Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.780759
Title: Functional analysis of POLE exonuclease domain mutations in cancer
Author: Rayner, Emily
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 2019
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Abstract:
POLE encodes the polymerase and exonuclease domains of the leading strand replicase, DNA polymerase ε. Somatic and germline POLE exonuclease domain mutations (EDMs) occur in colorectal and endometrial cancer and are associated with tumour ultramutation, a distinct mutation signature and an excellent prognosis. However, the mechanism by which POLE EDMs cause mutagenesis and the molecular consequences of this are unclear. Previous functional studies in several POLE EDM knock-in cell and mouse models have been performed, however they all have limitations regarding their clinical relevance. In addition, the timing of POLE EDMs in tumour development is based purely on genomic data from advanced POLE EDM tumours with no direct evidence of their occurrence in pre-cancers. The aim of this thesis was to study the function of POLE EDMs in clinically relevant human cell-line and mouse models and determine their timing in cancer development. Corrected somatic POLEP286R EDM isogenic colorectal cancer cell lines were generated using CRISPR-Cas9. Correction of the POLE mutation resulted in a striking reduction in the HPRT1 mutation rate (11-75-fold), consistent with a causal role in tumour ultramutation. Furthermore, the POLE EDM cell line somatic mutation spectra exhibited a significant bias towards POLE signature mutations: TCT>TAT, TCG>TTG and TTT>TGT (P<0.0001). Analysis of uterine and intestinal-specific tissue and constitutive germline PoleL424V EDM knock-in mice revealed no gross morphological or histopathological phenotype, however constitutive models demonstrated elevated Cleaved-Caspase-3 expression in the small intestine (P<0.05). Germline PoleL424V expression increased the colonic polyp burden in ApcMin mice (P<0.05), although there was no significant difference in the small intestinal polyp burden. Finally, a somatic POLEV411L EDM was identified in a colorectal pre-cancer with an elevated T-cell infiltrate, increased somatic mutation burden and a bias of POLE EDM-associated amino acid substitutions. Overall, this work provides strong evidence that POLE exonuclease domain mutations cause tumour ultramutation, a distinct mutation signature and are an early event in cancer. POLE EDM mice only developed a gross morphological phenotype when crossed with ApcMin mice, raising interesting questions regarding the mechanism of tumour initiation in cancers with POLE exonuclease domain mutations.
Supervisor: Church, David ; Tomlinson, Ian ; Kearsey, Stephen Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.780759  DOI: Not available
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