Dissecting splicing factor mutations in iPSCs

剖析 iPSC 中的剪接因子突变

• 批准号: 9893894
• 负责人: Eirini Papapetrou
• 金额: $ 78.89万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-05 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9893894
• 关键词: Acute leukemia; Affect; Affinity; Alleles; Biochemical; Biological Assay; Biology; Blood; Bone Marrow; C-terminal; CRISPR screen; CRISPR/Cas technology; Cell Differentiation process; Cell Line; Cell Proliferation; Cell Survival; Cells; Clonal Hematopoietic Stem Cell; Clustered Regularly Interspaced Short Palindromic Repeats; Development; Disease; Dysmyelopoietic Syndromes; Exhibits; Fractionation; Genes; Genetic; Genetic Heterogeneity; Genetic Predisposition to Disease; Genetic study; Genomics; Hematopoietic; Ineffective Hematopoiesis; Knock-out; Laboratories; Large-Scale Sequencing; Libraries; Mediating; Methods; Mutate; Mutation; Pathogenesis; Pathway interactions; Patients; Phenotype; Population; Protein Isoforms; RNA; RNA Binding; RNA Processing; RNA Splicing; RNA-Binding Proteins; Reporter; Risk; Role; SRSF2 gene; Sampling; Small Nuclear RNA; Specificity; System; Testing; Therapeutic; Time; Translations; U2 small nuclear RNA; cancer genome; crosslinking and immunoprecipitation sequencing; cytopenia; drug development; experimental study; hematopoietic differentiation; induced pluripotent stem cell; insight; knock-down; mRNA Stability; mutant; new therapeutic target; novel therapeutics; overexpression; peripheral blood; progenitor; ribosome profiling; small hairpin RNA; stem cell model; stem cells; therapeutic target; transcriptome sequencing

PROJECT SUMMARY/ABSTRACT Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis, peripheral blood cytopenias and increased risk of progression to acute leukemia. Therapeutic options and development of new drugs for patients with MDS are currently very limited and there is a great need for new therapeutic targets. A major recent discovery from large-scale sequencing studies was that over half of MDS patients harbor mutations in genes encoding splicing factors (SFs), with the 3 most commonly mutated being: splicing factor 3B, subunit 1 (SF3B1), serine/arginine-rich splicing factor 2 (SRSF2) and U2 small nuclear RNA auxiliary factor 1 (U2AF1). SF mutations are the most common class of mutations in MDS and occur early in the course of the disease. These strongly suggest that SF mutations are key to the pathogenesis of MDS and can likely provide new therapeutic opportunities. However the mechanisms by which they drive the disease are not understood. These mutations are heterozygous “hotspot” mutations, which strongly suggests a gain or alteration of function mechanism – corroborated by recent biochemical studies showing altered RNA binding specificities of the mutant SFs. Their mutual exclusivity provides yet another clue, as it suggests convergence in one or a few downstream targets. Identifying those targets could have tremendous implications for MDS, but presents a big challenge due to the cellular and genetic heterogeneity of primary patient samples and differences in gene isoforms among species. We (Papapetrou laboratory) have developed the first induced pluripotent stem cell (iPSC) models of MDS and provided proof-of-principle of their use for studying genetic mechanisms of the disease. For the current proposal, we have derived isogenic iPSC lines with the SRSF2 P95L mutation and shown that hematopoietic cells derived from them capture disease-relevant phenotypes and the altered RNA-binding affinity of mutant SRSF2. We (Yeo laboratory) have developed an enhanced CLIP-seq method (eCLIP) and used it to characterize for the first time in preliminary experiments the direct RNA binding of mutant SRSF2. In this multiple PI application we will join forces to identify common downstream effects of SF mutations that may constitute promising therapeutic targets. The proposed studies, which leverage the unique expertise of the Papapetrou lab in iPSC modeling of MDS, combined with the extensive expertise of the Yeo lab in RNA biology and genomics, will generate new insights into the pathogenesis of MDS with SF mutations and identify new therapeutic targets for drug development.

项目概要/摘要 骨髓增生异常综合征（MDS）是一种克隆性造血干细胞疾病，其特征是无效 造血功能、外周血细胞减少以及进展为急性白血病的风险增加。治疗性 目前针对MDS患者的新药选择和开发非常有限，而且还有很大的空间。 需要新的治疗靶点。 大规模测序研究最近的一项重大发现是，超过一半的 MDS 患者患有 编码剪接因子 (SF) 的基因发生突变，其中 3 种最常见的突变是： 剪接因子 3B，亚基 1 (SF3B1)，富含丝氨酸/精氨酸的剪接因子 2 (SRSF2) 和 U2 小核 RNA 辅助 因子 1 (U2AF1)。 SF 突变是 MDS 中最常见的一类突变，发生在病程早期 的疾病。这些强烈表明 SF 突变是 MDS 发病机制的关键，并且可能 提供新的治疗机会。然而，它们驱动疾病的机制并不明确。 明白了。这些突变是杂合的“热点”突变，强烈表明获得或 功能机制的改变——最近的生化研究证实了 RNA 结合的改变 突变体 SF 的特异性。它们的相互排他性提供了另一条线索，因为它表明了趋同性 一个或几个下游目标。确定这些目标可能对 MDS 产生巨大影响，但是 由于主要患者样本的细胞和遗传异质性，提出了巨大的挑战 物种间基因异构体的差异。 我们（Papapetrou 实验室）开发了第一个 MDS 的诱导多能干细胞 (iPSC) 模型， 提供了其用于研究疾病遗传机制的原理证明。对于当前的 根据建议，我们衍生出了具有 SRSF2 P95L 突变的同基因 iPSC 系，并表明造血功能 来自它们的细胞捕获与疾病相关的表型以及突变体改变的RNA结合亲和力 SRSF2。我们（Yeo 实验室）开发了一种增强型 CLIP-seq 方法（eCLIP），并将其用于 首次在初步实验中表征了突变体 SRSF2 的直接 RNA 结合。在这个 多个 PI 应用，我们将联手确定 SF 突变的常见下游影响，这些影响可能 构成有希望的治疗靶点。拟议的研究利用了独特的专业知识 Papapetrou 实验室进行 MDS 的 iPSC 建模，并结合 Yeo 实验室在 RNA 方面的丰富专业知识 生物学和基因组学，将对具有 SF 突变的 MDS 发病机制产生新的见解，并确定 药物开发的新治疗靶点。