83 Investigating the role of high motility group box 1 protein in H3-mutated gliomas

Abstract Introduction Paediatric high-grade gliomas (pHGGs) are aggressive, therapy-resistant CNS tumours that represent the leading cause of paediatric cancer deaths. Two classifications contain histone 3 mutations – H3K27-altered and H3G34-mutated. Our previous research using cell membrane proteomics indicates oncogenic protein HMGB1 is present in the cell membrane of H3G34V and H3G34R cells. However, little is known about HMGB1 expression patterns in pHGG or the functional effects of HMGB1 inhibition. Methods Cell extracts were obtained from H3-mutant, H3-wild type (WT) and control astrocyte and HeLa cell lines. HMGB1 expression was assessed by western blotting using anti HMGB1 antibodies, and band intensities were quantified using ImageJ. H3G34V, H3-WT and HeLa cells were incubated with HMGB1 inhibitor glycyrrhizin (20-400µM) for 36-72 hours and cell viability was assessed with MTT assays. Results HMGB1 showed significantly higher expression in H3G34V and H3-WT compared to astrocytes (p < 0.01) and in H3-WT compared to HeLa (p < 0.05). Expression did not differ significantly between H3G34V and H3 WT cells. Low concentrations of glycyrrhizin (20-60µM) increased cell viability and high concentrations (100-400µM) had no or minimally negative effects. Conclusion HMGB1 is significantly upregulated and localised to the cell membrane in pHGGs, indicating increased binding to cellular receptors. Consequent downstream activation of the NF-κB pathway is associated with oncogenic effects. HMGB1 could create feed-forward proangiogenic signalling loops in H3-mutated gliomas. Glycyrrhizin provides protection from HMGB1-induced apoptosis, potentially via inhibition of the mitochondrial pathway. HMGB1 may be an integral cell surface ligand for oncogenesis and therapeutic resistance in pHGGs via the NF-κB pathway, highlighting its potential as a therapeutic target.