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Mechanisms and therapeutic evaluation of splicing modulation for cohesin-mutant myelodysplastic syndromes

粘连蛋白突变型骨髓增生异常综合征剪接调节的机制和治疗评估

基本信息

批准号：
10612713
负责人：
Emily Wheeler
金额：
$ 2.92万
依托单位：
DANA-FARBER CANCER INST
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-09-01 至 2023-01-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10612713
关键词：
Alternative Splicing Area Binding Biological Bone marrow failure Cell Line Cells Chromatin Loop Clinical Trials Clonal Expansion Clone Cells Clustered Regularly Interspaced Short Palindromic Repeats Complex DNA Damage Dana-Farber Cancer Institute Data Development Diagnosis Disease Disease Progression Dysmyelopoietic Syndromes Enhancers Frameshift Mutation Genes Genetic Transcription Goals Hematopoiesis Hematopoietic stem cells Immunoprecipitation In Vitro Ineffective Hematopoiesis Institution Lead Lesion Link Mass Spectrum Analysis Mediating Modeling Molecular Mutate Mutation Outcome Patients Pharmaceutical Preparations Phenotype Principal Investigator Process Proteins RNA RNA Binding RNA Splicing Resources Running Site Spliceosomes Testing Therapeutic Time Training United States Work base clinical development clinically significant cohesin crosslink differential expression disease phenotype efficacy testing high risk in vivo in vivo Model in vivo evaluation insight interest loss of function mutation mouse model mutant mutant mouse model novel therapeutics patient subsets promoter protein TDP-43 research clinical testing targeted treatment therapeutic evaluation therapeutic target therapy development transcriptome sequencing

项目摘要

PROJECT SUMMARY Myelodysplastic syndromes (MDS) are the most common form of bone marrow failure disorders with more than 30,000 new cases diagnosed each year in the United States. These disorders are characterized by ineffective hematopoiesis as a result of mutations acquired in the hematopoietic stem and progenitor cells that clonally expand over time. Mutations in the cohesin complex, most commonly STAG2, are found in the high-risk subset of MDS patients with poor overall survival and for whom there are currently no targeted therapeutic options. Therefore, it is critical to gain a better understanding of the cellular mechanisms that drive cohesin-mutant MDS which can be therapeutically targeted. Our preliminary work has revealed that under normal conditions, the cohesin complex interacts with the spliceosome through an RNA-intermediate. Interestingly, this interaction is lost upon mutation of STAG2 in cells. Furthermore, STAG2-mutant cells are selectively killed compared to wild- type when treated with splicing modulators that target the SF3B complex, drugs that are currently undergoing clinical testing for splicing-factor mutant MDS. Based on our preliminary work, our central hypothesis is that regulatory RNAs mediate the interaction between cohesin and the spliceosome at enhancer-promoter loops that are either lost or altered in cohesin-mutant cells. Furthermore, we believe the loss of this interaction renders cohesin-mutant cells more sensitive to splicing modulation than normal, healthy cells. The overall objective of this work is to fully characterize the RNA and protein components that allow cohesin to interact with the spliceosome and test the in vivo efficacy of splicing modulation in cohesin-mutant MDS mouse models. In Aim1, we will determine the RNAs that mediate the interaction between cohesin and the spliceosome that are lost in cohesin-mutant MDS. We will perform enhanced cross-linking immunoprecipitation (eCLIP) on the cohesin complex to identify bound RNAs and use precision run-on sequencing (PRO-Seq) and total RNA-Seq to quantify both nascent and steady-state levels of bound RNAs in wild-type and STAG2-mutant cells. In Aim2, we will quantify the alternative splicing burden observed in cohesin-mutant MDS mouse models and determine the efficacy of splicing modulators to reverse disease phenotypes and expansion of mutant clones in vivo. Our in vivo model develops MDS phenotypes upon sequential acquisition of Tet2 and Stag2 mutations, a process that mimics disease progression in patients. Our long-term goal is to contribute to a mechanistic understanding of how the interaction between cohesin and the spliceosome is disrupted in disease and potentially offer cohesin- mutant MDS patients a new therapeutic option of splicing modulation. This work will be carried out under the guidance of Dr. Zuzana Tothova and Dr. Ben Ebert in the Dana-Farber Cancer Institute, with our local collaborators Dr. Karen Adelman and Dr. Chris Burge. Together, this team represents experts in all scientific areas necessary to complete this work in a world-class training institution with an abundance of resources to carry out high-quality scientific work with clinical significance.

项目概要骨髓增生异常综合征（MDS）是最常见的骨髓衰竭疾病，其发病率超过美国每年诊断出 30,000 例新病例。这些疾病的特点是无效造血干细胞和祖细胞中获得的突变导致造血作用随着时间的推移而扩大。粘连蛋白复合体中的突变（最常见的是 STAG2）出现在高风险子集中总体生存率较差且目前尚无针对性治疗选择的 MDS 患者。因此，更好地了解驱动粘连蛋白突变 MDS 的细胞机制至关重要可以作为治疗目标。我们的前期工作表明，在正常情况下，粘连蛋白复合物通过 RNA 中间体与剪接体相互作用。有趣的是，这个交互是细胞中 STAG2 突变后丢失。此外，与野生细胞相比，STAG2 突变细胞被选择性杀死。使用针对 SF3B 复合物的剪接调节剂治疗时的类型，目前正在接受的药物剪接因子突变MDS的临床测试。根据我们的初步工作，我们的中心假设是调节性 RNA 介导增强子-启动子环处的粘连蛋白和剪接体之间的相互作用在粘连蛋白突变细胞中要么丢失或改变。此外，我们相信这种相互作用的丧失会导致粘连蛋白突变细胞比正常健康细胞对剪接调节更敏感。总体目标这项工作是为了充分表征 RNA 和蛋白质成分，使粘连蛋白与剪接体并测试粘连蛋白突变 MDS 小鼠模型中剪接调节的体内功效。在目标 1 中，我们将确定介导粘连蛋白和剪接体之间相互作用的RNA，这些RNA在粘连蛋白突变MDS。我们将对粘连蛋白进行增强交联免疫沉淀 (eCLIP) 复杂的方法来识别结合的 RNA，并使用精确连续测序 (PRO-Seq) 和总 RNA-Seq 进行定量野生型和 STAG2 突变细胞中结合 RNA 的新生和稳态水平。在目标2中，我们将量化粘连蛋白突变 MDS 小鼠模型中观察到的选择性剪接负担，并确定剪接调节剂逆转疾病表型和体内突变克隆扩增的功效。我们在体内模型在连续获得 Tet2 和 Stag2 突变后形成 MDS 表型，这一过程模拟患者的疾病进展。我们的长期目标是促进对机械的理解粘连蛋白和剪接体之间的相互作用如何在疾病中被破坏并可能提供粘连蛋白- 突变MDS患者的剪接调节新的治疗选择。这项工作将在达纳法伯癌症研究所 Zuzana Tothova 博士和 Ben Ebert 博士的指导，以及我们当地的合作者：Karen Adelman 博士和 Chris Burge 博士。该团队共同代表了所有科学领域的专家在拥有丰富资源的世界一流培训机构中完成这项工作所需的领域开展高质量的具有临床意义的科研工作。

项目成果

期刊论文数量（2）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Genetic analysis of cancer drivers reveals cohesin and CTCF as suppressors of PD-L1.

DOI：
10.1073/pnas.2120540119
发表时间：
2022-02-15
期刊：
Proceedings of the National Academy of Sciences of the United States of America
影响因子：
11.1
作者：
Oreskovic E;Wheeler EC;Mengwasser KE;Fujimura E;Martin TD;Tothova Z;Elledge SJ
通讯作者：
Elledge SJ