Targeting Transcriptional Elongation in Pediatric Glioma

弥漫性桥脑胶质瘤(DIPG)是最具破坏性的肿瘤之一。 儿科癌症。几十年来的无数临床试验，涉及不同的化疗组合 药物和放射治疗在治疗DIPG方面一直无效。有效治疗靶点的确定 基于分子特征的研究对改善儿童白血病的治疗结果具有重要意义 DIPG。DIPG致癌性组蛋白基因突变的发现极大地提高了我们对 疾病发病机制的研究，并刺激了针对表观遗传学的新的治疗方法的发展 修饰符。我们最近发现靶向溴和末端外(BET)结构域蛋白4(BRD4) 使用JQ1抑制剂的活性可显著延缓肿瘤进展并延长动物存活时间 轴承DIPG患者来源的异种移植物(PDX)。由于具有良好的抗肿瘤活性，BRD4抑制剂 正在一些癌症患者的临床试验中进行测试。然而，最初对小肿瘤有反应的肿瘤 分子抑制疗法，如那些针对BRD4活性的疗法，最终会对单一疗法产生抗药性 治疗，确认需要更有效的治疗干预措施。为了找出新的有效 治疗靶点，并发现预防或延缓获得性抗药性的新组合方法 单一疗法，我们进行了基于CRISPR/Cas9的患者来源的全基因组无偏基因筛查 DIPG细胞。我们确定了在CRISPR目标中显著丰富的九个“网络模块”。这其中的一个 模块包括POLR2I，它编码参与转录的RNA聚合酶II(POL II)的一个亚单位 伸长率。我们随后观察到，通过短发夹状RNA敲除和 小分子Pol II抑制剂阻断DIPG转录延长并抑制其生长 在体外和体内。在这里，我们将测试假设，抑制POL II转录延长在 与抑制BRD4结合将进一步抑制基因转录，并将延缓或阻止DIPG 从获得抵抗力到单一疗法。这种双重抑制方式将干扰基因转录。 两个水平：转录起始(BRD4)和延伸(POL II)。本项目还将探索这些 靶向治疗与放射治疗DIPG相互作用，这一点很重要，因为在治疗中使用了放射治疗 几乎所有的儿童DIPG病例。成功完成提案研究对以下方面有重大影响 临床实践和从这项研究中积累的数据可以为早期临床奠定基础 考虑到DIPG的高度未得到满足的需求和孤儿疾病状况，对这种方法进行了试验。

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.