Glioblastoma : CIZ1 as a potential biomarker and target. Cytotoxicity screening of kinase inhibitors in monolayer and spheroid models

Fraser, Megan and Copeland, Nikki and Allinson, Sarah (2024) Glioblastoma : CIZ1 as a potential biomarker and target. Cytotoxicity screening of kinase inhibitors in monolayer and spheroid models. Masters thesis, Lancaster University.

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Abstract

Glioblastoma is the most aggressive and prevalent adult brain cancer, with poor prognosis and high rates of recurrence. Despite current therapeutic strategies and the extensive ongoing search for novel treatments, high rates of treatment resistance and low survival rates continue to pose a pressing problem. There is a clear need for biomarkers to improve early detection and diagnosis, as well as new individualised therapeutic strategies for glioblastoma. This work aimed to assess Cip1-interacting zinc-finger protein 1 (CIZ1) protein as a prognostic biomarker in neurological tumours, showing that CIZ1 overexpression is prognostically linked to poor survival outcomes in aggressive brain cancers. Here, the efficacy of small-molecule kinase inhibitors to reduce CIZ1 protein levels is interrogated, along with their in vitro effects on proliferation in established U87MG and primary BTNW914 glioblastoma cell lines. Finally, a glioblastoma spheroid model was utilised for further cytotoxicity assays, to better assess inhibitor activity in a more clinically relevant in vitro model. A panel of dbf4-dependent kinase (DDK) and cyclin dependent kinase (CDK) inhibitors were chosen to assess their modulation of CIZ1 protein levels. A significant reduction in CIZ1 protein was observed in U87MG cells treated with CDKi’s PD0332991 (PD) and CVT-313 (CVT), as well as DDKi PHA767491 (PHA). There was a significant CIZ1 reduction in BTNW914 cells treated with CDKi RO-3306 (RO), with pronounced CIZ1 reduction for CVT. These results demonstrate the potential for targeted kinase inhibition as an effective strategy for CIZ1 modulation in vitro. Application of these inhibitors in monolayer and spheroid cytoxocity assays, demonstrated clear dose dependent effects, reducing cellular viability in BTNW914 and U87MG monolayer cells, as well as U87MG spheroids. In BTNW914 monolayer assays, the lowest IC50’s for 72- hour treatment were observed for PHA (0.76 M) and RO (1.83 M). These results were reflected in monolayer U87MG cells, where IC50’s were also lowest for RO (2.21 M) and PHA (2.22 M). Further investigation of cytoxocity in U87MG spheroids determined PHA to have the lowest IC50 (1.14 M) and RO a much higher IC50 of 7.52 M at 48-hours. Interestingly by 120- and 144-hours the IC50’s of CVT and RO were lowest at 0.66 M and 2.24 M respectively, with the IC50 of PHA rising to 3.36 M. This work presents evidence of CIZ1’s value as a prognostic biomarker in neurological cancers, highlighting the correlation between high CIZ1 levels and lower OS in paediatric brain cancers, and its potential as a glioblastoma treatment target via kinase inhibitor modulation. The treatment of glioblastoma cells using a range of DDK and CDK inhibitors showed comparable cytotoxic potency in primary and established cells, and their potential treatment utilisation was further supported by U87MG spheroid data.