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

• 批准号: 10313199
• 负责人: Emily Wheeler
• 金额: $ 6.6万
• 依托单位: DANA-FARBER CANCER INST
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10313199
• 关键词: Mechanisms; therapeutic; evaluation; splicing; modulation

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.

项目摘要 骨髓增生异常综合征（MDS）是骨髓衰竭病症的最常见形式， 美国每年确诊3万例新病例。这些疾病的特点是无效的 造血干细胞和祖细胞中获得的突变的结果， 随着时间的推移而扩大。在高风险亚群中发现了粘附素复合物的突变，最常见的是STAG 2 MDS患者的总体生存率很低，目前还没有针对性的治疗选择。 因此，更好地了解细胞机制是至关重要的， 其可以是治疗靶向的。我们的初步工作表明，在正常情况下， 粘着蛋白复合物通过RNA中间体与剪接体相互作用。有趣的是，这种相互作用 在细胞中STAG 2突变后丢失。此外，与野生型相比，STAG 2突变型细胞被选择性地杀死。 当用靶向SF 3B复合物的剪接调节剂治疗时， 剪接因子突变型骨髓增生异常综合征的临床检测根据我们的初步工作，我们的中心假设是， 调节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， 在粘着蛋白突变细胞中丢失或改变。此外，我们认为，这种相互作用的丧失使 粘着蛋白突变细胞对剪接调节比正常健康细胞更敏感。的总体目标 这项工作是为了充分表征RNA和蛋白质成分，使粘蛋白相互作用， 剪接体和测试剪接调节在粘着蛋白突变型MDS小鼠模型中的体内功效。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 黏连蛋白突变型MDS。我们将对粘附素进行增强交联免疫沉淀（eCLIP）， 复杂的识别结合的RNA，并使用精确运行测序（PRO-Seq）和总RNA-Seq来定量 在野生型和STAG 2突变细胞中结合RNA的新生和稳态水平。在AIM 2中，我们将 量化在粘附素突变型MDS小鼠模型中观察到的可变剪接负荷，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 体内模型在相继获得Tet 2和Stag 2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 粘蛋白和剪接体之间的相互作用如何在疾病中被破坏，并可能提供粘蛋白- 突变型MDS患者的剪接调节的新的治疗选择。这项工作将在 在丹娜-法伯癌症研究所的Zuzana Tothova博士和Ben Ebert博士的指导下， Karen Adelman博士和Chris Burge博士。这个团队代表了所有科学领域的专家 在世界一流的培训机构完成这项工作所需的领域， 开展具有临床意义的高质量科研工作。