Investigations of Consequences of U2AF1 Mutations in MDS

MDS 中 U2AF1 突变后果的研究

• 批准号: 8666590
• 负责人: Jaroslaw P Maciejewski
• 金额: $ 38.83万
• 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/8666590
• 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（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异常综合征（MDS）的关键致病缺陷。分子技术的最新科学进展导致发现了新类型的复发性病变，并鉴定了与特定病理形态学特征相关的肿瘤发生或突变的新分子途径。该提案重点关注新发现的一类影响剪接体基因的新型突变，其中包括 U2AF1。影响该基因中 2 个锌指结构域的高度复发性杂合错义突变在 MDS 和 AML 中很常见，这些突变预示着加速进展和不良生存。由于剪接体突变在某些形式的MDS中似乎特别频繁，因此这种疾病将作为研究剪接体缺陷介导致癌作用机制的模型。我们的提议基于这样的假设：体细胞突变导致的剪接体基因缺陷会导致肿瘤抑制基因（TSG）不同组合的特定类型的错误剪接，因此最终导致与直接损伤这些基因所产生的致病后果相似的致病后果。 TSG。因此，剪接体突变可能是其他基因组缺陷的表型后果。在分子水平上，U2AF1突变通过特定剪接位点序列的差异排除而导致“功能改变”，从而导致特定错误剪接模式的产生。在这个项目中，我们将研究经常出现的错误剪接模式以及结构与功能的关系 MDS 中 U2AF1 突变导致的剪接缺陷，并识别受错误剪接影响的外显子。我们将确定在患者中观察到的剪接功能障碍和异常剪接模式是否可以在工程模型细胞系中重现。我们还将比较纯化的重组野生型和突变型 U2AF 的 RNA 结合特异性以及对错误剪接靶基因的体外剪接的影响。此外，我们将通过引入具有突变体结合位点的诱饵RNA来恢复细胞中的正常剪接，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。突变相关的表型和结果将与那些没有 U2AF1 突变但下游基因单倍体表达不足/亚形功能的患者进行比较，这些下游基因被发现受到 U2AF1 缺陷的影响。