MECHANISMS OF POL II ELONGATION IN DIFFUSE MIDLINE GLIOMA

项目摘要/摘要 弥漫性中线胶质瘤(DMG)是儿童和成人的侵袭性脑瘤，没有有效的治疗方法。 治疗。80%以上的儿童DMG和大多数成人DMG存在组蛋白非同义突变 H3(H3.3K27M和H3.1K27M)，导致染色质失调，导致脑内Pol II转录异常 肿瘤与正常组织相比。转录异常不仅是肿瘤发生的一个原因，而且是一种 可药物的脆弱性，因为有多项研究调查POL II和表观基因组靶向的效用 治疗癌症的药物。尽管最初承诺以转录失调为靶点的化合物 作为潜在的癌症治疗方法，临床试验中正在研究的药物没有一种能改善DMG患者的病情 到目前为止幸存下来。发现更有效、毒性更低的DMG治疗方法的一个关键障碍是我们缺乏 H3K27M突变组蛋白与肿瘤相关的分子通路的功能知识 开始和进展。我们最近进行了CRISPR筛查，确定了POL II的多个调节者 H3K27M突变细胞中转录延长的遗传依赖性，包括Elongin B(ELOB)及其 结合伙伴，冯·希佩尔-林道病蛋白和肿瘤抑制因子，VHL。进一步的初步研究 揭示了ELOB的遗传消融改变了H3K27M突变组蛋白的染色质结合模式，并且 干扰DMG基因的表达。这些数据支持我们的总体假设，即H3K27M突变组蛋白 特异性基因调控复合体诱导Pol II转录失控和触发DMG的活性 肿瘤发生学。 我们广泛的、长期的目标是确定驱动胶质瘤特异性的基因和分子通路 转录程序，并确定它们在脑癌发生和发展中的作用。我们建议 具体目的如下：1)建立Pol II延长调节剂在临床前小鼠模型中的作用 DMG和研究延伸机与H3K27M之间的遗传互作；2)应用刀刃 下一代测序分析剖析Pol II调节蛋白和Pol II之间的串扰机制 H3K27M，并确定它们在促进DMG基因调控异常中的作用；3)调查一个意外的 Elongin/VHL复合体在促进恶性基因表达模式和DMG肿瘤中的致癌作用 成长。这项工作的意义在于，这些研究将揭示H3K27M如何致癌的新见解 组蛋白共同选择特定的Pol II调节器来促进肿瘤的发生，并将揭示分子机制 在DMG和其他癌症中驱动染色质异常和基因调控。

Project Summary/Abstract Diffuse midline gliomas (DMGs) are aggressive brain tumors in both children and adults with no effective therapies. Over 80% of pediatric DMG and a majority of adult DMG harbor non-synonymous mutations in histone H3 (H3.3K27M and H3.1K27M), which dysregulate chromatin and result in aberrant Pol II transcription in brain tumors compared to normal tissue. Aberrant transcription is not only a cause of tumorigenesis, but also a druggable vulnerability as there are multiple studies investigating the utility of Pol II and epigenome-targeting drugs for the treatment of cancer. Despite the initial promise of compounds targeting transcriptional dysregulation as potential cancer therapies, none of the drugs under investigation in clinical trials have improved DMG patient survival thus far. A critical barrier to the discovery of more effective and less toxic therapies for DMG is our lack of functional knowledge regarding the molecular pathways connecting H3K27M mutant histones to tumor initiation and progression. We have recently conducted CRISPR screens identifying multiple regulators of Pol II transcriptional elongation as genetic dependencies in H3K27M mutant cells, including Elongin B (ELOB), and its binding partner, the Von Hippel-Lindau disease protein and tumor suppressor, VHL. Further preliminary studies reveal that genetic ablation of ELOB alters the chromatin binding patterns of H3K27M mutant histones, and disrupts DMG gene expression. These data support our overall hypothesis that H3K27M mutant histones enlist the activity of specific gene regulatory complexes to induce Pol II transcriptional deregulation and trigger DMG tumorigenesis. Our broad, long-term goal is to identity genes and molecular pathways driving glioma-specific transcriptional programs and to determine their roles in brain cancer development and progression. We propose the following specific aims: 1) Establish roles for Pol II elongation regulators in pre-clinical mouse models of DMG and investigate genetic interactions between the elongation machinery and H3K27M; 2) Apply cutting edge next generation sequencing analyses to dissect mechanisms of crosstalk between Pol II regulatory proteins and H3K27M and determine their roles in promoting abnormal gene regulation in DMG; 3) Investigate an unexpected oncogenic role for Elongin/VHL complexes in promoting malignant gene expression patterns and DMG tumor growth. The significance of this work is that these studies will reveal new insights into how H3K27M oncogenic histones co-opt specific Pol II regulators to promote tumorigenesis, and will uncover molecular mechanisms driving aberrant chromatin and gene regulation in DMG and other cancers.