Exploring the use of Arginine Methylation Inhibitors Against Brain Tumours in an ex vivo Perfusion System

Barry, Antonia

Abstract

Glioblastoma (GBM) is the most common primary malignancy in the central nervous system and the most devastating, with a median survival of 18 months from diagnosis. The development of models to research and understand GBM tumourigenesis and progression, as well as for drug screening is paramount to tackling this disease and determining new and effective therapeutic interventions. Microfluidics models have been developing over the past several years with the aim of maintaining tissue viability over several days and presenting a more patient-personalised and clinically relevant model of GBM. The University of Hull have driven the manufacture of a novel perfusion device which allows a micro-biopsy of the tissue to be maintained for up to 8 days for drug screening and evaluation of aspects of the tumour microenvironment.
Type I protein arginine methyltransferases (PRMTs) are responsible for the deposition of asymmetric dimethyl marks on arginine residues of both histones and proteins, whereas type II PRMTs deposit symmetric dimethylation marks and type III perform monomethylation, solely. Methylation is a very common and stable post-translational modification and can alter protein expression and function. PRMTs are upregulated in and have been associated with dysregulation of a variety of pathways in GBM and have been involved in clinical trials for a variety of cancers, including GBM. The overarching hypothesis for this study was that GSK3368715 could cause cell death in GBM patient tissues in the novel perfusion device. This was investigated by evaluating cell death through histology and cellular stress assays, determining transcriptomic changes as a result of treatment and comparing the outcomes of these results to healthy mouse brains maintained in the novel perfusion device.
Type I PRMT inhibition using GSK3368715 was evaluated in U87-MG cells and in GBM patient tissue in the novel perfusion device throughout this research. No significant cellular stress was found between individual time points in control GBM and healthy mouse or cichlid brain tissues, maintained in the perfusion device for 8- (GBM, F = 1.11, df = 7, p = 0.37; Mouse, F=1.45, df=5, p=0.22) or 12- days (GBM, F =1.38 , df =11 , p =0.22) after the first 48 hours. Similarly, no significant changes in apoptosis were determined between pre- and 8- (cleaved PARP: GBM, w=38, p=0.151, Mouse, t = 0.020, df = 2, p = 0.99; Annexin V: GBM, t = -0.064, df = 13.76, p = 0.95, Mouse, t = -0.23, df = 6, p = 0.8), or 12-day (cleaved PARP: GBM, t = -0.40, df = 4.75, p-value = 0.71; Annexin V: t = -0.064, df = 13.76, p = 0.95) post-perfused tissue in GBM, or healthy mouse brain tissues. Together with the presence of mitoses in histological staining and cytokine profile shifts, which showed greater changes in cytokine profiles after 12 days, this indicated that tissues could be maintained for up to 8 days on chip, but further optimization would be required to extend perfusion to 12 days. Cichlid brain tissues were also found to be able to maintained in the perfusion device through no significant change in cellular stress for up to 6 days (F=1.73, df=1, p=0.21), lack of significant cellular death up to 4 days (Χ2=5.50, df = 3, p = 0.14) and presence of live cells seen through histology.
GSK3368815 was found to cause a 2.17-fold increase in apoptotic cleaved PARP expression in GBM tissue in the perfusion device, after 8 days (t = −4.52, df = 9, p = 0.001), but did not show synergy with other drugs (X2 = -0.41, df = 14,42, p = 0.69). This was not recapitulated with the Annexin V apoptotic marker (t = -0.41, df = 14.42, p = 0.69). Variation between GBM response to treatment, as well as extension of time on-chip beyond 8 days requires further exploration and optimisation. GSK3368715 and TMZ also caused changes to cytokines, including decreased expression of matrix metalloproteinase 9 (logFC=-0.07, t=-43.32, padj=0.0046) and vascular endothelial growth factor (logFC=-0.08, t=-4.25, padj=0.0004), as well as an increase in mesenchymal cell phenotype marker chitinase 3 like 1 (logFC=0.05, t=-2.97, padj=0.0088), which has been implicated in successful treatment of GBM. Differential gene expression of treated tissue was also determined, with gene ontology indicating reductions in protein synthesis capacity and increase in cell death with GSK3368715 treatment, supporting apoptotic marker expression in the tissue. Hundreds of alternatively spliced genes and links with fused in sarcoma (FUS) suggested that this may be a mechanism by which PRMT inhibition functions to cause apoptosis. This data was then compared to healthy mouse brain controls, in which GSK3368715 did not cause any apoptotic effects (cleaved PARP: t=-1.20, df=5, p=0.28; Annexin V: t=-0.47, df=6, p=0.65).
In conclusion, GSK3368715 was found to cause apoptosis in 8-day post-perfused GBM patient tissue. This could be caused by PRMT-inhibition induced alternative splicing and involvement of FUS, which may lead cells to be driven to a more differentiated phenotype, resensitisation of cells to TMZ and reduction of tumour microenvironmental factors associated with aggressive GBM phenotypes.

Citation

Barry, A. (2025). Exploring the use of Arginine Methylation Inhibitors Against Brain Tumours in an ex vivo Perfusion System. (Thesis). University of Hull. https://hull-repository.worktribe.com/output/5133792

• Thesis Type: Thesis
• Deposit Date: Apr 28, 2025
• Publicly Available Date: Apr 28, 2025
• Keywords: Biomedical sciences
• Public URL: https://hull-repository.worktribe.com/output/5133792
• Additional Information: Biomedical Sciences; University of Hull
• Award Date: Apr 16, 2025