PP05. CHARACTERISATION OF DAXX IN THE DEPOSITION OF H3.3 IN PAEDIATRIC GLIOMA

Brain tumours are currently the leading cause of cancer-related mortality and morbidity in children. The cause and the pathobiology of childhood brain tumours is not fully understood. Interestingly the histone variant H3.3 and its chaperones DAXX and ATRX have recently been found to be mutated in a variety of cancers and particularly in paediatric glioblastoma, thus suggesting that defects of the chromatin architecture underlie paediatric glioblastoma pathogenesis. We aimed to describe the regulation of H3.3 deposition in paediatric glioblastoma and the involvement of DAXX expression and phosphorylation. We analysed DAXX phosphorylation level and expression of other histone chaperones in five different paediatric glioblastoma cell lines. We found that the phosphorylsated form of DAXX is mostly localised outside the areas of heterochtomatin in comparison to DAXX. Utilising CRISPR technology, cell models were created where endogenous DAXX was downregulated and overexpressed phospho-mutant forms of DAXX was introduced. The phospho-mutant clones produced a certain phenotype with irregular cell morphology and nuclear structure possibly due to chromosomal instability. Changes in telomere length were quantified between the cell models which showed little variance. However differences in cell cycle length were measured in between the different cell models by EdU staining. In summary, our data shows that DAXX expression is key to survival in peadriatic cell lines, that modulating DAXX phosphorylation is fundamental to its function and therefore its H3.3 chaperone activity, which has the potential to be a new target for paediatric glioblastoma treatment.