Use this URL to cite or link to this record in EThOS:
Title: Investigating the functional consequences of JAK/STAT mutations in Sézary Syndrome
Author: Butler, Rosie May
ISNI:       0000 0004 8500 2717
Awarding Body: King's College London
Current Institution: King's College London (University of London)
Date of Award: 2019
Availability of Full Text:
Access from EThOS:
Full text unavailable from EThOS. Restricted access.
Access from Institution:
Sézary syndrome (SS) is an aggressive, leukaemic sub-type of cutaneous T-cell lymphoma (CTCL). Deep sequencing screens have identified JAK and STAT gene perturbation and JAK-STAT signalling dysregulation in CTCL. Histone deacetylase inhibitors (HDIs), including FK228, indirectly inhibit STAT activity and are used therapeutically to treat CTCL. STAT3 and RAD23B are proposed determinants of HDI sensitivity in CTCL; RAD23B is a dual function protein with roles in DNA repair and proteasomal targeting. It was hypothesised that JAK/STAT SNVs detected in CTCL cause dysregulated signalling and that RAD23B and STAT3 modulate HDI response. This study aims to (i) identify pathogenic JAK/STAT variants, (ii) functionally interrogate JAK/STAT mutations, (iii) assess the effect of STAT mutation on response to HDI treatment using in vitro models and (iv) investigate RAD23B copy number in SS and its functional interaction with STAT3 during HDI response. Of the selected JAK/STAT variants detected in CTCL, 13/19 were identified as pathogenic using pathogenicity prediction algorithms. Immunoblot and luciferase reporter assays in HEK293T cells identified eight activating JAK/STAT mutants. A subset of the STAT3 and STAT5B mutants conferred partial resistance to FK228-mediated STAT transcriptional inhibition and cytotoxicity using in vitro models, whilst primary ex vivo SS tumour cells harbouring activating STAT mutants were partially resistant to FK228-induced cytotoxicity. RAD23B copy number variation was detected in 10% (12/119) of SS tumours by qPCR for the first time and was associated with reduced mRNA expression. FK228 treatment decreased activated phospho-STAT3 (pSTAT3) expression in primary SS tumour cells. Flow cytometry analysis revealed distinct patterns of RAD23B-pSTAT3 co-expression and immunoprecipitation experiments in HUT-78 SS cells identified a RAD23B-STAT3 interaction. To conclude, this study identified novel, activating JAK1 and STAT5 protein mutations and functionally characterised known gain-of-function mutants which are frequently perturbed in CTCL. These findings have potential clinical relevance as they suggest patient stratification based on STAT and RAD23B tumour genotype may be useful in predicting response to FKK28 treatment, highlighting the potential for personalised therapy in CTCL.
Supervisor: Mitchell, Tracey ; Whittaker, Sean Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available