85 Targeting the cell membrane protein, Flotillin-1 for therapy in H3-mutated paediatric high-grade gliomas

Abstract Introduction Paediatric high-grade gliomas (pHGGs) are aggressive with poor prognosis. Histone H3 mutations, including H3 G34R/V and H3 K27M, are characteristic of specific pHGG subgroups. Our previous research on cell membrane proteomics shows altered cell surface association of Flotillin-1 in H3- mutated pHGGs. Flotillin 1 are often overexpressed in various cancers and contributes to aberrant signalling. Here, we examined the localisation and expression of Flotillin 1 in H3 mutant and H3 wild type (WT) pHGG cell lines compared with non cancerous controls and assessed the impact of a Flotillin 1 inhibitor on cell viability. Method Immunofluorescence was used to assess Flotillin 1 localisation in H3 G34V and H3 WT cells compared with astrocytes and HeLa controls. Western blotting using anti Flotillin 1 antibodies was performed on protein extracts from all cell lines, and band intensities were quantified using ImageJ. MTT assays were performed by treating these cells, with PP121, Flotillin-1 inhibitor, at concentrations of 5, 15 and 20μM and a vehicle control, incubated for 36-72 hours. Results A higher Flotillin-1 expression in the cancerous cell lines compared to the non-cancerous control, astrocytes were observed. H3-G34V showed increased Flotillin-1 cell membrane localisation compared to H3-WT. PP121 treatment reduced cell viability in H3 G34V, H3 WT, and HeLa cells in a dose dependent manner. Conclusions Flotillin 1 overexpression and altered membrane localisation may contribute to dysregulated signalling, transcriptional changes, and tumour behaviour in pHGG. The mechanism PP121 induced reductions in cell viability requires further exploration, as it’s effect may extend beyond Flotillin-1 inhibition. Given the upregulation of Flotillin 1 in pHGGs, further exploration of Flotillin 1 as a potential therapeutic target is warranted.