Functional Genomic Dissection of Refractory Anemia

 描述（申请人提供）：骨髓增生异常综合征（MDS）的特征是无效造血，最常见的是红系造血，导致称为难治性贫血的表型。具有染色体5 q（Del（5 q））的杂合间质缺失的MDS患者具有高度可再现的临床表型，尽管这种表型的分子基础以前是未知的。在该资助的第一个资助期内，我们发现RPS 14（del（5 q）MDS常见缺失区域内的基因之一）的单倍不足导致红细胞生成阻滞，这是与这种遗传异常相关的主要临床表型，建立了del（5 q）MDS和Diamond Blackfan贫血之间以前未被认识的联系，一种先天性疾病，有相似的表现型，也是由编码核糖体蛋白质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CSNK 1A 1的单倍不足导致β-catenin水平升高，造血干细胞数量和功能增加，以及对药物来那度胺的敏感性。此外，我们在CSNK 1A 1中鉴定了复发性功能获得性体细胞突变，CSNK 1A 1是Del（5 q）常见缺失区中第一个鉴定为复发性体细胞突变的基因。在目前的建议中，我们的目标首先是确定CSNK 1A 1杂合缺失或突变的细胞克隆优势的机制，使用小鼠模型和磷酸化蛋白质组学。我们接下来的目标是确定CSNK 1A 1单倍不足如何与其他Del（5 q）基因的单倍不足相互作用，使用细胞条形码系统在单个小鼠模型中使用多种基因型进行竞争性再增殖测定。最后，我们将使用一种新的饱和诱变功能筛选，研究CRL 4CRBN泛素连接酶对来那度胺依赖性降解的生物化学。这些研究将为Del（5 q）MDS的生物学和治疗提供信息，更广泛地了解癌症中杂合缺失的机制，以及靶向泛素连接酶活性的新型治疗药物的开发。

 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.