Targeting Transcriptional Elongation in Pediatric Glioma

项目总结/摘要-弥漫性内在脑桥胶质瘤（DIPG）是最具破坏性的 儿科癌症几十年来的大量临床试验，涉及不同的化疗药物组合， 药物和放射治疗在治疗DIPG中无效。有效治疗靶点的确定 基于分子特征的治疗对于改善儿童的治疗结果非常重要， DIPG。DIPG中致癌组蛋白基因突变的发现极大地提高了我们的理解 疾病的发病机制，并刺激了新的治疗方法的发展，目标表观遗传 修饰语我们最近表明，靶向溴和额外末端（BET）结构域蛋白4（BRD 4） 使用JQ 1抑制剂的活性导致肿瘤进展的显著延迟和动物存活的延长 携带DIPG患者来源的异种移植物（PDX）。由于其有希望的抗肿瘤活性，BRD 4抑制剂 正在许多癌症患者的临床试验中进行测试。然而，最初对小细胞肺癌有反应的肿瘤 分子抑制剂疗法，如靶向BRD 4活性的那些，最终对单一疗法产生抗性 治疗，肯定需要更有效的治疗干预。为了确定新的有效 治疗目标，并发现新的组合方法，以防止或延迟获得性耐药性， 在单药治疗中，我们对患者来源的肿瘤细胞进行了无偏倚的全基因组CRISPR/Cas9基因筛查。 DIPG细胞。我们确定了9个在CRISPR靶点中显著富集的“网络模块”。其中一 模块包括POLR 2 I，其编码参与转录的RNA聚合酶II（Pol II）的亚基 伸长率我们随后观察到，通过短发夹RNA敲低靶向POLR 2 I活性， 小分子Pol II抑制剂的处理阻断转录延伸并抑制DIPG的生长 在体外和体内。在这里，我们将测试抑制Pol II转录延伸的假设， 与BRD 4抑制的组合将进一步抑制基因转录，并将延迟或预防DIPG 对单一疗法产生抗药性这种双重抑制方法将干扰基因转录， 两个水平：转录起始（BRD 4）和延伸（Pol II）。该项目还将探讨这些 靶向疗法在治疗DIPG中与放射相互作用，这是重要的，因为在治疗DIPG中使用放射。 几乎所有儿童DIPG病例。成功完成提案研究对以下方面有重大影响： 因此，临床实践和积累本研究的数据可以为早期临床研究奠定基础。 鉴于DIPG的高未满足需求和孤儿病状态，该方法的临床试验。

PROJECT SUMMARY/ABSTRACT - Diffuse intrinsic pontine glioma (DIPG) is one of the most devastating pediatric cancers. Numerous clinical trials in decades, involving different combinations of chemotherapeutic agents and radiation, have been ineffective in treating DIPG. The identification of efficacious therapeutic targets based on the molecular characteristic is of high importance for improving treatment outcomes for children with DIPG. The discovery of oncogenic histone gene mutations in DIPG has dramatically improved our understanding of disease pathogenesis, and stimulated the development of novel therapeutic approaches to target epigenetic modifiers. We have recently shown that targeted bromo- and extra-terminal (BET) domain protein 4 (BRD4) activity using JQ1 inhibitor results in a significant delay of tumor progression and prolonged survival of animals bearing DIPG patient-derived xenograft (PDX). Because of their promising anti-tumor activity, BRD4 inhibitors are being tested in a number of cancer patient clinical trials. However, tumors that initially respond to small molecule inhibitor therapies, such as those targeting BRD4 activity, eventually become resistant to monotherapy treatment, affirming the need for more effective therapeutic interventions. In order to identify new effective therapeutic targets, and discover novel combinatorial approaches to prevent or delay acquired resistance to monotherapy, we performed an unbiased genome-wide CRISPR/Cas9-based genetic screen of patient-derived DIPG cells. We identified nine “network modules” that are significantly enriched in CRISPR targets. One of these modules includes POLR2I, which encodes a subunit of RNA polymerase II (Pol II) that is involved in transcription elongation. We subsequently observed that targeting POLR2I activity through short-hairpin RNA knockdown and treatment of the small-molecule Pol II inhibitors block transcriptional elongation and inhibit the growth of DIPG in vitro and in vivo. Here, we will test the hypothesis that inhibition of Pol II transcriptional elongation in combination with BRD4 inhibition will further suppress gene transcription and will either delay or prevent DIPG from acquiring resistance to monotherapy. This dual inhibition approach will interfere with gene transcription at two levels: transcriptional initiation (BRD4) and elongation (Pol II). This project will also explore how these targeted therapies interact with radiation in treating DIPG, which is important due to the use of radiation in treating nearly all cases of DIPG in children. The successful completion of proposal study has significant impact on clinical practice and accumulating data from this research could therefore lay the foundation for early clinical trials of this approach, given the high unmet need and orphan disease status of DIPG.