[{"key":"dc.contributor.author","value":"Baillie, Katherine Eva","language":null},{"key":"dc.date.accessioned","value":"2025-04-25T21:19:41Z","language":null},{"key":"dc.date.available","value":"2026-04-30T07:00:00Z","language":null},{"key":"dc.date.issued","value":"2025","language":"en"},{"key":"dc.identifier.uri","value":"http:\/\/hdl.handle.net\/2429\/90831","language":null},{"key":"dc.description.abstract","value":"Despite advances in cancer treatment, resistance still presents a significant barrier. Cancers are genetically unstable, relying on the replication stress response to tolerate uncontrolled cell division. These response factors are emerging targets for precision medicine. One such target is the DNA replication ATP-dependent helicase\/nuclease DNA2, which has roles in replication stress responses and DNA repair. DNA2 is upregulated across multiple tumors and is important for tolerating oncogene-induced replication stress and TP53 mutations. However, multiple redundancies challenge treatment efficacy and drive resistance. To overcome this, we look to the example of PARP inhibitors, a clinically successful DNA repair therapy. These inhibitors trap PARP on DNA, sabotaging function and creating barriers to replication and repair. Certain PARP mutations mimic inhibitors, providing a roadmap to expand this mechanism to other targets. Here, I identify and study dominant negative mutations in DNA2 to guide development of inhibitors and identification of specific cancer contexts for DNA2 therapies.  \r\n\r\nCharacterization of a DNA2 mutant panel revealed nuclease-inactivating mutants cause DNA damage and cell cycle arrest even in the presence of the endogenous protein. Further experiments showed this phenotype depends on DNA2\u2019s helicase activity and DNA binding capability, supporting a trapping mechanism. Comparison of mutant effects across a panel of cell lines discovered the alternative lengthening of telomeres (ALT) phenotype increased sensitivity to the mutant. \r\n\r\nI next used homology models and in silico virtual ligand screening to identify candidate DNA2 inhibitors. Biochemical and cytotoxicity profiling revealed seven compounds which reduced cleavage activity and caused cell death. The most potent of these compounds demonstrated selectivity for ALT cancer lines. Finally, I explored a high-throughput screening method to screen a library of DNA2 mutants for dominant DNA damage phenotypes. \r\n\r\nOverall, this work contributes to our understanding of the interplay between nuclease and helicase functions of DNA2. I have also shown that cells exhibiting the ALT phenotype are differentially sensitive to DNA2 inhibition. ALT is critical in 10-15% of cancers and associated with poor prognosis. The identification of lead molecules for DNA2 inhibition contributes to the future development of a targeted treatment approach for ALT cancers.","language":"en"},{"key":"dc.language.iso","value":"eng","language":"en"},{"key":"dc.publisher","value":"University of British Columbia","language":"en"},{"key":"dc.rights","value":"Attribution-NonCommercial-NoDerivatives 4.0 International","language":"*"},{"key":"dc.rights.uri","value":"http:\/\/creativecommons.org\/licenses\/by-nc-nd\/4.0\/","language":"*"},{"key":"dc.title","value":"The DNA2 nuclease as an ALTernative target in cancer","language":"en"},{"key":"dc.type","value":"Text","language":"en"},{"key":"dc.degree.name","value":"Doctor of Philosophy - PhD","language":"en"},{"key":"dc.degree.discipline","value":"Interdisciplinary Oncology","language":"en"},{"key":"dc.degree.grantor","value":"University of British Columbia","language":"en"},{"key":"dc.contributor.supervisor","value":"Stirling, Peter C.","language":null},{"key":"dc.date.graduation","value":"2027-05","language":"en"},{"key":"dc.type.text","value":"Thesis\/Dissertation","language":"en"},{"key":"dc.description.affiliation","value":"Medicine, Faculty of","language":"en"},{"key":"dc.degree.campus","value":"UBCV","language":"en"},{"key":"dc.description.scholarlevel","value":"Graduate","language":"en"}]