Investigations of Consequences of U2AF1 Mutations in MDS

描述（申请人提供）：体细胞突变、染色体缺陷和表观遗传改变是骨髓增生异常综合征（MDS）的主要致病缺陷。最近在分子技术方面的科学进展导致发现了新的复发性病变类别，并确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。本提案的重点是新发现的一类影响剪接体基因的突变，其中包括U2AF1。影响该基因2个锌指结构域的高复发性杂合错义突变在MDS和AML中很常见，它们是加速进展和低生存率的预后因素。由于剪接体突变似乎在某些形式的MDS中特别频繁，因此这种疾病将作为研究剪接体缺陷介导致癌作用的机制的模型。我们的建议是基于这样的假设，即由于体细胞突变导致剪接体基因缺陷导致特定类型的肿瘤抑制基因（TSG）不同组合的错误剪接，因此最终导致类似于这些TSG直接病变所产生的病理后果。因此，剪接体突变可能是其他基因组缺陷的表型后果。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”，从而导致特异性错剪接模式的产生。在这个项目中，我们将研究基因的重复错误拼接模式和结构-功能关系

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.