O18 Molecular crosstalk between PLCγ1 and STAT3 in cutaneous T-cell lymphoma

Abstract Cutaneous T-cell lymphoma (CTCL) is a non-Hodgkin lymphoma of skin-homing T lymphocytes that is difficult to diagnose and treat due to a high degree of clinical and genomic heterogeneity. Mutations in PLCG1/PLCγ1 occur in 10% of CTCL tumours. Meanwhile, signal transducer and activator of transcription 3 (STAT3) – a common driver of lymphomagenesis – is frequently activated (pY705-STAT3) in CTCL in the absence of mutation. Activation of STAT3 may also be mediated by serine phosphorylation (pS727-STAT3), and there is a lack of data on the role of this modification in CTCL. We aimed to identify if gain-of-function PLCG1 mutations in CTCL activate STAT3, either directly or indirectly, via downstream mitogen-activated protein kinase (MAPK) signalling. Flow cytometry was used to study STAT3 phosphorylation and subcellular localization in ex vivo CD4+-enriched tumour cell populations from patients with CTCL and healthy donors. STAT3 phosphorylation was also studied in the J.gamma1 T-cell line (PLCγ1-null) and primary ex vivo CD4+ cells upon lentiviral overexpression of wild-type (WT) and mutant (p.R48W, p.S345F) PLCγ1 constructs. To investigate the role of MAPK signalling, the CTCL-derived Hut78 and SeAx cell lines were treated with MAPK inhibitors (trametinib), and STAT3 phosphorylation assessed by Western blotting. Significantly increased basal levels of pS727-STAT3 (P < 0.05) and pY705-STAT3 (P < 0.001) were observed in CD4+-enriched populations from patients with Sézary syndrome compared with healthy donors. Mitochondrial translocation of STAT3 was also increased in CD4+-enriched populations from patients with Sézary syndrome (P < 0.05). In transduced J.gamma1 cells, p.S345F-PLCγ1 overexpression activated pS727-STAT3 (P < 0.05), while overexpression of both WT and mutant PLCγ1 constructs activated pS727-STAT3 in primary ex vivo CD4+ cells (P < 0.05 and P < 0.01, respectively). Inhibition of MAPK kinase (MEK)–extracellular signal regulated kinase (ERK) signalling did not significantly modulate STAT3 phosphorylation or subcellular localization in CTCL cell lines, suggesting that pS727-STAT3 phosphorylation is not mediated via ERK in CTCL. This work extends our understanding of PLCγ1 signalling and identifies pS727-STAT3 as a potential target of interest for further study in CTCL.