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

项目总结/摘要 越来越多的证据表明，核糖体比以前描述的更具活力和异质性。 我们对核糖体功能的理解的进展现在描绘了核糖体作为关键调节因子的画面。 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。 异常核糖体活性已在许多肿瘤类型和核糖体蛋白的复发性突变中评估 已经在许多癌症和与癌症相关的核糖体病中报道，如T-ALL和 Diamond-Blackfan贫血这可能包括翻译的保真度和速度的变化，优先翻译 某些转录本的变化，替代性起始和终止密码子的利用，以及转录本内的总体变化。 细胞核糖体含量538例慢性淋巴细胞性白血病患者的全外显子组测序研究 白血病（CLL）已在核糖体蛋白S15热点区域鉴定出复发性错义突变（~5% （RPS 15）。RPS 15突变通常与TP 53畸变（36%）同时发生，并在del（17 p）CLL中富集（12%） - 这些异常通常具有较差的耐受性和对化学免疫疗法的难治性。的确， RPS 15突变与化疗后不良预后和复发风险增加相关。 免疫疗法，甚至在没有TP 53或del（17 p）缺失的情况下。然而，尽管这些令人信服的临床 研究结果表明，关于RPS 15突变如何影响CLL发病机制的信息很少。我们有 为研究这种必需蛋白质的提议产生了一系列独特的工具，这些工具有望解开这一难题。 机制这包括人CLL细胞系，其被充分表征并携带遗传突变， 反映常见的CLL病变，如del（13 q）和TP 53，并已被编辑以表达RPS 15突变 与CLL有关。我们还产生了生理上忠实的Rps 15基因敲入小鼠， 表达至B细胞。使用这些模型来表征这种突变对CLL的功能影响 发展和进步肯定会为治疗干预提供新的方向，并将增加我们的 核糖体病理学的基础知识。拟议的研究旨在彻底询问 RPS 15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，MDM 2-P53核糖体监视途径）;以及 2)确定其对癌症标志物的影响，如体外增殖和生长因子独立性， 对体内致瘤性的影响。因为RPS 15突变参与mRNA解码位点，我们假设它们 通过mRNA的异常翻译导致发病。提出的实验方法 包括复杂和新颖技术，例如核糖体表征、核糖体谱、FISH、核糖体 通过北方印迹进行RNA表征，以及使用体外和体内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.