Modeling Splicing Factor Mutations in MDS Using Human iPSCs

使用人类 iPSC 模拟 MDS 中的剪接因子突变

• 批准号: 9901476
• 负责人: Shailee Vora
• 金额: $ 4.39万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-04-16 至 2021-04-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9901476
• 关键词: Acute Myelocytic Leukemia; Acute leukemia; Affect; Alleles; Alternative Splicing; Automobile Driving; Biochemical; Biological; Biological Assay; Biological Models; Biology; Blood; CRISPR/Cas technology; Cell Line; Cell Proliferation; Cell Survival; Cell model; Cells; Clinical; Clonal Evolution; Disease; Dysmyelopoietic Syndromes; Ectopic Expression; Epitopes; Event; Fibrinogen; Flow Cytometry; Gene Mutation; Genes; Genetic; Genetic Population Study; Genetic study; Goals; Hematological Disease; Hematopoietic; Hematopoietic stem cells; Human; In Vitro; Ineffective Hematopoiesis; Investigation; Mediating; Modeling; Molecular; Morphology; Mus; Mutate; Mutation; Outcome; Pathogenesis; Patients; Phenotype; Population; Protein Isoforms; RNA Binding; RNA Splicing; RNA-Binding Proteins; RUNX1 gene; Recurrence; Role; SRSF2 gene; Small Nuclear RNA; Somatic Mutation; Specificity; Spliced Genes; Testing; U2 small nuclear RNA; Work; base; crosslinking and immunoprecipitation sequencing; cytopenia; genome editing; hematopoietic differentiation; induced pluripotent stem cell; mouse model; mutant; next generation sequencing; novel; novel therapeutics; outcome forecast; peripheral blood; stem cell model; therapeutic target; transcriptome; transcriptome sequencing

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.

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)的突变。这些血液病的特点是无效 表现为外周血细胞减少和发育异常的形态改变的造血 进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 在剪接中的已知作用，通常称为剪接因子(最常见的三个是 SF3B1 K700E、U2AF1 S34F和SRSF2 P95L)是最常见的突变类型 MDS影响了超过一半的患者。SF突变发生在疾病的早期，因此很可能 对其发病机制和吸引人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的模型和细胞系中的异位表达表明，突变改变了rna结合。 突变体SFS的特异性。然而，对疾病发病机制至关重要的下游事件 仍然难以捉摸，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突变共存的不同突变与 不同的临床结果(预后差或好)。DNMT3A与SF3B1突变共存 预后良好，而SF3B1与RUNX1或ASXL1共突变导致预后不良 预后。然而，这些遗传相互作用背后的机制尚不清楚。这个 Papapetrou实验室率先使用诱导多能干细胞(IPSC)模型对MDS进行建模。 作为选择性剪接，人类IPSC模型对于SF突变的研究特别有吸引力 同源异构体在老鼠和人类之间显示出很差的保守性。 我建议开发基于IPSC的SF突变和协同突变的新模型，以研究 这些突变及其在MDS发病机制中的遗传交互作用。具体地说，我建议：(1)产生 具有三个最常见的典型SF突变SRSF2 P95L、SF3B1 K700E和U2AF1的IPSC系 S34F使用CRISPR/Cas9，并使用RNA-seq和eCLIP-2对它们进行表型和分子鉴定。 序列)和(2)产生带有DNMT3A、RUNX1或 ASXL1，并鉴定它们的造血表型和转录本。这项工作可以产生新的 关于SF突变驱动MDS机制的假说和揭示突变原理 MDS发病机制中的协同性。