Dysregulated transcription processes in Ewing sarcoma

尤文肉瘤中转录过程失调

基本信息

批准号：
10617256
负责人：
Alexander James Bishop
金额：
$ 41.81万
依托单位：
UNIVERSITY OF TEXAS HLTH SCIENCE CENTER
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-01 至 2026-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10617256
关键词：
Acute Alternative Splicing BRCA1 Protein Binding Biological Assay Biology Cell Cycle Cell Cycle Progression Cell physiology Cells ChIP-seq Chemicals Childhood Chimeric Proteins DNA Damage DNA Repair Data Data Analyses Defect EWSR1 gene Ewings sarcoma FLI1 gene Genes Genetic Genetic Transcription Genome Genome Stability Genomic Instability Genomics Goals Growth Impairment Intervention Lesion Link Location Malignant Childhood Neoplasm Malignant Neoplasms Measures Modeling Molecular Monitor Nature Oncogenes Paper Pathologic Pathway interactions Pharmacologic Substance Phenotype Physiological Process Proteins Publishing RNA RNA Interference RNA Polymerase II RNA Processing RNA Splicing RNA helicase A RNA metabolism Reporter Reporting Resolution Role SETX gene Site Small Interfering RNA Techniques Testing Text Therapeutic Time Toxic effect Transcription Process Uridine Work cDNA Expression cell type comparison control efficacy testing fitness genome-wide global run on sequencing helicase in vivo inhibitor insight interest prevent programs protein function replication stress response single cell sequencing stem cells targeted treatment transcriptome sequencing tumor tumor growth tumor xenograft young adult

项目摘要

Abstract Ewing sarcoma (EwS) is a pediatric and young adult cancer that is driven by the EWSR1-FLI1 translocation. Despite decades of work, this cancer is still an enigma, with poorly understood biology and no targeted treatments. Our recent work published in Nature demonstrated a previously overlooked consequence of EWSR1-FLI1, that this fusion causes hyperphosphorylated RNA polymerase II (pRNAPII) due to loss of EWSR1 inhibition of CDK7 and CDK9. We observed high levels of transcription, with high levels of R-loops present in locations that R-loops normally (physiologically) occur. Based upon these findings, we began to reconsider cellular phenotypes of EwS to identify the molecular basis of these phenotypes and ask whether these changes provide a fundamental defect in all EwS. One phenotype that was previously identified in EwS is that these cells display altered splicing profiles. In recent years there were several reports linking R-loops to splicing, with splicing defects causing R-loop accumulation and R-loops being associated with sites of alternative splicing. Further, it was reported that the splicing machinery inhibits DHX9 (aka RNA helicase A; RHA) from causing accumulation of toxic R-loops. Also, of interest, is that EWSR1-FLI1 interacts with and impairs DHX9 activity. By performing a genomic RNAi viability screen, we determined that EwS is acutely sensitive to a loss of RNA processing capability. These collective observations led us to the hypothesis that Ewing sarcoma is dependent upon RNA processing machinery to prevent accumulation of toxic R- loops. If our hypothesis is correct, then it suggests that there may be a therapeutic opportunity to target splicing components, converting the high levels of physiological R-loops in EwS into pathological R-loops to drive toxic genomic instability. We propose to test our hypothesis with two Aims. In Aim 1, we will investigate the mechanistic relationship between transcription levels, R-loops and splicing in EwS. For this we will modulate splicing components by siRNA depletions, cDNA expression or use of pharmaceutical inhibitors, examining transcription activity (Gro-Seq and uridine incorporation), splicing (reporters and RNA-Seq analysis) and R-loops (DRIP-Seq). In Aim 2, we will examine whether EwS is particularly reliant on splicing components or RNA:DNA helicases to block toxic conversion of R-loops and how targeting these processes impacts EwS viability, DNA damage response and/or cell cycle progression. We will ask if these modulations effect EwS cells at a particular time during cell cycle or stem cell state using single cell sequencing techniques. We will also assess how these various components of R-loop biology interact with one another, with pRNAPII and with R-loops in EwS. Finally, based upon these results, we will extend our findings to test efficacy of removing the R-loop metabolizing program that EwS is most reliant upon as a means to inhibit EwS tumor growth. Overall, this work should provide critical insight into the biology of Ewing sarcoma and provide new avenues for treatment beyond the standard chemotherapeutics currently used.

抽象的 Ewing肉瘤（EWS）是由EWSR1-FLI1易位驱动的儿科和年轻成人癌症。尽管工作了数十年，但这种癌症仍然是一个谜，知识渊博，没有针对性治疗。我们在自然界发表的最新作品证明了以前被忽视的结果 eWSR1-FLI1，由于损失的 EWSR1抑制CDK7和CDK9。我们观察到高水平的转录，R-loops高水平存在于正常（生理上）的R-Loops的位置。根据这些发现，我们开始重新考虑EWS的细胞表型，以识别这些表型的分子基础，并询问是否是否这些变化在所有EWS中都提供了根本缺陷。以前在EWS中鉴定的一种表型是这些单元显示改变的剪接曲线。近年来，有几份报道将R环与拼接，带有剪接缺陷，导致R环的积累，R环与替代剪接。此外，据报道，剪接机械抑制了DHX9（又称RNA解旋酶A； rha）引起有毒的R环的积累。同样，有趣的是，ewsr1-fli1与损害DHX9活动。通过执行基因组RNAi生存力屏幕，我们确定EWS是敏锐的对RNA处理能力的丧失敏感。这些集体观察使我们提出了这样的假设尤因肉瘤取决于RNA加工机制，以防止有毒R-积累循环。如果我们的假设是正确的，则表明可能有一个治疗机会来靶向剪接组件，将EWS中的高水平的R-Loops转化为病理R-loops 驱动有毒的基因组不稳定性。我们建议用两个目标检验我们的假设。在AIM 1中，我们将调查 EWS中转录水平，R环和剪接之间的机械关系。为此，我们将通过siRNA耗竭，cDNA表达或药物抑制剂的使用来调节剪接成分，检查转录活性（GRO-SEQ和URIDINE掺入），剪接（记者和RNA-Seq分析）和R-loops（Drip-Seq）。在AIM 2中，我们将检查EWS是否特别依赖剪接组件或RNA：DNA解旋酶阻断R环的有毒转化率以及靶向这些过程如何影响EWS 生存力，DNA损伤反应和/或细胞周期进程。我们将询问这些调制是否影响EWS 使用单细胞测序技术，在细胞周期或干细胞状态的特定时间细胞。我们将还应评估R-loop Biology的这些各个组成部分如何与Prnapii相互相互作用，并与 EWS中的R-loops。最后，根据这些结果，我们将扩展我们的发现以测试删除的功效 EWS最依赖的R环代谢程序是抑制EWS肿瘤生长的一种手段。全面的，这项工作应提供对尤因肉瘤生物学的批判性见解，并为当前使用的标准化学治疗剂以外的治疗。