Impact of RPS15 mutation on development and progression of chronic lymphocytic leukemia

项目摘要/摘要 越来越多的证据表明，核糖体比之前描述的更具动态化和异质性。 我们对核糖体功能的了解取得了进展，现在描绘了一幅核糖体作为关键调控因子的图景 基因表达具有独特的组成和高度专业化的功能，可以在不同的组织类型中有所不同。 核糖体活性异常在许多肿瘤类型和核糖体蛋白的反复突变中被评估 已经报道在一些癌症和与癌症相关的核糖体疾病中，如T-ALL和 戴蒙德-布莱克凡贫血。这可以包括翻译的保真度和翻译速度的变化、优先翻译 某些转录本优于其他转录本，替代起始和终止密码子的使用，以及内部的总体变化 细胞核糖体含量。538例慢性淋巴细胞患者近期全外显子组测序研究 白血病(CLL)已发现核糖体蛋白S15热点区域的反复错义突变(~5%) (RPS15)。RPS15突变经常与TP53异常共存(36%)，并富含del(17P)CLL(12%) -通常预后不良的异常和对化学免疫治疗的抵抗力。的确， RPS15基因突变与化疗后不良预后和复发风险增加相关 免疫治疗，即使在没有TP53或del(17P)缺失的情况下也是如此。然而，尽管有这些令人信服的临床 研究发现，关于RPS15突变如何影响CLL发病的信息很少。我们有 产生了一系列独特的工具，用于对这种基本蛋白质的拟议研究，这些工具有望解开这一点 机制。这包括特征良好的人类CLL细胞株，它们携带的基因突变 反映常见的CLL病变，如del(13q)和TP53，并已编辑为表达RPS15突变 与CLL相关。我们还产生了生理上忠实的Rps15敲入小鼠，这些小鼠限制了突变 表达到B细胞。使用这些模型来表征这种突变对CLL的功能影响 发展和进步必将为治疗干预提供新的方向，并将增加我们的 对核糖体病理学的基本了解。这项拟议的研究试图彻底询问 RPS15突变通过以下目的对CLL生物学的影响：1)表征其对核糖体的影响 蛋白质功能(如核糖体RNA合成、核糖体组装、翻译和核糖体外 在健康和恶性组织中的功能--即MDM2-p53核糖体监测途径)；以及 2)体外测定其对肿瘤标志物如增殖和生长因子独立性的影响，以及 体内致瘤性的研究。因为RPS15突变与信使核糖核酸的解码位点接触，所以我们假设它们 通过mRNA的异常翻译参与发病。建议的实验方法 包括复杂和新颖技术，例如核糖体特征、核糖体图谱、FISH、核糖体 通过Northern印迹鉴定RNA，并使用体外和体内CLL模型进行致瘤性研究。

Project Summary/Abstract There is mounting evidence that ribosomes are more dynamic and heterogenous than previously described. Advances in our understanding of ribosomal functions now paint a picture of ribosomes as critical regulators of gene expression with unique compositions and highly specialized functions that can differ across tissue types. Aberrant ribosome activity has been assessed in many tumor types and recurrent mutations in ribosomal proteins have been reported in a number of cancers and ribosomopathies associated with cancer, such as T-ALL and Diamond-Blackfan anemia. This can include changes in the fidelity and rate of translation, preferential translation of certain transcripts over others, utilization of alternative start and stop codons, and overall changes in intra- cellular ribosome content. Recent whole exome sequencing studies of 538 patients with chronic lymphocytic leukemia (CLL) have identified recurrent missense mutations (~5%) at a hotspot region of ribosomal protein S15 (RPS15). RPS15 mutations often co-occur with TP53 aberrations (36%) and are enriched in del(17p) CLL (12%) – abnormalities that are often carry poor prognoses and refractoriness to chemoimmunotherapy. Indeed, mutation of RPS15 is associated with adverse prognosis and an increased risk of relapse following chemo- immunotherapy, even in the absence of TP53 or del(17p) deletions. However, despite these compelling clinical findings, there is a paucity of information regarding how RPS15 mutation influences CLL pathogenesis. We have generated an array of unique tools for the proposed study of this essential protein that promise to unravel this mechanism. This includes human CLL cell lines that are well characterized and carry genetic mutations that reflect common CLL lesions, such as del(13q) and TP53, and that have been edited to express RPS15 mutations relevant in CLL. We have also generated physiologically faithful Rps15 knock-in mice that restrict mutant expression to B cells. Using these models to characterize the functional impact of this mutation on CLL development and progression will certainly provide new directions for therapeutic intervention and will add to our fundamental understanding of ribosome pathology. The proposed study seeks to thoroughly interrogate the impact of RPS15 mutations on CLL biology through the following aims: 1) Characterize its influence on ribosomal protein functions (such as ribosomal RNA synthesis, ribosome assembly, translation and extra-ribosomal functions – namely, the MDM2-P53 ribosome surveillance pathway) in both healthy and malignant tissue; and 2) Determine its impact on cancer hallmarks such as proliferation and growth factor independence in vitro and on tumorigenicity in vivo. Because RPS15 mutations engage the mRNA decoding site, we hypothesize that they contribute to pathogenesis through aberrant translation of mRNA. The proposed experimental approaches include complex and novel techniques such as ribosome characterization, ribosome profiling, FISH, ribosomal RNA characterization through northern blotting, and tumorigenicity studies using in vitro and in vivo CLL models.