Neuro-Oncology
86 Investigating the role of NPM1 protein in H3-Mutated pHGG
By Aimee Grey, Rob Layfield and Farhana Haque.
Abstract
Abstract Introduction Paediatric high grade gliomas (pHGGs) are aggressive brain tumours and the leading cause of cancer related mortality in children and adolescents. Histone H3 mutations define key molecular subgroups of pHGGs. Nucleophosmin (NPM1) is a multifunctional nucleolar shuttling protein implicated in several malignancies. Our previous research on H3 interacting protein identified that NPM1interaction is altered with H3-mutated protein. Here, we investigated NPM1 expression and subcellular localisation in pHGG cell lines, its co-localisation with glial fibrillary acidic protein (GFAP), and the impact of NPM1 inhibition on cell viability. Methods Cell lines H3-G34V mutant, H3-wild type (WT), control HeLa and astrocytes were used. NPM1 expression in these cell line extracts were assessed by western blotting with anti-NPM-1 antibody. Immunofluorescence analysis was performed to determine subcellular localisation of NPM1 and its colocalization with GFAP, further analysed by image J. Cell viability was evaluated using MTT assays following pharmacological inhibition of NPM1 with NSC348884 at varying concentrations for 72 hours. Results Western blot analysis demonstrated increased NPM1 expression in all cancerous cell lines compared to control astrocytes. Immunofluorescence analysis revealed aberrant cytoplasmic localisation in H3-G34V cells and partial colocalization with GFAP. NSC348884 treatment reduced cell viability in a dose dependent manner across glioma cell lines. Conclusion The findings of this study suggest that NPM1 is overexpressed and aberrantly localised in pHGG cell lines, supporting a role in tumour cell maintenance and survival. Partial colocalization with GFAP indicates a potential association between NPM1 and the cytoskeleton, in H3G34V-mutant glioma cells. NPM1 inhibition highlights the potential of targeting NPM1 in pHGG. Further investigation is required to elucidate NPM1’s functional role in pHGGs and its suitability as a therapeutic target.