ID #542 Histone 3 mutant protein interactome analyses identifies YB1 and NPM1 as potential targets in pHGG

Abstract

Paediatric high-grade gliomas (pHGGs) are aggressive brain tumours representing the leading cause of cancer-related mortality in children and adolescents. Current treatment strategies remain largely ineffective, resulting in a poor prognosis. Histone H3 mutations, specifically H3.3K27M and H3.3G34R/V, are defining molecular features of pHGGs and are known to drive widespread epigenetic dysregulation.

Our previous proteomic studies investigating H3 interactomes, using recombinant wild type and mutant H3 proteins in precipitation/pull down assays, identified common and unique interacting binding partners of H3-G34 mutant proteins relative to wild type H3. Among these were YB-1 and Nucleophosmin 1 (NPM1). YB-1 is a multifunctional DNA/RNA-binding oncoprotein over-expressed in multiple cancers, while NPM1 is a multifunctional nucleolar shuttling protein implicated in several malignancies.

To investigate the functional relevance of YB-1 and NPM1 in H3-mutant pHGGs and assess their potential as therapeutic targets, we examined their expression and localisation using comparative immunofluorescence microscopy and western blotting analyses in H3-mutant and H3 wild-type glioma cell lines, alongside non-cancerous astrocyte controls

Both YB-1 and NPM1 were upregulated with increased expression in glioma cells in comparison to astrocytes. Notably, NPM1 exhibited increased and aberrant cytoplasmic localization in H3 mutated cells compared with H3 WT and control cells. Immunofluorescence analysis further revealed partial colocalization of NPM1 with GFAP in H3-G34 mutant glioma cells.

Functional inhibition studies were conducted using NSC348884 to target NPM1 and SU056 to inhibit YB-1. Both inhibitors reduced glioma cell viability/proliferation as determined by MTT assays.

Results from these functional analyses will be presented.