MOLECULAR PATHOPHYSIOLOGY OF HISTONE 3.3 G34R MUTANT CHILDHOOD BRAIN TUMOURS; TOWARDS THE DEVELOPMENT OF NOVEL TARGETED THERAPIES

There have been no significant improvements in the treatments for childhood and adolescent High-Grade Glioma (pHGG) and Diffuse Intrinsic Pontine Glioblastoma (DIPG), which have a very poor prognosis. These cancers harbour mutations affecting histone 3 proteins, 80% of DIPGs harbour histone H3.1 and H3.3 K27M somatic mutations whilst 30% of pHGGs exhibit H3.3 G34R or G34V mutations. Several studies have highlighted the epigenetic changes associated with these mutations, however their precise role in tumourigenesis is still unknown. We have generated and validated a Histone 3.3 (H3.3) G34R antibody, and investigated the downstream effects of H3.3 G34R mutations in pHGG. In order to identify the genes that may be deregulated by G34R mutant histone expression, we have performed chromatin immunoprecipitation (ChIP) assays with our H3.3 G34R and wild type H3 antibodies, using pHGG H3 G34R mutant and wild type cell lines. Initial analyses of ChIP data have implicated deregulation of various cell-signaling pathways including Notch1, Hedgehog, PPAR-1, PLC-beta and Androgen, in H3 G34R mutated pHGG. We are currently determining the effects of altered expression of Notch pathway components, HES1, HES5 and Notch intracellular domain (NICD) on tumorigenesis of H3 G34R mutated pHGG, through gene and protein expression and inhibition assays. In parallel, we are also determining the effect of Hedgehog signalling pathway components Gli2, PTHLH, TGFβ2 in H3 G34R mutated pHGG and if there is cross talks between these two pathways. These analyses would enable us to find potential novel therapeutic targets. Results of these analyses/experiments will be presented.