OP12NEW TOOLS TO STUDY HISTONE H3.3 MUTATIONS IN DIPG AND PHGG

Abstract

INTRODUCTION: Diffuse intrinsic pontine glioma (DIPG) is an aggressive malignant astrocytoma in children, with poor survival rate and few treatment options. Recently, K27M somatic mutations have been identified in histone H3.1 and H3.3 proteins in 80% of DIPGs and 20% of non-brain stem paediatric gliomas. In addition, 30% of paediatric high grade gliomas (pHGG) exhibit G34R or G34V H3.3 mutations. Several studies have highlighted the epigenetic changes associated with these mutations, however their precise role in tumourigenesis is still unknown. METHOD: We have generated novel polyclonal antibodies that can discriminate H3.3 G34R and G34V mutant proteins from their wild-type counterparts. The antibodies were validated by western blot, immunofluorescence and immunohistochemistry. RESULTS: The antibodies exhibit specific reactivity against recombinant H3.3 proteins, transfected mammalian cells, DIPG and pHGG H3.3 mutant cell lines, and patient samples. CONCLUSION: These antibodies can now be used to study the consequences of single amino acid substitutions in H3.3, for example by providing insights into spatial/anatomical aspects of the mutations in DIPG. They may find prognostic utility in the rapid identification of H3.3 mutant DIPG/pHGG cases and will be used to identify novel downstream targets and pathways of the different mutants in DIPG/pHGG genesis and progression.