- Title
- Will DNA repair inhibitors improve survival of patients with brain cancer?
- Creator
- Lozinski, Mathew
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2023
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- Glioblastoma is an aggressive and highly invasive disease that often recurs and resists standard treatment. Tumour cells can restrict the DNA-damaging effects of temozolomide (TMZ) and radiation therapy (RT) using the DNA damage response (DDR) mechanism which activates cell cycle arrest and DNA repair pathways. Three proteins play major roles in the recognition of treatment induced DNA damage, including DNA-dependent protein kinase (DNA-PK), Ataxia-telangiectasia mutated (ATM) protein, and ATM- and Rad3-Related (ATR) protein. Chapter 1 introduced glioblastoma, the complexity of its biology and the relationship between DNA repair and standard treatment resistance. The chapter then discussed the strategy of inhibiting DNA repair proteins to enhance standard treatment response, one which is relatively unexplored and has potential therapeutic use in glioblastoma. In Chapter 2, the relationship between baseline DDR expression and resistance to standard treatment was examined. Furthermore, the response of DDR pathways was observed after treatment with TMZ and/or RT in patient-derived glioblastoma cell lines to elucidate the extent of DDR response after treatment. It was apparent that high nucleotide excision repair (NER) and low mismatch repair (MMR) expression associated with TMZ resistance, while high homologous recombination (HR) expression associated with resistance to RT. No significant survival difference was observed between distinct patient groups based on DDR transcriptomic expression, however high expression of three DDR genes (ATP23, RAD51C and RPA3) associated with poor patient survival. When observing DDR expression after treatment in glioblastoma cell lines, DDR pathways were predominantly up-regulated within 24hr after standard treatment. This data thus suggested that targeting DDR components in combination with standard treatment would be optimal within a 24hr timeframe after treatment with TMZ and RT. Chapter 3 investigated the inhibition of DDR in 12 patient-derived glioblastoma cell lines using two ATR inhibitors (M4344 and M6620) and a DNA-PK inhibitor (M3814) that are being used in clinical trials in solid and haematological tumours. Testing the single agent and combined activity with TMZ and/or RT, ATR inhibition using M4344 was potent and synergised strongly with TMZ and moderately with RT. M4344 was subsequently examined in greater depth in Chapters 4 and 5. Although not examined further within this thesis, M3814 showed strong synergy with RT across most glioblastoma cell lines and is a potentially effective strategy to enhance the cell killing abilities of RT in glioblastoma. In Chapter 4, the effect of ATR inhibition was further investigated within 12 glioblastoma cell lines using M4344. The single agent activity of ATR inhibitors can be influenced by genomic alterations within tumour cells where a higher dependence on ATR activity is required to repair DNA damage. Using baseline genomic and transcriptomic data, M4344 sensitivity was enhanced in cell lines displaying higher frequency of DDR mutations and expression of cell cycle and NER pathways. Sensitivity was also higher in MGMT unmethylated and standard treatment resistance cells. Using live-cell imaging, M4344 was shown to significantly enhance markers of apoptosis and cell death when combined with TMZ and/or RT in glioblastoma cell lines. DNA damage may enhance inflammatory responses in tumour cells, while potentially increasing mutation load and consequently the expression of neoantigens that can stimulate immunogenic responses and activate antitumour immune responses. In Chapter 4, an upregulation of pro-inflammatory and innate immune signalling pathways was observed after M4344 treatment, alone and in combination with TMZ and RT. Chapter 5 aimed to explore in further depth the transcriptomic changes after M4344 treatment, as well as somatic mutations induced by TMZ+RT and M4344+TMZ+RT treatment. ATR inhibition produced a predominantly immunomodulating response, including increased PD-L1 gene expression. Reduction in glioma signalling pathways, including hypoxia, was also observed suggesting ATR inhibition may modulate the tumour microenvironment, a major factor in glioblastoma aggressiveness and treatment resistance. Higher expression changes in G1 cell cycle were correlated with M4344 sensitivity, while higher hypoxia and metabolic pathway expression after standard treatment associated with sensitivity to TMZ or RT treatment. Furthermore, frequency of somatic mutations formed after standard treatment positively correlated with TMZ and RT sensitivity in glioblastoma cell lines. Surprisingly, the addition of M4344 to TMZ+RT did not significantly increase the frequency of somatic mutations in glioblastoma cell lines. However, distinct mutation signatures were identified when M4344 was combined with TMZ and RT, specifically the presence of a hypermutation-associated signature which may produce highly immunogenic neoantigens. Overall, this thesis provides support for DDR as one mechanism leading to the treatment-resistance in glioblastoma. Targeting key DDR components was shown to enhance the cell killing ability of standard treatment, as well as inducing a potentially immunogenic response that could further enhance antitumour responses against glioblastoma.
- Subject
- DNA; brain cancer; Glioblastoma; repair
- Identifier
- http://hdl.handle.net/1959.13/1468859
- Identifier
- uon:48119
- Rights
- Copyright 2023 Mathew Lozinski
- Language
- eng
- Full Text
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | ATTACHMENT01 | Thesis | 6 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 213 KB | Adobe Acrobat PDF | View Details Download |