Functional Genomic Dissection of Refractory Anemia

 描述(申请人提供)：骨髓增生异常综合征(MDS)的特征是无效的造血，最常见的是红系，导致一种称为难治性贫血的表型。具有染色体5q杂合性间质缺失(Del(5q))的MDS患者具有高度可重复性的临床表型，尽管该表型的分子基础先前尚不清楚。在这笔赠款的第一个资助期，我们发现RPS14基因的单倍体不足，这是del(5q)MDS共同缺失区域内的基因之一，导致红细胞生成受阻，红细胞生成是与这种遗传异常相关的主要临床表型，在del(5q)MDS和钻石黑扇贫血之间建立了以前未知的联系，钻石黑扇贫血是一种具有相似表型的先天性疾病，也是由编码核糖体蛋白的基因的一个等位基因的遗传失活引起的。在最近的资助期间，我们发现CSNK1A1的单倍体不足导致β-连环蛋白水平升高，造血干细胞的数量和功能增加，以及对来那度胺药物的敏感性。此外，我们在CSNK1A1中发现了重复的、功能获得的体细胞突变，这是Del(5q)共同缺失区域中的第一个基因，被确认为重复的体细胞突变。在目前的方案中，我们首先利用小鼠模型和磷酸蛋白质组学来确定具有CSNK1A1杂合缺失或突变的细胞克隆优势的机制。我们的下一个目标是确定CSNK1A1单倍性不足如何与其他Del(5q)基因的单倍性不足相互作用，使用细胞条形码系统在单个小鼠模型中执行多个基因类型的竞争性再种群分析。最后，我们将使用一种新的饱和诱变功能筛选，研究CRL4CRBN泛素连接酶对来那度胺依赖降解的生物化学。这些研究将为Del(5q)MDS的生物学和治疗、更广泛的癌症杂合缺失的机制以及针对泛素连接酶活性的新疗法的开发提供信息。

 DESCRIPTION (provided by applicant): Myelodysplastic syndrome (MDS) is characterized by ineffective hematopoiesis, most commonly of the erythroid lineage, resulting in a phenotype termed refractory anemia. Patients with MDS who have a heterozygous, interstitial deletion of chromosome 5q (Del (5q)), have a highly reproducible clinical phenotype, though the molecular basis of this phenotype was previously unknown. In the first funding period of this grant, we found that haploinsufficiency for RPS14, one of the genes within the common deleted region for del (5q) MDS, causes a block in erythropoiesis, the dominant clinical phenotype associated with this genetic abnormality, establishing a previously unrecognized link between del (5q) MDS and Diamond Blackfan anemia, a congenital disorder with a similar phenotype that is also caused by genetic inactivation of one allele of genes encoding ribosomal proteins. In the most recent funding period, we found that haploinsufficiency for CSNK1A1 causes elevated β-catenin levels, an increase in the number and function of hematopoietic stem cells, and sensitivity to the drug lenalidomide. Moreover, we identified recurrent, gain-of-function somatic mutations in CSNK1A1, the first gene within the Del (5q) common deleted region identified with recurrent somatic mutations. In the current proposal, we first aim to determine the mechanisms underlying clonal dominance of cells with CSNK1A1 heterozygous deletion or mutation using both murine models and phosphoproteomics. We next aim to determine how CSNK1A1 haploinsufficiency interacts with haploinsufficiency for other Del (5q) genes using a cellular barcoding system to perform competitive repopulation assays with multiple genotypes in a single mouse model. Finally, we will investigate the biochemistry of lenalidomide-dependent degradation by the CRL4CRBN ubiquitin ligase, using a novel saturation mutagenesis functional screen. These studies will inform the biology and treatment of Del (5q) MDS, the mechanisms underlying heterozygous deletions in cancer more broadly, and the development of novel therapeutics targeting ubiquitin ligase activity.