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

儿童高级别胶质瘤是最具侵袭性的儿童脑肿瘤，预后极差，总存活率不到15%。在30-40%的PedHGG患者中，存在涉及组蛋白3赖氨酸27(H3K27M)突变的特定基因改变，导致受影响患者的预后更差。虽然已知H3K27M-突变导致H3K27三甲基化(H3K27me3)的全球缺失和H3K27的高乙酰化，但其潜在的生物学机制仍不清楚。我们推测，H3K27M通过组蛋白乙酰转移酶(=表观遗传学编写者)，如CREB结合蛋白(CBP)，诱导H3K27ac的超乙酰化(H3K27ac)，可能伴随着乙酰化依赖的表观遗传学读取器的更大影响。事实上，阅读器溴结构域和额外末端结构域蛋白(BET)BRD4被发现与H3K27M突变的核小体强烈结合。因此，针对H3K27ac特异的表观遗传学读者和作者，可能是治疗H3K27M突变peHGG的有效治疗组合。我们以前的研究已经证明，与H3.3野生型peDHGG细胞相比，BRD4抑制H3K27M peDHGG细胞的肿瘤相关特性更强。BET和CBP抑制剂的联合应用甚至增强了抗肿瘤作用。然而，BRD4和CBP在H3K27M pedHGG细胞中潜在的基因调控功能尚不清楚。本项目旨在通过研究CBP和BET作为表观遗传H3K27ac相关的写入者和读取者在H3K27M和H3K27野生型peHGG细胞中的影响，进一步阐明H3K27ac相关的基因调控过程。为此，我们将确定H3K27M/CBP/BET依赖的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、侵袭和茎。最后，我们的目标是通过寻找更多的BET/CBP抑制剂，将所获得的关于H3.3K27M突变peHGG中与H3K27ac相关的表观遗传调控事件的知识转化为体外优化的靶向治疗方法，这将有助于改善H3K27M peHGG儿童的临床情况。

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.