Functional investigation of molecular crosstalk between PLCy1 and STAT3 in Cutaneous T-cell Lymphoma

Cutaneous T-cell lymphoma (CTCL) is a rare non-Hodgkin's T-cell lymphoma which shows a high degree of clinical, pathological, and genomic heterogeneity. Due to this heterogeneity it is often difficult to diagnose and treat; current therapeutic optionsare generally palliative. Genomic sequencing studies including work in our laboratory (1) have identified frequent aberrations inthe T-Cell Receptor (TCR) signalling pathway in CTCL, particularly in the gene encoding phospholipase C gamma 1 (PLCy1). PLCG1 is the second most frequently mutated gene in CTCL and is commonly mutated in other T-cell malignancies. Our previous work has shown that the majority of PLCG1 mutations occurring in CTCL tumour cells are bone fidegain of function mutations that confer constitutive activation of PLCy1 and drive expression of key transcription factors NFkB, AP-1 and NFAT (2). We have also shown that overexpression and constitutive activation of signal transducer and activation of transcription 3 (STAT3) is also a common feature of CTCL (3). However, STAT3 mutations in CTCL are rare and the mechanism(s) driving aberrant STAT3 activation are unknown. TCR and STAT3 signalling are known to regulate cell survival and apoptosis and therefore these pathways are key players in the pathogenesis of CTCL. Work leading up to this proposal suggests that there may be molecular crosstalk between PLCy1 and STAT3. We have shown that in vitro, PLCy1 upregulates STAT3 expression and have preliminary evidence for a direct interaction between PLCy1 and STAT3. This thesis will investigate the hypothesis that activating mutations of PLCG1can drive STAT3 activation in CTCL. Using lentiviral overexpression in PLCG1-null J.gamma1 T-cells, we will investigate the effect of PLCy1 and its activating mutations p.R48W and p.S345F on the regulation of STAT3. This will include investigation of changes to STAT3 expression, phosphorylation and activation in response to WT, R48W, and S345F PLCy1 expression. We will investigate PLCy1-driven activation of both canonical and non-canonical STAT3 signalling pathways, via phosphorylation at Y705 and S727 respectively. Additionally, we will investigate a potential direct PLCy1-STAT3 protein-protein interaction. Overall, this work will evaluate molecular crosstalk between PLCy1 and STAT3, potentially confirming the convergence of the TCR and JAK-STAT signalling pathways in CTCL. This investigation will also confirm the validity of PLCy1 as a therapeutic target and support the development of PLCy1 inhibitors as a treatment option for CTCL and other mature T-cell malignancies harbouring PLCG1mutations.

We hypothesise that activating mutations in PLCy1 may drive the aberrant activation of STAT3 in CTCL. Mutations in PLCy1 and STAT3 are mutually exclusive in CTCL, and accumulating evidence suggests that STAT3 expression and activation can be regulated by downstream effectors ofPLCy1. The following aims will be addressed1)Investigate the effect of PLCy1 and its activating mutations on STAT3 expression, activation, and subcellular localisation.2)Determine the effect of PLCy1 and its activating mutations on the canonical and non-canonical STAT3 signalling pathways.3)Validate findings in primary T-cells and CTCL tumour cells.