Defining protein synthesis demands and specificities in multiple myeloma

项目摘要 多发性骨髓瘤（MM）是分化的B淋巴细胞（或“浆细胞”）的不可治愈的癌症， 通常在生命晚期出现，并且从诊断时起的中位预期寿命为约4-7年。MM细胞 在大多数其他细胞谱系中是不寻常的，包括其他类型的癌症，因为它们合成了大量的 单个无功能蛋白质的量。尽管有这种独特的特征，蛋白质合成的调节 细胞的机器，核糖体，仍然很少在MM研究。最近的证据已经推进了我们的研究。 理解核糖体下游触发的应激反应级联反应，其范围从 核糖体介导的质量控制（RQC）的单一mRNA的“核糖毒性”应激反应和全球 完全激活时平移关闭。重要的是，初步数据表明，在375种癌症中， 细胞系中，MM细胞是表达最高基线水平的上游因子的细胞之一， 级联，EDF 1.总之，这表明RQC和核糖毒性应激反应可能被普遍激活 与其他细胞类型相比，MM细胞的基线水平。除了RQC和核糖毒性应激反应，MM细胞 对细胞翻译机制的特定成分的抑制也有不同的敏感性， 包括真核起始因子4F复合物的所有三个亚基。文献证据和初步 数据表明MM细胞可能高度依赖于eIF 4F复合物来翻译特定的 促进其增殖或存活的mRNA，但这些mRNA的身份和机制的见解 为什么他们特别依赖于eIF 4F水平，无论是一般还是在MM中，尚不清楚。在这一提议中， 我试图将这些想法联系起来，以了解MM细胞如何调节翻译机制， 满足其独特的蛋白质合成需求。这一建议的核心假设是，翻译是 在MM中广泛失调，使蛋白质合成和特定mRNA的翻译速率高， 促进生存和增殖。我建议通过以下具体目标来解决这一假设： 目标1：表征MM中的翻译、质量控制和核糖毒性应激反应;目标2：定义 MM中的eIF 4F敏感性翻译景观;目的3：多聚体相关CAGE-seq（paCAGE）以询问 这些目标将通过生物化学和生物化学的结合来实现。 在永生化MM细胞系中的测序方法，包括开发一种新技术， 对于鉴定eIF 4F敏感性mRNA中的5 'UTR序列基序是必不可少的。这项工作将具有重大意义 因为它可能揭示多发性骨髓瘤的新治疗靶点，同时阐明重要的核糖体 生物学可能更普遍地涉及其他模型系统或疾病状态。

PROJECT SUMMARY Multiple Myeloma (MM) is an incurable cancer of differentiated B lymphocytes (or “plasma cells”) which generally presents late in life and has a median life expectancy of ~4-7 years from time of diagnosis. MM cells are unusual among most other cell lineages, including other types of cancer, in that they synthesize large amounts of a single, nonfunctional protein. Despite this unique signature, the regulation of the protein synthesis machinery of the cell, the ribosome, remains poorly studied in MM. Recent evidence has advanced our understanding of a stress response cascade triggered downstream of the ribosome, which ranges from ribosome-mediated quality control (RQC) of single mRNAs to a “ribotoxic” stress response and global translational shutdown when fully activated. Importantly, preliminary data demonstrate that among 375 cancer cell lines, MM cells are among those that express the highest baseline levels of an upstream factor in this cascade, EDF1. Together, this suggests that RQC and the ribotoxic stress response may be generally activated at baseline in MM cells compared to other cell types. Beyond RQC and the ribotoxic stress response, MM cells are also differentially sensitive to inhibition of specific components the translational machinery of the cell, including all three subunits of the eukaryotic initiation factor 4F complex. Literature evidence and preliminary data suggest that MM cells may be hyper-dependent on the eIF4F complex to translate specific subsets of mRNAs which promote their proliferation or survival, but the identity of these mRNAs and mechanistic insight into why they are particularly dependent on eIF4F levels, both generally and in MM, is unclear. In this proposal, I seek to connect these ideas in order to understand how MM cells modulate the translational machinery to meet their unique protein synthesis demands. The central hypothesis of this proposal is that translation is broadly dysregulated in MM to allow high rates of protein synthesis and the translation of specific mRNAs which promote survival and proliferation. I propose to address this hypothesis through the following specific Aims: Aim 1: Characterize translation, quality control, and the ribotoxic stress response in MM; Aim 2: Define the eIF4F-sensitive translational landscape in MM; Aim 3: polysome-associated CAGE-seq (paCAGE) to interrogate translational control by 5'UTRs in MM. These aims will be achieved through a combination of biochemical and sequencing approaches in immortalized MM cell lines, including the development of a new technology which will be essential to identify 5'UTR sequence motifs within eIF4F-sensitive mRNAs. This work will be significant because it may reveal new therapeutic targets in multiple myeloma while elucidating important ribosomal biology which may be more generally involved in other model systems or disease states.