Model systems for hematologic disorders caused by ribosomal protein deficiency

描述(由申请人提供)：本申请涉及广泛的挑战领域(15)翻译科学和特殊挑战主题15-DK-106：翻译血液学基本概念。核糖体蛋白(RP)基因的单倍体不足是遗传性骨髓衰竭综合征钻石-布莱克凡贫血(DBA)和5q-骨髓增生异常综合征(5q-MDS)的基础。虽然已知核糖体蛋白表达缺陷会导致核糖体生物发生中断，但这种缺陷导致骨髓衰竭和MDS的分子机制尚不清楚。这种缺乏了解限制了我们辨别当前治疗方法的作用机制的能力，并阻碍了针对这些疾病的新疗法的开发。人们普遍认为，细胞在细胞周期中进步的能力与新生核糖体的生物发生密切相关。新生核糖体生物发生的上调有两个主要功能：(I)它提供了细胞达到进入细胞周期所需的临界量所需的增加的翻译能力，(Ii)它确保每个子细胞获得适当数量的核糖体。鉴于核糖体与信使核糖核酸比率的改变最终会改变翻译模式和遗传程序，导致异常的生长形式，越来越多的证据表明，检查点的存在是为了感知和响应新生核糖体生物发生的状态。事实上，早期的研究已经暗示了p53肿瘤抑制基因的作用，最近的研究结果表明，p53是在RPS产生不足的情况下被激活的，由于60rpL11和rpL5结合和抑制人类双分钟2(Hdm2)而导致细胞周期停滞。已经证明，这种检查点反应是由rpL11和rpL5通过不同的机制介导的，这取决于40s或60s的RP是否被耗尽。这项研究将检验的假设是，rpL11/rpL5检查点对RP应激的反应在DBA和5q-MDS的造血失败中起着因果作用，目前的药物治疗针对的是这一途径以获得治疗收益。为了解决这一假设，将实现以下目标：目标1：表征rpL5和rpL11在耗尽小或大核糖体亚单位蛋白的人造血细胞中的翻译状态；AIM2：确定地塞米松、来那度胺和亮氨酸对P53反应以及对原代缺乏核糖体蛋白的人造血细胞中rpL5和rpL11翻译的影响；以及目标3：在核糖体蛋白缺乏的体内模型中评估地塞米松、来那度胺和亮氨酸的作用，并在表型患者样本中验证这些发现。从这些研究中获得的知识对于确定新的治疗靶点和开发新的合理设计的治疗方法至关重要。 与公共卫生相关：核糖体蛋白基因的突变影响核糖体的产生，核糖体是细胞内负责蛋白质合成的机械，导致造血系统紊乱，如贫血，这是生产红细胞的缺陷。虽然这些疾病有治疗方法，但它们的作用机制尚不清楚。很有可能 核糖体蛋白基因突变患者的贫血是由于 核糖体生产中的缺陷会导致细胞分裂停滞，从而导致细胞 死亡。拟议项目的目的是确定用于治疗的疗法的效果。 由核糖体蛋白基因突变引起的贫血，并确定 当核糖体产生受损时，会导致细胞增殖停滞和细胞死亡。这将是 允许识别损害红细胞产生的细胞过程和 在新疗法的设计中将是必不可少的。

DESCRIPTION (provided by applicant): This application addresses broad Challenge Area (15) Translational Science and specific Challenge Topic 15-DK-106: Translating basic hematology concepts. Haploinsufficiency of ribosomal protein (rp) genes underlies the inherited marrow failure syndromes Diamond-Blackfan anemia (DBA) and the 5q- myelodysplastic syndrome (5q- MDS). Although it is known that deficiencies in ribosomal protein expression result in disrupted ribosome biogenesis, the molecular mechanisms by which such defects lead to marrow failure and MDS are unclear. This lack of understanding limits our ability to discern the mechanism of action of current therapies and hampers the development of new therapies for these disorders. It is accepted that the ability of cells to progress through the cell cycle is tightly linked to nascent ribosome biogenesis. The upregulation of nascent ribosome biogenesis serves two major functions: (i) it provides the increased translational capacity required for a cell to achieve the critical mass to enter the cell cycle, and (ii) it ensures that each daughter cell obtains the appropriate number of ribosomes. Given that a change in the ratio of ribosomes to mRNA would eventually modify the pattern of translation and the genetic program, resulting in aberrant forms of growth, evidence is mounting that checkpoints exist to sense and respond to the status of nascent ribosome biogenesis. Indeed, earlier studies had suggested a role for the p53 tumor suppressor gene, and more recent results show that p53 is activated in response to deficiencies in the production of rps, leading to cell- cycle arrest due to the binding and inhibition of human double minute 2 (HDM2) by 60S rpL11 and rpL5. It has been demonstrated that this checkpoint response is mediated by rpL11 and rpL5 by distinct mechanisms, depending on whether a 40S or 60S rp is depleted. The hypothesis that will be tested in this study is that the rpL11/rpL5 checkpoint response to rp stress plays a causal role in hematopoietic failure in DBA and 5q- MDS and that current medical therapies target this pathway for therapeutic gain. To address this hypothesis, the following aims will be carried out: Aim 1: Characterize rpL5 and rpL11 translation status in human hematopoietic cells depleted of either small or large ribosomal subunit proteins; Aim2: Determine the effects of dexamethasone, lenalidomide, and leucine on the p53 response and on rpL5 and rpL11 translation in primary human hematopoietic cells depleted of ribosomal proteins; and Aim 3: Assess the effects of dexamethasone, lenalidomide, and leucine in an in vivo model of ribosomal protein deficiency and validate those findings in phenotyped patient samples. The knowledge derived from these studies will be critical for identifying novel therapeutic targets and for the development of new rationally designed therapies. PUBLIC HEALTH RELEVANCE: Mutations in ribosomal protein genes affect the production of ribosomes, the machinery inside the cell which is responsible for the synthesis of proteins, resulting in disorders of the hematopoietic system, such as anemia, a defect in the production of red blood cells. Although therapies are available for these diseases, their mechanism of action is not known. It is likely that the anemia of patients with mutations in genes for ribosomal proteins is due to the fact that defects in the production of ribosomes cause an arrest in cell division which can result in cell death. The aim of the proposed project is to determine the effects of therapies used to treat anemias caused by mutations in ribosomal protein genes and to identify mechanisms that trigger arrest in cell proliferation and cell death when ribosome production is impaired. This will allow for the identification of the cellular processes that impair production of red blood cell and will be essential in the design of new therapies.