Investigations of Consequences of U2AF1 Mutations in MDS

描述（由申请人提供）：体细胞突变、染色体缺陷和表观遗传学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已经导致发现新的复发性病变类别，并鉴定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。这项建议的重点是一个新发现的一类新的突变影响剪接体基因，其中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中常见，它们是加速进展和不良生存的预后因素。由于剪接体突变似乎是特别频繁的某些形式的MDS，这种疾病将作为一个模型的机制，剪接体缺陷介导的致癌作用的调查。我们的建议是基于这样的假设，即由于体细胞突变引起的剪接体基因缺陷导致肿瘤抑制基因（TSG）的不同组合的特定类型的错误剪接，因此最终导致与这些TSG的直接损伤所产生的那些类似的病理后果。因此，剪接体突变可能是其他基因组缺陷的表型复制结果。在分子水平上，U2AF1突变通过特异性剪接位点序列的差异排除导致"功能改变"，从而导致产生特异性错误剪接模式。在这个项目中，我们将研究反复出现的错误剪接模式和结构与功能的关系， MDS中由于U2AF1突变导致的剪接缺陷，并鉴定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配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PP1/PP2磷酸酶的抑制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。将突变相关表型和结局与无U2AF1突变但下游基因单倍表达不足/亚型功能受U2AF1缺陷影响的患者进行比较。

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.