http://theses.ncl.ac.uk/jspui/handle/10443/102 2026-02-04T14:16:15Z 2026-02-04T14:16:15Z Mould, Emily Victoria Alinda http://theses.ncl.ac.uk/jspui/handle/10443/6425 2025-03-28T18:59:13Z 2014-01-01T00:00:00Z

Title: The role of DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) kinase in the cellular response to microtubule-targeting drugs Authors: Mould, Emily Victoria Alinda Abstract: DNA-dependent protein kinase (DNA-PK) and ataxia telangiectasia mutated (ATM) kinase are DNA-damage activated kinases that play central roles in the non homologous end joining (NHEJ) and homologous recombination (HR) DNA double strand break repair pathways, respectively. DNA-PK and ATM have both been shown to have roles in addition to DNA repair, involving localisation at the centrosome during mitosis and mitotic regulation. Pilot studies demonstrated that the selective DNA-PK and ATM inhibitors, NU7441 and KU55933, respectively, caused greater sensitisation to DNA-damaging and microtubule-targeting agents in multidrug-resistant cells compared with parental cells. This observation led to the hypothesis that inhibition of DNA-PK and ATM using NU7441 and KU55933, respectively, or loss of DNA-PK function in DNA-PK deficient cells, would sensitise cells to agents that interfere with the formation of the mitotic spindle, e.g. microtubule-targeting agents such as vincristine, docetaxel and paclitaxel. Growth inhibition assays in four different paired parental sensitive and multidrug resistant cell lines (resistant through overexpression of the drug efflux transporter, MDR1) demonstrated that 1 µM NU7441 and 10 µM KU55933 sensitised the multidrug-resistant cells to vincristine and either docetaxel or paclitaxel to a significantly greater extent than in parental cells. Phosphorylation of DNA-PK at a DNA-damage-associated autophosphorylation site (Ser2056) was observed in response to vincristine, which did not cause DNA damage as determined using the COMET assay. Unexpectedly, three MDR1-overexpressing multidrug-resistant cell lines were found to be not only chemo-resistant but also radio-resistant. Investigations into the effects of NU7441 and KU55933 on drug transport demonstrated that in MDR1-overexpressing canine kidney MDCKII-MDR1 cells, 1µM NU7441 significantly increased doxorubicin cellular accumulation, measured by fluorescence microscopy, via an MDR1-dependent mechanism. NU7441 (1 µM), three structurally-related compounds (NU7742 (an inactive NU7441 analogue), DRN1 and DRN2 (DNA-PK-inhibitory and DNA-PK non-inhibitory atropisomeric NU7441 derivatives, respectively)) at 1 µM, and KU55933 at 1 µM and 10 µM, all increased intracellular vincristine accumulation in the MDR1-overexpressing CCRF-CEM VCR/R cells to a level similar to that induced by verapamil, as measured by LC-MS. Growth inhibition and cytotoxicity studies using an isogenic panel of DNA-PK proficient and deficient cell lines, with varying DNA-PK catalytic subunit expression levels (parental DNA-PK +/+, DNA-PK +/-, DNA-PK -/- and DNA-PK -/- cells with PRKDC cDNA re-expression (DNA-PK RE)) established that DNA-PK -/- cells were more sensitive to ionising radiation, vincristine and docetaxel, thereby demonstrating a role for DNA-PK in the response of cells to all of these agents. KU55933 (10 µM) caused significant sensitisation in the HCT116 DNA-PK +/+, DNA-PK +/- and DNA PK -/- cells to ionising radiation, vincristine and docetaxel, suggesting an additional role for ATM. The combination of vincristine and ionising radiation was significantly more active in the absence of DNA-PK or following inhibition of DNA-PK. Confocal microscopy studies demonstrated that phosphorylated DNA-PK localised to mitotic structures and that lack or inhibition of DNA-PKcs caused an increase in aberrant mitotic events, such as chromosome misalignment, increases in centrosome number and multipolar spindle formation. Overall, the studies described in this thesis demonstrate that DNA-PK and ATM play a role in mitosis and in the response of cancer cells to microtubule-targeting agents. Dual DNA-PK and MDR1 inhibitors, and dual ATM and MDR1 inhibitors, were identified. These results extend the clinical potential of targeted inhibition of DNA-PK and ATM to use in combination with microtubule-targeting agents Description: PhD Thesis

2014-01-01T00:00:00Z Hannaway, Nicola Louise http://theses.ncl.ac.uk/jspui/handle/10443/5767 2023-08-18T09:42:06Z 2022-01-01T00:00:00Z

Title: The investigation of circulating biomarkers and potential mechanisms of resistance in the ATR/CHK1 signalling pathway in response to CHK1 inhibitor therapy Authors: Hannaway, Nicola Louise Abstract: The DNA damage response is a network of cell checkpoints leading to cellular repair and genome integrity when DNA insults have occurred. In cancers, enhanced tumour replication stress and genomic instability along with mutations in oncogenes and tumour suppressor genes can lead to disruption of the DNA damage response. Tumour survival becomes dependant on critical checkpoint controls. Drugs to target intact pathways, such as CHK1 inhibitors are therefore desirable and are being evaluated in clinical trials as prospective anticancer therapies. Cancer treatments have moved to a personalised approach based on tumour profiling and circulating biomarkers, such as circulating cell free DNA to predict patient response to targeted treatments. The mechanisms underpinning sensitivity and resistance to CHK1 inhibitor treatment are still unclear. This project explores three distinct strands of research to examine both primary and acquired mechanisms of CHK1i resistance. Firstly, investigating gene and protein expression changes in Eμ-Myc mouse models of B-Cell lymphoma. Wild type Eμ-Myc mice are sensitive to CHK1i treatment, however by contrast Eμ-Myc NF-κB c-Rel -/- and RelA T505A lymphomas are CHK1i resistant. Secondly, the generation and characterisation of U2OS osteosarcoma cells with acquired CHK1i resistance. Lastly, investigating if DNA damage response mutations can be isolated from patient cfDNA samples on a CHK1 inhibitor clinical trial. Results have shown that resistance to CHK1 is complex and that there are multiple resistance mechanisms in place. The Eμ-Myc NF-κB c-Rel -/- mice completely downregulate the DNA damage response and this pattern is shared with some resistant cell lines, but alternative mechanisms developed in other models. DDR mutations can be detected and tracked in cfDNA samples and show a large degree of heterogeneity between patients. This study highlights the importance of the ATR/CHK1 DNA damage checkpoint and how drug resistance mechanisms can vary between diverse tumour mutational profiles. Description: Ph. D. Thesis.

2022-01-01T00:00:00Z Hunter, James Henry http://theses.ncl.ac.uk/jspui/handle/10443/5478 2022-06-30T09:32:48Z 2021-01-01T00:00:00Z

Title: Expanding the scope of DNA compatible chemistry for the application within DNA encoded libraries Authors: Hunter, James Henry Abstract: The discovery of potential lead like molecules is a crucial phase for any drug discovery program. Current methods to identify lead molecules are often resource intensive, often requiring millions of compounds to be screened in biological assays. Reducing the time and cost taken to generate, store and screen large compound libraries would have a positive impact on academia and small pharmaceutical companies. DNA encoded libraries (DELs) aim to improve upon this by screening an entire library in a single vessel against a target and utilising the DNA tag to identify potential inhibitors. Current methods of preparing DELs are limited to chemistry that is compatible with DNA. These chemical methods are often limited to simple chemical reactions, such as cross-couplings and amide bond formation, which are used combinatoriality to generate vast libraries. As analogous chemical reactions are used, current libraries are often populated with compounds with similar physical properties and have limited structural diversity. The reactions used to generate DELs are often low yielding, or are limited in substrate scope, further reducing the diversity of potential libraries. Development of new approaches with DEL synthesis will increase the ability to synthesise libraries with greater chemical diversity and improved physical property profiles. Products produced from the encoded transformation paradigm. A new paradigm termed “encoded transformations” was introduced. This technique involves encoding a specific chemical transformation of a reactive core molecule instead of coding the addition of a building block. This would lead to a potential library of compounds with reduced overall molecular weight and more lead-like physical properties. A common reactive 2-oxobetenamide core was used and several chemical transformations have been successfully employed. These chemical transformations were developed off-DNA and provide a means to prepare libraries with significant scaffold diversity using DNA compatible chemistry. Micellar solvents have been shown to improve normal phase organic chemical reactions. Applying this technique to on-DNA synthesis vastly improved the scope and diversity of the reactants used. Both amide couplings and Suzuki-Miyaura reactions were successfully optimised utilising micellar media as a solvent. Optimisation was carried out in both examples by factorial experimental design (FED), which revealed second order relationships between variables tested that would not likely be discovered using conventional techniques. Products produced from novel micellar promoted Suzuki-Miyaura and amide reactions. These novel reactions were used to synthesise a prototype 6x6 library, which was PCR amplified and sequenced proving that reactions in micellar media do not cause DNA damage. The reverse amide coupling was then used to create a 99,405-member library, including compounds designed to specifically target the SARs-CoV-2 main protease (Mpro) binding site, using an array of both covalent and non-covalent inhibitors. Description: PhD Thesis

2021-01-01T00:00:00Z Gamie, Zakareya Esame Khalil http://theses.ncl.ac.uk/jspui/handle/10443/5446 2022-06-15T08:51:24Z 2020-01-01T00:00:00Z

Title: Preclinical testing of a targeted TRAIL therapeutic for bone sarcoma Authors: Gamie, Zakareya Esame Khalil Abstract: Background: TNF-related apoptosis-inducing ligand (TRAIL) can induce cell death in cancer cells after binding to its TRAIL receptors [TRAILR, Death Receptor 4 (DR4) and Death Receptor 5 (DR5)] while sparing non-malignant cells. The application of TRAIL provides an approach that can potentially overcome drug resistance and toxicity associated with high doses of conventional therapies. It could be administered alone or in combination with conventional therapies and, therefore, may offer a promising new approach to bone sarcoma treatment. Enhancing the cytotoxic effect of TRAIL involves targeting a tumour associated antigen (TAA). Here, the aim was to characterise bone sarcoma cells for TRAILR expression and to assess the effectiveness, both in vitro and in vivo of a novel TRAIL construct, neural/glial antigen 2 (NG2) targeted TRAIL (ScFvNG2-Fc-scTRAIL). Methods: Bone sarcoma cell lines were characterised for TRAILR and NG2 expression on RNA and protein level. Together with non-malignant cell lines, they were exposed to the novel TRAIL therapeutic (ScFvNG2-Fc-scTRAIL) in vitro and then tested in vivo in a newly developed xenograft model of dedifferentiated chondrosarcoma. Results: Surface DR5 was expressed in all cell lines examined (very high: HT1080, MG63; moderate: SW153, U2OS, TC71). NG2 was also expressed (very high: SW1353, MG63; moderate: U2OS, HT1080). ScFvNG2-Fc-scTRAIL demonstrated enhanced cytotoxicity in DR5- and NG2-expressing cell lines (MG63>HT1080>U2OS), which increased with doxorubicin and was also found in vivo when engrafting a luciferase expressing HT1080 cell line in a dedifferentiated chondrosarcoma mouse model. Conclusion: I demonstrate that a novel targeted TRAIL therapeutic, ScFvNG2-FcscTRAIL, has a selective and significant cytotoxic effect on cell lines expressing both cell surface DR5 and NG2, and these cytotoxic effects can be enhanced further with doxorubicin. Such combinations could minimise the risk of treatment failure due to drug resistance, a common problem of single agent approaches. Furthermore, these findings provide a framework for the clinical development of ScFvNG2-Fc-scTRAIL and could potentially be used in the neoadjuvant setting, which would be a shift from the usual convention of prioritising excision of the sarcoma. Description: PhD Thesis

2020-01-01T00:00:00Z