The epigenetic mechanisms of high-grade pediatric glioblastoma

弥漫性桥脑胶质瘤是一种原发于儿童的侵袭性脑肿瘤。的 DIPG患者的中位生存期为从诊断开始约一年，没有治疗的迹象。最近的研究 已经发现了DIPG的驱动突变，特别是H3 F3 A基因中的体细胞突变， 在大多数DIPG病例中观察到。H3 F3 A编码组蛋白H3变体H3.3，该变体H3.3在细胞凋亡中起重要作用。 在发育过程中调节基因表达。H3 F3 A的主要突变导致氨基酸 H3.3的赖氨酸（K）27残基变为蛋氨酸（M）（H3 K27 M）。在人类细胞中，有两种基因 编码H3.3，其在非复制依赖性核小体组装中组装成核小体 通路此外，有13个基因编码典型组蛋白H3.1和H3.2，它们与H3.3的差异在于： 4或5个氨基酸，并在复制偶联过程中组装成核小体。赖氨酸27是 在所有这些组蛋白H3蛋白中是保守的。因此，目前尚不清楚H3.3 K27 M突变， 发生在H3 F3 A基因的一个等位基因上，促进肿瘤发生。 H3 K27通过乙酰化或甲基化进行后修饰。H3 K27甲基化由以下物质催化： PRC 2赖氨酸甲基转移酶，并在干细胞分化过程中的基因沉默中起重要作用 和维护我们和其他人已经表明，在DIPG细胞中发生H3 K27甲基化的整体丢失， 含有K27 M突变，这是由于H3.3M27突变蛋白抑制PRC 2酶活性。 除了与基因激活相关的H3 K27 me 3的整体丢失外，我们还观察到， H3 K27 me 3存在于约800个基因的基因启动子处。这些基因在很大程度上是沉默的， 富含与肿瘤发生相关的途径。此外，我们进行了一个shRNA筛选，以确定 当耗尽时抑制DIPG细胞增殖的基因。我们发现Wnt 5a，一种参与 非经典Wnt信号通路，抑制DIPG细胞增殖。基于这些令人兴奋的结果，我们 假设H3.3M27突变蛋白重编程表观遗传状态和基因表达是一个关键 调节子/途径，从而促进肿瘤发生。在这个建议中，我们将阐明分子 800个基因与H3 K27 me 3的作用机制，并确定H3 K27 me 3在多大程度上“获得”， 这些基因促进肿瘤发生。此外，我们还将阐明Wnt 5a Wnt 5a信号通路的几个组分是DIPG细胞增殖所必需的， 假设抑制Wnt 5a信号通路的组分是未来的可行方法， DIPG治疗。总之，拟议的研究不仅会发现肿瘤发生的分子基础， 同时也为这种致命疾病的分子靶向治疗奠定了基础。

Diffuse intrinsic pontine glioma (DIPG) is an aggressive primary brain tumor found exclusively in children. The median survival for DIPG patients is about one year from diagnosis, with no treatment in sight. Recent studies have uncovered driver mutations for DIPG, specifically, somatic mutations in the H3F3A gene have been observed in majority of DIPG cases. H3F3A encodes histone H3 variant H3.3 that plays an important role in regulating gene expression during development. The predominant mutation at H3F3A leads to amino acid change at lysine (K) 27 residue of H3.3 to methionine (M) (H3K27M). In human cells, there are two genes that encode H3.3, which is assembled into nucleosomes in a replication-independent nucleosome assembly pathway. In addition, there are 13 genes encoding canonical histones H3.1 and H3.2, which differ from H3.3 by 4 or five amino acids and are assembled into nucleosomes in a replication-coupled process. Lysine 27 is conserved among all these histone H3 proteins. Therefore, it is unknown how H3.3 K27M mutation, which occurs at one allele of H3F3A gene, promotes tumorigenesis. H3K27 is modified post-translationally by either acetylation or methylation. H3K27 methylation is catalyzed by the PRC2 lysine methyltransferase and plays an important role in gene silencing during stem cell differentiation and maintenance. We and others have shown that a global loss of H3K27 methylation occurs in DIPG cells containing K27M mutation, which is due to inhibition of PRC2 enzymatic activity by H3.3M27 mutant proteins. In addition to the global loss of H3K27me3 that is associated with gene activation, we also observed that H3K27me3 is present at the gene promoters of about 800 genes. These genes are largely silenced and are enriched in pathways associated with tumorigenesis. In addition, we performed an shRNA screen to identify genes that when depleted inhibit the proliferation of DIPG cells. We found that Wnt5a, a protein involved in non-canonical Wnt signaling pathway, inhibits proliferation of DIPG cells. Based on these exciting results, we hypothesize that H3.3M27 mutation proteins reprogram epigenetic states and gene expression of a key regulator(s)/pathway(s) and thereby promotes tumorigenesis. In this proposal, we will elucidate the molecular mechanism whereby the 800 genes with H3K27me3 and determine to what extent “gain” of H3K27me3 at these genes promotes tumorigenesis. In addition, we will elucidate the molecular mechanisms whereby Wnt5a and several components of Wnt5a signaling pathway are required for the proliferation of DIPG cells and test the hypothesis that inhibition of components of Wnt5a signaling pathway is a viable approach for the future treatment of DIPG. Together, the proposed studies will not only discover the molecular basis of tumorigenesis of DIPG, but also lay the foundation for molecularly targeted therapies for this deadly disease.