title: Investigating mechanisms and indicators of sensitivity to replication stress-targeting therapies in glioblastoma
Creators: name: Clough, Emily Heather
Publisher: name: University of Glasgow
Year: 2023

description: Introduction Evidence suggests a subpopulation of treatment resistant glioblastoma (GBM) cancer stem cells (GSCs) is responsible for tumour recurrence, an almost universally deadly characteristic of this cancer of extreme unmet need. Current treatments fail to eradicate GSCs and novel GSC targeting therapies are a clinical priority. Elevated DNA replication stress (RS) in GSCs has been described, leading to constitutive DNA damage response activation and treatment resistance and targeting RS with combined ATR and PARP inhibition (CAiPi) has provided potent GSC cytotoxicity. Nevertheless, there are a relative lack of studies investigating the underlying mechanisms of response to CAiPi in GBM and a lack of robust transcriptional signatures or genomic biomarkers correlated with CAiPi response in GSCs. Aims This thesis aims to investigate RS as a targetable vulnerability of GSCs. It aims to achieve this by studying the mechanisms of sensitivity to inhibition of the RS response to inform transcriptional indicators of sensitivity. Lastly, it aims to investigate the feasibility of this therapeutic strategy in a preclinical model. Methods Paired GSC-enriched and GSC-depleted, differentiated (‘bulk’) populations, derived from resected GBM specimens, were maintained in serum-free, stemenriching conditions or differentiating conditions respectively. WGS and RNAseq were utilised to characterise the genomic and transcriptomic landscape of the cell line panel. Responses to CAiPi were assessed by clonogenic and cell viability assays and validated in a CD133 sorted population by neurosphere assay. Replication dynamics in paired GSC and bulk cells were investigated by a DNA fibre assay. Dysregulated S phase was analysed by quantification of 53BP1 nuclear bodies (53BP1NB), indicative of under-replication of the genome, and quantification of re-replicating cells by flow cytometry. Chromosomal instability was interrogated by quantification of chromatin bridges and micronuclei. Novel mechanistic discoveries prevalent in GSCs with potent CAiPi-sensitivity were used to curate a transcriptional marker of sensitivity for interrogation in GBM cell lines and in published clinical datasets. Lastly the feasibility of CAiPi was investigated in an in vivo preclinical model, assessing tolerability and tumour penetration. Results CAiPi was potently cytotoxic to a population of GSCs but highly heterogenous responses to CAiPi were observed across a panel of seven paired GSCs and bulk cells. Sensitivity was not predicted by elevated RS in GSCs or any previously defined biomarkers of RS or CAiPi sensitivity. Differential sensitivity was exploited for further investigations which identified transcriptional dysregulation of DNA replication, specifically in a CAiPi-responsive GSC line. Subsequent analysis of DNA replication identified PARPi-induced increase in origin firing, associated with PARP trapping. GSCs with this origin firing phenotype also exhibited an increase in both under-replicated DNA and re-replication in response to CAiPi, with an increase in chromosomal aberrations and instability. A curated transcriptional signature, based on mechanistic discoveries in CAiPisensitive GSCs, predicted GSC sensitivity and identified populations of GBM patients with poor survival who may respond to CAiPi treatment. In vivo studies demonstrated murine blood brain barrier (BBB) penetration of a PARPi and an ATRi with minimal toxicity, however optimal dosing and scheduling remains a challenge. Conclusions We propose that CAiPi-sensitivity is marked by loss of replication coordination leading to chromosomal damage as cells move through S phase. Additionally, we propose a model whereby under-replication and re-replication can occur due to spatial and temporal uncoupling during S phase. Targeting RS via CAiPi represents a promising therapeutic strategy for selectively targeting recurrence driving GSCs to improve clinical outcomes in GBM.