An investigation of mechanistic and prognostic aspects of BAP1 in uveal melanoma



Farquhar, N
(2019) An investigation of mechanistic and prognostic aspects of BAP1 in uveal melanoma. PhD thesis, University of Liverpool.

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Abstract

BRCA1-associated protein 1 (BAP1) is a deubiquitylase (DUB) that is inactivated through mutation in several cancers, including uveal melanoma (UM). BAP1 inactivation is associated with significantly poorer prognoses in UM, though the mechanisms underpinning this are unclear. BAP1 has diverse roles, both through its DUB activity and as a transcriptional regulator. In addition, it has recently been shown that BAP1-deficiency may sensitise UM cells to therapeutic compounds called histone deacetylase inhibitors (HDACi), and this presents a novel therapeutic opportunity for UM treatment. To address these questions, one approach was to generate novel isogenic models of BAP1-deficient UM using recombinant adeno-associated viral-mediated gene editing (rAAV). A clonogenic UM cell line (92.1C1) was utilised and after intensive screening, 120 cell populations were identified that appeared to have incorporated the targeting vector at the BAP1 locus. Unfortunately, none of these could be propagated, suggesting that the cells cannot tolerate the mutation. The gene editing project was ultimately terminated, but it left us with insights into the essential nature of BAP1 function in certain cell models. In addition, a comprehensive characterisation of the prognostic role of BAP1 in UM was performed. The correlation between BAP1 immunohistochemistry (IHC) scores on 165 UM patients and both clinical and genetic parameters was analysed. Loss of nuclear BAP1 (nBAP1) expression occurred in 53% (88/165) UMs, and was the most significant independent prognostic parameter studied (log-rank; P < 0.001). Strikingly, an nBAP1 positive-monosomy 3 (M3) sub-group was identified that had significantly prolonged survival relative to nBAP1 negative-M3 UMs (log-rank; P = 0.014), suggesting that BAP1 is the key mediator of prognosis in M3 UM. These results suggest that BAP1 IHC should be incorporated into the routine prognostic work-up for UM patients. I further characterised, for the first time, cytoplasmic BAP1 (cBAP1) expression in UM. A subset of 26 UMs had all BAP1 exons sequenced, and cBAP1 expression patterns were linked to mutation status in these cases. I identified a distinctive ‘focal perinuclear’ cBAP1 phenotype that was associated with loss-of-function BAP1 mutations. cBAP1 expression did not affect survival in this study (log-rank; P = 0.925), indicating that cBAP1 expression cannot be used as a prognostic indicator in UM. Building on a previous study that identified a positive correlation between BAP1 and histone deacetylase 2 (HDAC2) in vitro, I performed HDAC2 IHC on 155 primary UMs, correlating HDAC2 and BAP1 expression. The majority of UM tumours had a high percentage of HDAC2 expression. BAP1 and HDAC2 expression did not correlate in UM tissues, providing a contradictory result to in vitro data. There was a strong trend towards reduced HDAC2 expression in patients with loss of chromosome 6q compared to patients with normal 6q; whilst patients with loss of 1p had significantly higher HDAC2 expression compared to patients with normal 1p (Chi-Square; P = 0.038). Patients with loss of 1p and 6q tended to have significantly lower HDAC2 expression compared to patients with loss of 1p and normal 6q (Fisher’s Exact Test; P = 0.017), suggesting patients with 1p and 6q deficiencies perhaps cannot compensate for class I HDAC deficiency by upregulating HDAC2. Together, the data in this thesis provide insights into BAP1 biology, demonstrate the crucial prognostic role of BAP1 in UM, and characterise cBAP1 and HDAC2 expression in UM tissues for the first time.

Item Type: Thesis (PhD)
Divisions: Fac of Health & Life Sciences > Institute of Translational Medicine
Depositing User: Symplectic Admin
Date Deposited: 21 Aug 2019 12:49
Last Modified: 03 Mar 2021 17:00
DOI: 10.17638/03034446
Supervisors:
URI: https://livrepository.liverpool.ac.uk/id/eprint/3034446