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.

项目摘要/摘要 骨髓增生综合征（MDS）是克隆造血干细胞疾病，其特征是无效 造血，周围血细胞质减少症和急性白血病的风险增加。治疗性 目前，针对MDS患者的新药的期权和开发新药非常有限，而且有一个很好的 需要新的治疗靶标。 大规模测序研究最近发现的主要发现是，超过一半的MDS患者藏有 编码剪接因子（SFS）的基因突变，最常见的突变为：剪接因子 3B，亚基1（SF3B1），丝氨酸/精氨酸富含剪接因子2（SRSF2）和U2小核RNA辅助 因子1（U2AF1）。 SF突变是MD中最常见的突变类别，并且在课程初期发生 疾病。这些强烈表明SF突变是MD发病机理的关键，并且很可能 提供新的治疗机会。但是，它们驱动疾病的机制不是 理解。这些突变是杂合的“热点”突变，强烈暗示增益或 功能机制的改变 - 通过最近的生化研究证实了RNA结合的改变 突变体SF的特异性。他们的相互排他性提供了另一个线索，因为它表明了收敛性 在一个或几个下游目标中。确定这些目标可能对MD具有巨大影响，但 由于原代患者样本的细胞和遗传异质性和 物种之间基因同工型的差异。 我们（Papapetou Laboratory）已经开发了MD和MD的第一个诱导的多能干细胞（IPSC）模型 提供了用于研究疾病遗传机制的原理证明。对于电流 提案，我们用SRSF2 P95L突变得出了等源IPSC线，并表明造血 源自它们的细胞捕获与疾病相关的表型和突变体的RNA结合亲和力改变 SRSF2。我们（YEO实验室）已经开发了一种增强的夹式方法（Eclip），并将其用于 在初步实验中首次表征突变体SRSF2的直接RNA结合。在这个 多个PI应用我们将联合起来确定可能的SF突变的常见下游效应 构成承诺的治疗靶标。提出的研究，利用了独特的专业知识 MDS的IPSC建模中的Papapetou Lab，结合RNA中YEO实验室的广泛专业知识 生物学和基因组学将对具有SF突变的MD的发病机理产生新的见解并识别 药物开发的新治疗靶标。