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Title: Utilization of high throughput screening to identify therapeutic targets for defective MCPH1/BRIT1 function-induced premature chromosome condensation in breast and ovarian cancer
Author: Awaji, Aeshah
ISNI:       0000 0004 5921 2761
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2016
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Mutations in the N-terminal region of MCPH1/BRIT1 cause premature chromosome condensation (PCC), whereby cells enter mitosis before completing DNA replication. 792 chemical compounds (CC) were selected based on the crystal structure of the N-terminus of MCPH1/BRIT1 and assayed using high throughput- high content imaging to identify CC that induced PCC. Hit validation revealed 4 potential CC, 2 of which induced high PCC at low concentrations. A screen using a human protein kinase (hPK) siRNA sub-library was performed to identify genes that induced PCC. Four hits were selected for validation, however PCC induction was not confirmed. A complementary hPK siRNA screen combined with MCPH1/BRIT1 siRNA knockdown was performed. The cell number outputs from both hPK siRNA screens were analysed to identify synthetic lethal (SL) genes in MCPH1/BRIT1-deficient cells. CDK1/CDC2, STK39, VRK1 and TTK/MPS1 were subsequently validated as potential MCPH1/BRIT1 SL genes. The expression of MCPH1/BRIT1 was examined by immunostaining in breast cancer (BC) tissue pre and post neoadjuvant chemotherapy (NACT) to determine its effect on response and survival. MCPH1/BRIT1 expression increased in response to NACT with high expression in 51.4% (36/70) of cases pre-NACT compared to 81.4% (57/70) post-NACT (p = 0.0002). Reduced MCPH1/BRIT1 expression correlated with longer overall survival (OS) pre- but not post-NACT (p = 0.017). Change in MCPH1/BRIT1 expression (from low-high) post-NACT was significantly correlated with better OS (p = 0.010). MCPH1/BRIT1 has previously been found to regulate p53 stability in BC cell lines. Notably, in this study a significant increase in MCPH1/BRIT1 staining was accompanied by a decrease in p53 staining in post-NACT samples (p < 0.0001). In conclusion, these data support the idea that CC inhibitors targeting MCPH1/BRIT1 may sensitize BC cells to chemotherapy. Additionally, genes whose inhibition could promote cell death in MCPH1/BRIT1–deficient cells have been identified as potential therapeutic targets in tumours where MCPH1/BRIT1 expression or function has been compromised.
Supervisor: Bell, Sandra ; Bond, Jacquelyn ; Morrison, Ewan ; Cookson, Victoria Sponsor: Government of Saudi Arabia
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available