Dissecting splicing factor mutations in iPSCs

项目摘要/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、外周血细胞减少和进展为急性白血病的风险增加。治疗性的 目前用于MDS患者的新药选择和开发非常有限，有很大的 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 剪接因子编码基因的突变，最常见的3种突变是：剪接因子 3B、亚基1(SF3B1)、富含丝氨酸/精氨酸的剪接因子2(SRSF2)和辅助的U2小核RNA 因子1(U2AF1)。SF突变是MDS中最常见的一类突变，发生在病程的早期 这种疾病的危害。这些都强烈地表明SF突变是MDS发病机制的关键，并且很可能 提供新的治疗机会。然而，它们驱动疾病的机制并不是 明白了。这些突变是杂合的“热点”突变，这强烈地表明了一种获得或 作用机制的改变-最近的生化研究表明RNA结合的改变证实了这一点 突变型SFS的特异性。它们的相互排他性提供了另一条线索，因为它暗示着趋同 在一个或几个下游目标。确定这些目标可能会对MDS产生巨大影响，但 由于原发患者样本的细胞和遗传异质性以及 不同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，并 为研究疾病的遗传机制提供了使用它们的原则证据。就目前而言 建议，我们已经获得了具有SRSF2 P95L突变的等基因IPSC系，并表明 从它们获得的细胞捕获与疾病相关的表型和突变的RNA结合亲和力改变 SRSF2.我们(Yeo实验室)开发了一种增强的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 多个PI应用我们将联合起来识别SF突变的常见下游影响，这些突变可能 构成了有希望的治疗靶点。拟议的研究，利用了 Papapetrou实验室对MDS进行IPSC建模，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，将对带有SF突变的MDS的发病机制产生新的见解，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.