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

儿科高级别胶质瘤（pedHGG）是最具侵袭性的儿科脑肿瘤实体，预后极差，总体生存率低于15%。在30-40%的pedHGG中存在涉及组蛋白3的赖氨酸27（H3 K27 M）突变的特定遗传改变，导致受影响患者的预后更差。尽管已知H3 K27 M突变导致H3 K27三甲基化（H3 K27 me 3）的整体丧失以及H3 K27的超乙酰化，但潜在的生物学机制仍然未知。我们推测，H3 K27 M诱导的超乙酰化（H3 K27 ac）的组蛋白乙酰转移酶（=表观遗传作家），如CREB结合蛋白（CBP），可能伴随着更大的影响乙酰化依赖的表观遗传读者。事实上，发现读取器溴结构域和额外末端结构域蛋白（BET）BRD 4被H3 K27 M突变的核小体强烈结合。因此，靶向H3 K27 ac特异性表观遗传读取器和写入器两者可以代表治疗H3 K27 M突变的pedHGG的有效治疗组合。我们以前的研究已经证明，H3 K27 M pedHGG细胞的肿瘤相关特征比H3.3野生型pedHGG细胞的特征更强地被BRD 4抑制所降低。BET和CBP抑制的组合甚至增强了抗肿瘤作用。然而，BRD 4和CBP在H3 K27 M pedHGG细胞中的潜在基因调控功能尚未真正了解。本项目旨在通过研究CBP和BET作为H3 K27 M和H3 K27野生型pedHGG细胞中表观遗传H3 K27 ac相关的写入器和读取器的影响，进一步阐明H3 K27 ac相关的基因调控过程。为此，我们将确定H3 K27 M/CBP/BET依赖性表观基因组和转录组以及由此产生的肿瘤表型特征，如增殖，侵袭和干性。最后，我们的目标是通过鉴定BET/CBP的进一步抑制剂，将所获得的关于H3. 3 K27 M突变pedHGG中H3 K27 ac相关表观遗传调控事件的知识转化为优化的体外靶向治疗方法，这将有助于改善H3 K27 M pedHGG儿童的临床状况。

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.