Dissecting splicing factor mutations in iPSCs

项目总结/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，其特征是无效的造血干细胞移植。 造血、外周血细胞减少和进展为急性白血病的风险增加。治疗 用于MDS患者的新药的选择和开发目前非常有限， 需要新的治疗靶点。 最近大规模测序研究的一个主要发现是，超过一半的MDS患者携带有 编码剪接因子（SF）的基因突变，其中3种最常见的突变是：剪接因子 3B，亚基1（SF 3B 1），富含丝氨酸/丝氨酸的剪接因子2（SRSF 2）和U2小核RNA辅助 因子1（U2 AF 1）。SF突变是MDS中最常见的一类突变，发生在病程早期， 疾病。这些结果强烈提示SF突变是MDS发病机制的关键， 提供新的治疗机会。然而，它们驱动疾病的机制并不 明白这些突变是杂合的“热点”突变，这强烈表明获得或减少了基因突变。 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SF的特异性。它们的相互排斥提供了另一条线索，因为它表明了趋同 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， 由于原始患者样本的细胞和遗传异质性， 物种间基因异构体的差异。 我们（Papapetrou实验室）已经开发了第一个MDS的诱导多能干细胞（iPSC）模型， 提供了它们用于研究疾病遗传机制的原理证明。为当前 根据这一提议，我们已经衍生出具有SRSF 2 P95 L突变的同基因iPSC系，并表明造血干细胞的表达与SRSF 2 P95 L突变无关。 来源于它们的细胞捕获疾病相关的表型和突变体的改变的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 在初步实验中首次表征突变体SRSF 2的直接RNA结合。在这 多个PI应用程序，我们将联手确定SF突变的常见下游效应， 构成有希望的治疗靶点。拟议的研究利用了 Papapetrou实验室在MDS的iPSC建模方面，结合Yeo实验室在RNA方面的广泛专业知识 生物学和基因组学，将产生新的见解MDS的发病机制与SF突变，并确定 药物开发的新治疗靶点。

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.