title: Glioblastoma
title: CIZ1 as a potential biomarker and target. Cytotoxicity screening of kinase inhibitors in monolayer and spheroid models
titleType: Subtitle
Creators: name: Fraser, Megan
Publisher: name: Lancaster University
Year: 2024

description: 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.