Abstract
Cutaneous T-cell lymphomas (CTCLs) are a diverse group of rare non-Hodgkin lymphomas caused by a malignant proliferation of skin-homing T-lymphocytes. Data previously published by our group identified mitogen-activated protein kinase (MAPK) perturbations in 56/101 Sézary Syndrome tumours. Following variant prioritisation analysis, this thesis functionally investigated identified mutations in CTCL in two genes involved in MAPK signalling: Protein Phosphatase 5 (PP5) and Phospholipase C Gamma 1 (PLCG1). PP5 regulates RAF-MEK-ERK signalling by dephosphorylating RAF-1. PLCγ1 (encoded by PLCG1) is a key component in T-cell receptor signalling, triggering activation of RAS and signalling pathways that induce NFκB, NFAT and AP-1 activity.The Jurkat T-cell lymphoma cell line was transduced with wild-type or variant PP5 constructs and stimulated with PMA/Ionomycin to investigate the effect on pRAF-1 using immunoblot assays. The presence of HSP90 complexes was also ascertained, using immunoprecipitation and immunoblot assays. Jurkat cells transduced with PP5 variants showed an increase in pRAF-1 and a decrease in abundance of complexed protein to HSP90 compared to wild-type. Binding ability to HSP90 was supported in CD4+ tumour cells.
Somatic mutations in PLCγ1 frequently occur in CTCL, with p.R48W and p.S345F being the most commonly reported. NFAT, AP-1, NFκB transcriptional activity was determined by luciferase reporter assays in the J.gamma1 (PLCγ1 null) T-cell lymphoma line, reconstituted with wild-type or variant PLCG1 constructs. Flow cytometry was used to determine cellular apoptosis in response to the calcineurin inhibitor Tacrolimus and the MEK-1/2 inhibitor Trametinib. In basal conditions, both mutations confer PLCγ1 gain-of-function activity through downstream activation of AP-1 and NFAT transcriptional activity and p.S345F alone increased activity of NFκB. p.R48W or p.S345F transduced cells were significantly less apoptotic than wild-type PLCγ1 in response to Tacrolimus treatment. p.R48W also displayed an increase in MEK-1/2 activity as well as resistance to Trametinib, potentially involving increased STAT3 activity.
These results suggest PP5 and PLCγ1 may have a functional role in CTCL pathogenesis via dysregulation of MAPK signalling. These findings also suggest the possibility of patient stratification based on PLCG1 tumour genotype and highlight it as a potential new therapeutic target. In conclusion, this highlights the importance of MAPK dysregulation and personalised therapies in CTCL.
| Date of Award | 1 Mar 2021 |
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| Original language | English |
| Awarding Institution |
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| Supervisor | Tracey Mitchell (Supervisor) & Sean Whittaker (Supervisor) |