Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.797819
Title: Functional interrogations of Phospholipase C Gamma 1 (PLCG1) mutations in Sézary Syndrome
Author: Patel, Varsha Maheshkumar
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2019
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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 Tcell 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.
Supervisor: Mitchell, Tracey ; Whittaker, Sean Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.797819  DOI: Not available
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