Modeling Splicing Factor Mutations in MDS Using Human iPSCs

项目摘要 该项目的目标是研究剪接因子对细胞和分子的影响 (SF)骨髓增生异常综合征（MDS）中的突变。这些血液病的特点是无效的 造血表现为外周血细胞减少和异型增生形态学改变， 发展为急性白血病的倾向增加。最近，RNA结合蛋白的体细胞突变 在剪接中具有已知的作用，通常称为剪接因子（其中三种最常见的是 SF3B1 K700 E、U2AF1 S34 F和SRSF2 P95 L）被揭示为最常见的突变类型。 MDS影响超过一半的患者。SF突变发生在疾病的早期，因此， 对于其发病机制和有吸引力的治疗靶点至关重要。使用原代患者细胞的早期研究，小鼠 使用RNA-seq的细胞系模型和异位表达表明，突变改变了RNA结合， 突变SF的特异性。然而，对于疾病发病机制至关重要的下游事件 仍然是难以捉摸的，突变因子的RNA结合特异性还有待直接测试。人口 遗传学研究表明，与SF突变共同发生的不同突变与 不同的临床结果（不良或良好的预后）。DNMT3A与SF3B1突变的共现是 与良好的预后相关，而SF3B1与RUNX1或ASXL1的共突变导致不良的预后。 预后然而，这些遗传相互作用的机制尚不清楚。的 Papapetrou实验室率先使用诱导多能干细胞（iPSC）模型对MDS进行建模。 人类iPSC模型对于SF突变的研究特别有吸引力，因为选择性剪接 同种型在小鼠和人之间显示出较差的保守性。 我建议开发新的基于iPSC的SF突变和协作突变模型，以研究 这些突变及其在MDS发病机制中的遗传相互作用。具体而言，我建议：（1）生成 具有三种最常见的典型SF突变SRSF2 P95L、SF3B1 K700E和U2AF1的iPSC系 S34F，并使用RNA-seq和eCLIP在表型和分子上表征它们。 seq）和（2）产生在DNMT3A、RUNX1或RUNX2中具有额外突变的SF3B1 K700E iPSC系。 ASXL1，并表征其造血表型和转录组。这项工作可以产生新的 关于SF突变驱动MDS的机制的假说，并揭示了突变的原理。 MDS发病机制的协同性。

Project Summary The goal of the proposed project is to investigate the cellular and molecular consequences of splicing factor (SF) mutations in myelodysplastic syndromes (MDS). These blood diseases are characterized by ineffective hematopoiesis manifested as peripheral blood cytopenias and dysplastic morphological changes and increased propensity for progression to acute leukemia. Recently, somatic mutations in RNA binding proteins with known roles in splicing, commonly referred to as splicing factors (with the three most common being SF3B1 K700E, U2AF1 S34F and SRSF2 P95L), were revealed as the most common class of mutations in MDS affecting more than half of patients. SF mutations occur early in the disease and are, therefore, likely critical for its pathogenesis and attractive therapeutic targets. Early studies using primary patient cells, mouse models and ectopic expression in cell lines using RNA-seq suggest that the mutations alter the RNA binding specificity of the mutant SFs. However, the downstream events that are critical for the disease pathogenesis are still elusive and the RNA binding specificity of the mutant factors has yet to be directly tested. Population genetics studies have shown that different mutations co-occurring with SF mutations are associated with different clinical outcomes (poor or favorable prognosis). Co-occurrence of DNMT3A with SF3B1 mutations is associated with good prognosis, whereas co-mutations of SF3B1 with either RUNX1 or ASXL1 confer poor prognosis. However, the mechanisms underlying these genetic interactions are not understood. The Papapetrou lab has pioneered the modeling of MDS using induced pluripotent stem cell (iPSC) models. Human iPSC models are particularly attractive for the investigation of SF mutations, as alternative splicing isoforms show poor conservation between mice and humans. I propose to develop novel iPSC-based models of SF mutations and cooperating mutations to study the role of these mutations and their genetic interactions in MDS pathogenesis. Specifically, I propose to: (1) generate iPSC lines with the three most common canonical SF mutations SRSF2 P95L, SF3B1 K700E and U2AF1 S34F using CRISPR/Cas9 and characterize them phenotypically and molecularly using RNA-seq and eCLIP- seq) and (2) generate SF3B1 K700E iPSC lines with additional mutations in either DNMT3A, RUNX1, or ASXL1 and characterize their hematopoietic phenotypes and transcriptomes. This work can generate new hypotheses about the mechanisms by which SF mutations drive MDS and reveal principles of mutational cooperativity in MDS pathogenesis.