| Abstract |
Sezary 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. PLCgamma1 (encoded by PLCG1) is
fundamental in Tcell receptor (TCR) signalling as it hydrolyses a plasma
membrane component to trigger pathways that induce NFkappaB, NFAT and AP-1
transcriptional activity. This thesis aimed to functionally interrogate
nine PLCgamma1 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 PLCgamma1
activity by elevating inositol phosphate production and significantly
enhanced downstream NFkappaB and NFAT activity, demonstrating bona fide
gain-of-function properties. The hotspot p.R48W protein required
stimulation to significantly elevate NFkappaB activity. Four activating
mutations mapped to the PLCgamma2 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 PLCgamma1 phosphorylation residue did not influence
the elevated NFkappaB, NFAT and AP-1 activity induced by gain-of-function
proteins in basal conditions or NFkappaB activity in stimulated cells,
suggesting that the mutant proteins act in a phosphorylation-independent
manner. The indel in the C2 domain of PLCgamma1 reduced total protein
expression but importantly mediated gain-of-function, proposing a novel
and critical role for this domain in regulating protein activity. An
IKKbeta inhibitor was ineffective at reducing PLCgamma1-induced NFkappaB
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 PLCgamma1 inhibitors.
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