Investigations of Consequences of U2AF1 Mutations in MDS

MDS 中 U2AF1 突变后果的研究

• 批准号: 8482808
• 负责人: Jaroslaw P Maciejewski
• 金额: $ 37.69万
• 依托单位: CLEVELAND CLINIC LERNER COM-CWRU
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2018-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8482808
• 关键词: Affect; Alleles; Alternative Splicing; Binding Sites; Bone Marrow Cells; Cell Line; Cells; Clinical; Data; Defect; Disease; Dysmyelopoietic Syndromes; Elderly; Engineering; Epigenetic Process; Evolution; Exclusion; Exons; Fingers; Frequencies; Functional disorder; Gene Expression; Gene Proteins; Gene Targeting; Genes; Genomics; Heterogeneity; Human; Hypermethylation; In Vitro; Ineffective Hematopoiesis; Investigation; Laboratories; Lead; Lesion; Life Expectancy; Link; Mediating; Medical; Messenger RNA; Missense Mutation; Modeling; Molecular; Molecular Profiling; Mutate; Mutation; Myeloid Cells; Myeloproliferative disease; Oncogenic; Outcome; Pathogenesis; Pathway interactions; Patients; Pattern; Peptide Signal Sequences; Phenocopy; Phenotype; Phosphoric Monoester Hydrolases; Population; Process; RNA Binding; RNA Processing; RNA Sequences; RNA Splicing; Recombinants; Recurrence; Reporter; Sampling; Scientific Advances and Accomplishments; Site; Somatic Mutation; Specificity; Spliced Genes; Spliceosomes; Structure-Activity Relationship; Technology; Testing; Tumor Suppressor Genes; Zinc Fingers; base; cancer type; cytopenia; exome sequencing; improved; in vivo; leukemia; leukemogenesis; mutant; new therapeutic target; next generation sequencing; novel; prognostic; promoter; public health relevance; small hairpin RNA; socioeconomics; transcriptome sequencing; tumorigenesis

DESCRIPTION (provided by applicant): Somatic mutations, chromosomal defects and epigenetic changes constitute the key pathogenic defects in myelodysplastic syndrome (MDS). Recent scientific advances in molecular technologies have led to the discovery of new classes of recurrent lesions and the identification of novel molecular pathways of oncogenesis or mutations associated with specific pathomorphologic features. This proposal focuses on a newly discovered novel class of mutations affecting spliceosomal genes and among them U2AF1. Highly recurrent, heterozygous missense mutations affecting 2 zinc finger domains in this gene are frequent in MDS and AML where they are prognostic for accelerated progression and poor survival. Because spliceosomal mutations appear to be particularly frequent in certain forms of MDS, this disease will serve as a model for investigation of mechanisms by which spliceosomal defects mediate oncogenic effects. Our proposal is based on the hypothesis that defects in spliceosomal genes due to somatic mutations lead to specific types of mis- splicing of distinct combinations of tumor suppressor genes (TSG) and therefore ultimately result in pathogenetic consequences similar to those produced by direct lesions to these TSG. Hence, spliceosomal mutations may phenocopy consequences of other genomic defects. On the molecular level, U2AF1 mutations result in "change of function" through differential exclusion of specific splice site sequences and thereby result in creation of specific mis-splicing patterns. In this project, w will investigate the recurrent mis-splicing patterns and the structure-function relationship of the splicing defects due to U2AF1 mutations in MDS and identify exons affected by mis-splicing. We will determine whether splicing dysfunction and the aberrant splicing patterns observed in patients can be recapitulated in engineered model cell lines. We will also compare the RNA binding specificities of purified recombinant wild type and mutant U2AF as well as the effects on in vitro splicing of mispliced target genes. Furthermore, we will restore normal splicing in cells y introducing decoy RNAs with binding sites for the mutant and investigate whether the inhibition of PP1/PP2 phosphatases can improve spliceosomal function. Finally, we will analyze the clinical consequences of U2AF1 mutations. Mutation-associated phenotypes and outcomes will be compared to those seen in patients without U2AF1 mutations but with the haploinsufficient expression/hypomorphic function of downstream genes found to be otherwise affected in by U2AF1 defects.

描述（由申请人提供）：体细胞突变，染色体缺陷和表观遗传变化构成骨髓增生综合征（MDS）的关键致病性缺陷。分子技术的最新科学进步导致发现了新的复发性病变，并鉴定了与特定的病原体特征相关的肿瘤发生或突变的新型分子途径。该提案着重于一种新发现的新型突变类，这些突变影响了剪接体基因，其中包括U2AF1。在MDS和AML中，高度复发的，杂合的错义突变经常在该基因中，在该基因中，它们在加速进展和存活率不佳的情况下预后具有预后。由于剪接体突变在某些形式的MDS中似乎特别频繁，因此该疾病将作为研究机制的模型，通过这种机制，剪接体缺陷会介导致癌作用。我们的建议是基于以下假设：跨体突变引起的剪接基因缺陷导致特定类型的肿瘤抑制基因（TSG）组合的错误剪接（TSG），因此最终导致了与这些TSG直接病变产生的病毒后果。因此，剪接突变可能会导致其他基因组缺陷的表现后果。在分子水平上，U2AF1突变通过特定的剪接位点序列的差异排除导致“函数变化”，从而导致创建特定的错误分解模式。在这个项目中，W将研究复发的错误拼写模式和结构 - 功能关系 由于MDS中的U2AF1突变引起的剪接缺陷，并识别出因误导而影响的外显子。我们将确定在工程模型细胞系中是否可以概括在患者中观察到的剪接功能障碍和异常剪接模式。我们还将比较纯化的重组野生型和突变体U2AF的RNA结合特异性，以及对错误绘制靶基因的体外剪接的影响。此外，我们将在细胞中恢复带有结合位点的诱饵RNA的正常剪接，并研究抑制PP1/PP2磷酸酶是否可以改善剪接体功能。最后，我们将分析U2AF1突变的临床后果。将突变相关的表型和结局与没有U2AF1突变的患者中看到的表型和结局进行比较，但发现下游基因的单倍弹性表达/低形态功能受到U2AF1缺陷的影响。