Functional analysis of POLE exonuclease domain mutations in cancer
<p><em>POLE</em> encodes the polymerase and exonuclease domains of the leading strand replicase, DNA polymerase ε. Somatic and germline <em>POLE</em> exonuclease domain mutations (EDMs) occur in colorectal and endometrial cancer and are associated with tumour ultramutat...
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| Format: | Thesis |
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2019
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| _version_ | 1826313226725359616 |
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| author | Rayner, E |
| author2 | Church, D |
| author_facet | Church, D Rayner, E |
| author_sort | Rayner, E |
| collection | OXFORD |
| description | <p><em>POLE</em> encodes the polymerase and exonuclease domains of the leading strand replicase, DNA polymerase ε. Somatic and germline <em>POLE</em> 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. </p> <p>Previous functional studies in several <em>POLE</em> EDM knock-in cell and mouse models have been performed, however they all have limitations regarding their clinical relevance. In addition, the timing of <em>POLE</em> EDMs in tumour development is based purely on genomic data from advanced <em>POLE</em> EDM tumours with no direct evidence of their occurrence in pre-cancers. The aim of this thesis was to study the function of <em>POLE</em> EDMs in clinically relevant human cell-line and mouse models and determine their timing in cancer development.</p> <p>Corrected somatic <em>POLE</em><sup>P286R</sup> EDM isogenic colorectal cancer cell lines were generated using CRISPR-Cas9. Correction of the <em>POLE</em> mutation resulted in a striking reduction in the <em>HPRT1</em> mutation rate (11-75-fold), consistent with a causal role in tumour ultramutation. Furthermore, the <em>POLE</em> EDM cell line somatic mutation spectra exhibited a significant bias towards <em>POLE</em> signature mutations: TCT>TAT, TCG>TTG and TTT>TGT (P<0.0001).</p> <p>Analysis of uterine and intestinal-specific tissue and constitutive germline <em>Pole</em><sup>L424V</sup> 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 <em>Pole</em><sup>L424V</sup> expression increased the colonic polyp burden in Apc<sup>Min</sup> mice (P<0.05), although there was no significant difference in the small intestinal polyp burden. </p> <p>Finally, a somatic <em>POLE</em><sup>V411L</sup> EDM was identified in a colorectal pre-cancer with an elevated T-cell infiltrate, increased somatic mutation burden and a bias of <em>POLE</em> EDM-associated amino acid substitutions. </p> <p>Overall, this work provides strong evidence that <em>POLE</em> exonuclease domain mutations cause tumour ultramutation, a distinct mutation signature and are an early event in cancer. <em>POLE</em> EDM mice only developed a gross morphological phenotype when crossed with <em>Apc</em><sup>Min</sup> mice, raising interesting questions regarding the mechanism of tumour initiation in cancers with <em>POLE</em> exonuclease domain mutations.</p> |
| first_indexed | 2024-03-07T06:03:29Z |
| format | Thesis |
| id | oxford-uuid:ed08e93f-3807-4dd0-84b9-63935abaa420 |
| institution | University of Oxford |
| last_indexed | 2024-09-25T04:09:44Z |
| publishDate | 2019 |
| record_format | dspace |
| spelling | oxford-uuid:ed08e93f-3807-4dd0-84b9-63935abaa4202024-06-14T08:10:56ZFunctional analysis of POLE exonuclease domain mutations in cancerThesishttp://purl.org/coar/resource_type/c_db06uuid:ed08e93f-3807-4dd0-84b9-63935abaa420ORA Deposit2019Rayner, EChurch, DTomlinson, IKearsey, S<p><em>POLE</em> encodes the polymerase and exonuclease domains of the leading strand replicase, DNA polymerase ε. Somatic and germline <em>POLE</em> 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. </p> <p>Previous functional studies in several <em>POLE</em> EDM knock-in cell and mouse models have been performed, however they all have limitations regarding their clinical relevance. In addition, the timing of <em>POLE</em> EDMs in tumour development is based purely on genomic data from advanced <em>POLE</em> EDM tumours with no direct evidence of their occurrence in pre-cancers. The aim of this thesis was to study the function of <em>POLE</em> EDMs in clinically relevant human cell-line and mouse models and determine their timing in cancer development.</p> <p>Corrected somatic <em>POLE</em><sup>P286R</sup> EDM isogenic colorectal cancer cell lines were generated using CRISPR-Cas9. Correction of the <em>POLE</em> mutation resulted in a striking reduction in the <em>HPRT1</em> mutation rate (11-75-fold), consistent with a causal role in tumour ultramutation. Furthermore, the <em>POLE</em> EDM cell line somatic mutation spectra exhibited a significant bias towards <em>POLE</em> signature mutations: TCT>TAT, TCG>TTG and TTT>TGT (P<0.0001).</p> <p>Analysis of uterine and intestinal-specific tissue and constitutive germline <em>Pole</em><sup>L424V</sup> 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 <em>Pole</em><sup>L424V</sup> expression increased the colonic polyp burden in Apc<sup>Min</sup> mice (P<0.05), although there was no significant difference in the small intestinal polyp burden. </p> <p>Finally, a somatic <em>POLE</em><sup>V411L</sup> EDM was identified in a colorectal pre-cancer with an elevated T-cell infiltrate, increased somatic mutation burden and a bias of <em>POLE</em> EDM-associated amino acid substitutions. </p> <p>Overall, this work provides strong evidence that <em>POLE</em> exonuclease domain mutations cause tumour ultramutation, a distinct mutation signature and are an early event in cancer. <em>POLE</em> EDM mice only developed a gross morphological phenotype when crossed with <em>Apc</em><sup>Min</sup> mice, raising interesting questions regarding the mechanism of tumour initiation in cancers with <em>POLE</em> exonuclease domain mutations.</p> |
| spellingShingle | Rayner, E Functional analysis of POLE exonuclease domain mutations in cancer |
| title | Functional analysis of POLE exonuclease domain mutations in cancer |
| title_full | Functional analysis of POLE exonuclease domain mutations in cancer |
| title_fullStr | Functional analysis of POLE exonuclease domain mutations in cancer |
| title_full_unstemmed | Functional analysis of POLE exonuclease domain mutations in cancer |
| title_short | Functional analysis of POLE exonuclease domain mutations in cancer |
| title_sort | functional analysis of pole exonuclease domain mutations in cancer |
| work_keys_str_mv | AT raynere functionalanalysisofpoleexonucleasedomainmutationsincancer |