Identification and characterisation of suppressors of synthetic lethality resulting from loss of SETD2 and WEE1
<p>Our lab has recently identified a conserved synthetic lethality between loss of SETD2-dependent H3K36 trimethylation and inhibition of WEE1 kinase (Pfister et al., 2015). In order to explore the possible mechanisms of resistance to WEE1 inhibitor-treatment in H3K36me3-deficient cells, a yea...
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| Format: | Thesis |
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2017
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| _version_ | 1826296709312937984 |
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| author | van Bijsterveldt, L |
| author2 | Humphrey, T |
| author_facet | Humphrey, T van Bijsterveldt, L |
| author_sort | van Bijsterveldt, L |
| collection | OXFORD |
| description | <p>Our lab has recently identified a conserved synthetic lethality between loss of SETD2-dependent H3K36 trimethylation and inhibition of WEE1 kinase (Pfister et al., 2015). In order to explore the possible mechanisms of resistance to WEE1 inhibitor-treatment in H3K36me3-deficient cells, a yeast genetic screen was performed to isolate mutants that suppressed the <em>set2Δ wee1-50</em> temperature sensitivity. Whole-genome sequencing of 23 spontaneous suppressor mutants revealed mutations in eight genes involved in cell cycle regulation (<em>bub1, cdc2, cut2, cut1, gtb1, myo52, slp1</em> and <em>wee1</em>), five genes involved in cell communication (<em>gpa1, shk1, ssp2, ste20</em> and <em>wis1</em>), and seven genes involved in transcription (<em>bdf1, brf1, bye1, epl1, lsm4, paf1</em> and <em>taf10</em>). </p> <p>Further analysis in mammalian cells revealed that knockdown of RNA Polymerase II-associated factor 1 (PAF1) partially rescued cell viability at higher concentrations (300 and 600 nM) of the WEE1 inhibitor AZD1775 in both <em>SETD2</em> wild-type and <em>SETD2</em> CRISPR knockout U2OS cells. Data were verified in fission yeast as well as in the renal cell carcinoma cell lines 786-O (<em>SETD2</em> wild-type) and A498 (bearing a truncating mutation in <em>SETD2</em>), indicating functional conservation of Paf1/PAF1-dependent resistance to Wee1/WEE1 inhibition from fission yeast to mammalian cells. </p> <p>Investigations into the molecular mechanisms responsible for PAF1-mediated suppression in human cells suggested involvement of the CDK inhibitor p21<sup>Cip1/Waf1</sup>. Knockdown of PAF1 induced relocalisation of p21<sup>Cip1/Waf1</sup> to the cytoplasm where it is thought to suppress caspase-3 dependent apoptosis as well as the degradation of RRM2 via CDK2 inhibition in the presence of AZD1775. These findings suggest that targeting cytoplasmic p21<sup>Cip1/Waf1</sup> may offer new strategies for the treatment of resistance to WEE1-inhibition and knowledge of this pathway should be taken into account in the design of trials of targeted therapies for H3K36me3-deficient cancers.</p> |
| first_indexed | 2024-03-07T04:20:31Z |
| format | Thesis |
| id | oxford-uuid:cadc1392-a031-496e-8ad1-def942ab4893 |
| institution | University of Oxford |
| last_indexed | 2024-03-07T04:20:31Z |
| publishDate | 2017 |
| record_format | dspace |
| spelling | oxford-uuid:cadc1392-a031-496e-8ad1-def942ab48932022-03-27T07:10:58ZIdentification and characterisation of suppressors of synthetic lethality resulting from loss of SETD2 and WEE1Thesishttp://purl.org/coar/resource_type/c_bdccuuid:cadc1392-a031-496e-8ad1-def942ab4893ORA Deposit2017van Bijsterveldt, LHumphrey, T<p>Our lab has recently identified a conserved synthetic lethality between loss of SETD2-dependent H3K36 trimethylation and inhibition of WEE1 kinase (Pfister et al., 2015). In order to explore the possible mechanisms of resistance to WEE1 inhibitor-treatment in H3K36me3-deficient cells, a yeast genetic screen was performed to isolate mutants that suppressed the <em>set2Δ wee1-50</em> temperature sensitivity. Whole-genome sequencing of 23 spontaneous suppressor mutants revealed mutations in eight genes involved in cell cycle regulation (<em>bub1, cdc2, cut2, cut1, gtb1, myo52, slp1</em> and <em>wee1</em>), five genes involved in cell communication (<em>gpa1, shk1, ssp2, ste20</em> and <em>wis1</em>), and seven genes involved in transcription (<em>bdf1, brf1, bye1, epl1, lsm4, paf1</em> and <em>taf10</em>). </p> <p>Further analysis in mammalian cells revealed that knockdown of RNA Polymerase II-associated factor 1 (PAF1) partially rescued cell viability at higher concentrations (300 and 600 nM) of the WEE1 inhibitor AZD1775 in both <em>SETD2</em> wild-type and <em>SETD2</em> CRISPR knockout U2OS cells. Data were verified in fission yeast as well as in the renal cell carcinoma cell lines 786-O (<em>SETD2</em> wild-type) and A498 (bearing a truncating mutation in <em>SETD2</em>), indicating functional conservation of Paf1/PAF1-dependent resistance to Wee1/WEE1 inhibition from fission yeast to mammalian cells. </p> <p>Investigations into the molecular mechanisms responsible for PAF1-mediated suppression in human cells suggested involvement of the CDK inhibitor p21<sup>Cip1/Waf1</sup>. Knockdown of PAF1 induced relocalisation of p21<sup>Cip1/Waf1</sup> to the cytoplasm where it is thought to suppress caspase-3 dependent apoptosis as well as the degradation of RRM2 via CDK2 inhibition in the presence of AZD1775. These findings suggest that targeting cytoplasmic p21<sup>Cip1/Waf1</sup> may offer new strategies for the treatment of resistance to WEE1-inhibition and knowledge of this pathway should be taken into account in the design of trials of targeted therapies for H3K36me3-deficient cancers.</p> |
| spellingShingle | van Bijsterveldt, L Identification and characterisation of suppressors of synthetic lethality resulting from loss of SETD2 and WEE1 |
| title | Identification and characterisation of suppressors of synthetic lethality resulting from loss of SETD2 and WEE1 |
| title_full | Identification and characterisation of suppressors of synthetic lethality resulting from loss of SETD2 and WEE1 |
| title_fullStr | Identification and characterisation of suppressors of synthetic lethality resulting from loss of SETD2 and WEE1 |
| title_full_unstemmed | Identification and characterisation of suppressors of synthetic lethality resulting from loss of SETD2 and WEE1 |
| title_short | Identification and characterisation of suppressors of synthetic lethality resulting from loss of SETD2 and WEE1 |
| title_sort | identification and characterisation of suppressors of synthetic lethality resulting from loss of setd2 and wee1 |
| work_keys_str_mv | AT vanbijsterveldtl identificationandcharacterisationofsuppressorsofsyntheticlethalityresultingfromlossofsetd2andwee1 |