Effects of Histone 3.3 K27M mutation on CBP- and BET-mediated epigenetic gene regulation in pediatric diffuse intrinsic pontine gliomas (DIPG). Towards a combined epigenetic therapy approach for DIPG

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Pediatric high-grade gliomas (pedHGG) represent the most aggressive pediatric brain tumor entity with very poor prognosis and overall-survival rates of less than 15 %. There are specific genetic alterations involving mutations in lysine 27 of histone 3 (H3K27M) in 30-40% of pedHGG resulting in even worse prognosis of affected patients. Although it is known that H3K27M-mutation leads to global loss of H3K27 trimethylation (H3K27me3) combined with hyperacetylation of H3K27, the underlying biological mechanism is still unknown. We hypothesize that the H3K27M-induced hyperacetylation (H3K27ac) by histone acetyltransferases (= epigenetic writer), such as CREB Binding Protein (CBP), might be accompanied by a greater impact of acetylation-dependent epigenetic readers. Indeed, the reader Bromodomain and Extra Terminal domain protein (BET) BRD4 was found to be strongly bound by H3K27M-mutated nucleosomes. Thus, targeting both, H3K27ac-specific epigenetic readers and writers, could represent an effective therapeutic combination to treat H3K27M mutated pedHGG. Our previous investigations already proved that tumor related characteristics of H3K27M pedHGG cells are stronger decreased by inhibition of BRD4 than characteristics of H3.3 wildtype pedHGG cells. Combinations of BET and CBP inhibition even enhanced anti-tumor effects. However, the underlying gene regulatory functions of BRD4 and CBP in H3K27M pedHGG cells are not yet really known. The present project aims at further elucidating the H3K27ac-associated gene regulatory processes by investigating the impact of CBP and BET as epigenetic H3K27ac-associated writers and readers in H3K27M and H3K27 wildtype pedHGG cells. To this end, we will determine the H3K27M/CBP/BET-dependent epigenome and transcriptome together with resulting tumor phenotype features, such as proliferation, invasion, and stemness. Finally, we aim to translate the gained knowledge about H3K27ac-associated epigenetic regulatory events in H3.3K27M-mutated pedHGG into an optimized targeted treatment approach in vitro by identifying further inhibitors for BET/CBP which will help to improve the clinical situation of children with H3K27M pedHGG.