteins potently activate proximal and distal signalling independently of extracellular stimuli and contribute to the dysregulated TCR signalling that is characteristic of CTCL. The data presented here provides compelling evidence for the development of novel mutation-specific PLCγ1 inhibitors. 3 Table of Contents Chapter 1 Introduction ...................................................................................... 19 1.1 Cutaneous T-cell lymphomas ........................................................................... 19 1.2 Diagnosis of CTCL ........................................................................................... 19 1.2.1 Clinical manifestation ......................................................................... 19 1.2.2 Histological analysis ........................................................................... 21 1.2.3 Immunophenotypic characterisation .................................................. 22 1.3 CTCL staging.................................................................................................... 23 1.4 Treatment modalities ........................................................................................ 25 1.4.1 Skin-directed therapies ....................................................................... 25 1.4.2 Systemic therapies .............................................................................. 25 1.5 Differentiation of CD4+ T-cells ........................................................................ 30 1.6 Immunopathogenesis of CTCL ........................................................................ 33 1.6.1 Neoplastic cells in CTCL have central and effector memory T-cell immunophenotypes............................................................................. 33 1.6.2 Advanced stage CTCL has a Th2 predominant cytokine expression profile ................................................................................................. 33 1.6.3 Activated T-cells in CTCL display an exhausted phenotype ............. 35 1.6.4 Neoplastic Tfh cells are detected in CTCL ........................................ 36 1.6.5 Dysregulation of Tregs in CTCL ........................................................ 36 1.6.6 NFκB is constitutively active in CTCL .............................................. 37 1.6.7 Dysregulated expression of the AP-1 transcription factor family ...... 38 1.6.8 CTCL cells are resistant to apoptosis ................................................. 39 1.6.9 Dysregulated JAK/STAT signalling ................................................... 39 1.6.10 Loss of cell cycle regulators ............................................................... 40 1.6.11 Genomic instability is a key feature of CTCL.................................... 40 1.7 Characteristic features of cancer cells .............................................................. 42 1.8 Driver and passenger mutations and tumour mutation burden ......................... 43 1.8.1 Driver and passenger mutations ......................................................... 43 1.8.2 Tumour mutation burden .................................................................... 44 1.9 Genetic landscape of CTCL.............................................................................. 47 1.9.1 CTCLs have prevalent UV signature mutations ................................. 47 1.9.2 CTCLs harbour mutations in genes that alter key cellular processes and pathways ............................................................................................. 48 1.9.3 Copy number alterations frequently occur in CTCL .......................... 51 1.9.4 Chromosomal translocations are infrequent in CTCL........................ 53 1.9.5 Frequently perturbed pathways in CTCL ........................................... 54 4 1.9.6 Limitations of NGS analyses in CTCL .............................................. 56 1.10 Phospholipases .................................................................................................. 58 1.11 The Phospholipase C family ............................................................................. 59 1.12 An introduction to PLCγ1 and PLCγ2 .............................................................. 60 1.13 The pivotal role of PLCγ1 in TCR signalling ................................................... 63 1.13.1 The T-cell receptor ............................................................................. 63 1.13.2 TCR activation and signalling to PLCγ1 ............................................ 64 1.13.3 PLCγ1 auto-inhibition and activation by tyrosine phosphorylation ... 66 1.13.4 Activation of secondary messengers and downstream signalling by PLCγ1 ................................................................................................. 67 1.13.5 Common target genes of the NFκB, NFAT and AP-1 transcription factor families ..................................................................................... 69 1.14 The crucial role of PLCγ2 in BCR signalling ................................................... 70 1.15 Mutations and overexpression of PLCγ1 in cancer .......................................... 72 1.15.1 Somatic PLCG1 mutations in cancer ................................................. 72 1.15.2 CNV of PLCG1 .................................................................................