Exploiting myeloma proteome remodeling to extend proteasome inhibitor efficacy

利用骨髓瘤蛋白质组重塑来延长蛋白酶体抑制剂的功效

基本信息

批准号：
10341162
负责人：
Arun P. Wiita
金额：
$ 27.82万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-03-01 至 2023-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10341162
关键词：
Active Sites Address Alternative Splicing Apoptosis Apoptotic Architecture Biochemical Biological Assay Bortezomib CRISPR interference Cell Death Cell Line Cells Cellular biology Clinical Clinical Trials Complex Data Diagnosis Disease Event Exons Exposure to FDA approved Family Genetic Genetic Transcription Goals Heat shock proteins Heat-Shock Proteins 70 Hematologic Neoplasms Hematopoietic Neoplasms Immunoglobulins Introns Investigation Kinetics Lead Longevity Malignant - descriptor Malignant Neoplasms Mass Spectrum Analysis Mediating Methods Modification Molecular Chaperones Monitor Multiple Myeloma Mutation Outcome Pathway interactions Patients Pharmaceutical Preparations Pharmacology Phosphorylation Physiologic pulse Plasma Cells Post-Translational Protein Processing Proteasome Inhibition Proteasome Inhibitor Protein Biosynthesis Proteins Proteome Proteomics RNA Splicing Refractory Relapse Resistance Role Sampling Signal Transduction Spliceosomes System Therapeutic Time Transcript Treatment Efficacy Ubiquitin base cell type clinically relevant combat follow-up functional genomics genomic data improved in vivo Model inhibitor insight knock-down mRNA Precursor mouse model multicatalytic endopeptidase complex neoplastic cell novel novel therapeutic intervention novel therapeutics phosphoproteomics pre-clinical protein degradation protein folding proteostasis response small molecule inhibitor synergism therapy resistant tool transcriptome sequencing treatment strategy valosin-containing protein

项目摘要

PROJECT SUMMARY/ABSTRACT Multiple myeloma is an aggressive hematologic malignancy that remains incurable despite recent progress. This disease of malignant plasma cells is fundamentally associated with aberrant protein homeostasis, defined by an extremely high burden of immunoglobulin synthesis. Proteasome inhibitors (PIs), a widely-used first-line therapy in myeloma, are thought to directly take advantage of this aberrancy by increasing unfolded protein stress leading to cell death. However, this mechanism is not fully proven, and further insight into PI-induced cell death may lead to more effective combination strategies. In addition, PI resistance is a major clinical problem in myeloma, and new strategies are needed to overcome this condition. Here, we hypothesize that the remodeling of the plasma cell proteome after therapy is central to both PI response and resistance. We specifically propose that proteome remodeling is mediated through rewiring of proteostasis pathways involving chaperones, the VCP/p97 complex, and the ubiquitin-proteasome system, as well as through changes to the alternative splicing landscape, as mediated by post-translational modification of the splicing machinery. To explore this hypothesis we will take advantage of novel pharmacologic and genetic perturbation tools, cellular and biochemical assays, in vivo models, clinical trial genomic data, primary sample analysis, RNA sequencing, and mass spectrometry approaches. The overall goals of this proposal are 1) develop new therapy strategies either in combination with PIs or in the PI-refractory setting, and 2) describe a new, systematic approach to probe the architecture of proteostasis networks. Importantly, our preliminary results challenge existing paradigms related to PI efficacy. In Aim 1, we address paradoxical findings relating the unfolded protein response, the interaction between the p97 degradation machinery and PIs, and the relevance of inducible HSP-family chaperones. We will take advantage of novel pharmacology available to us, including active site and allosteric inhibitors of p97 and allosteric inhibitors of HSP70, in combination with functional genetics by CRISPR interference, to define the role of central protein homeostasis nodes defining PI response and resistance. Furthermore, we will use our unique expertise in pulsed-SILAC proteomics to determine specific substrates of the p97 machinery and the proteasome in the presence of clinically-relevant resistance modifications. Toward Aim 2, our preliminary studies using unbiased mass spectrometry have revealed significant phosphorylation of the spliceosome after PI treatment. We first aim to characterize the relationship between specific alternative splicing events and proteome remodeling after PIs. We then aim to extend our promising preliminary data demonstrating the efficacy of splicing inhibitors as a new anti-myeloma therapy. Overall, the studies here will have a direct impact on delineating the surprisingly broad range of PI-mediated effects in plasma cells, validate the novel therapeutic strategy of splicing inhibition, and reveal new mechanistic approaches to dissect proteostasis networks and alternative splicing that could extend far beyond myeloma.

项目摘要/摘要多发性骨髓瘤是一种侵略性的血液系统恶性肿瘤，尽管最近进展，但仍无法治愈。这种恶性血浆细胞的疾病从根本上与异常蛋白稳态有关，定义为由于免疫球蛋白合成的负担极高。蛋白酶体抑制剂（PIS），一条广泛使用的一线骨髓瘤的疗法被认为可以通过增加展开的蛋白质直接利用这种异常压力导致细胞死亡。但是，这种机制尚未得到充分证明，并进一步了解了PI诱导的细胞死亡可能会导致更有效的组合策略。此外，PI耐药性是主要的临床骨髓瘤的问题以及需要新的策略来克服这种情况。在这里，我们假设治疗后血浆细胞蛋白质组的重塑对于PI反应和抗性既关键。我们特别建议蛋白质组重塑是通过重新布线涉及蛋白质的途径来介导的伴侣，VCP/p97复合物和泛素 - 蛋白酶体系统，以及通过更改通过剪接机械的翻译后修饰介导的替代剪接景观。到探索这一假设，我们将利用新型的药理学和遗传扰动工具，细胞和生化测定，体内模型，临床试验基因组数据，主要样本分析，RNA测序，和质谱方法。该提案的总体目标是1）制定新的治疗策略与PI结合或在PI-RACRACTORY环境中，以及2）描述一种新的系统方法探测蛋白质抗体网络的体系结构。重要的是，我们的初步结果挑战现有与PI功效有关的范例。在AIM 1中，我们解决了与展开蛋白有关的矛盾发现响应，p97降解机械与PI之间的相互作用以及诱导的相关性 HSP家庭伴侣。我们将利用我们可以使用的新型药理学，包括活跃的网站以及HSP70的P97和变构抑制剂的变构抑制剂，结合功能遗传学 CRISPR干扰，定义了定义PI响应和反抗。此外，我们将使用我们在脉冲 - 硅胶蛋白质组学方面的独特专业知识来确定特定 p97机械和蛋白酶体的底物在临床上与抗电阻相关的情况下修改。对于目标2，我们使用无偏质谱法的初步研究表明 PI处理后，剪接体的显着磷酸化。我们首先要表征这种关系在PI后特定的替代剪接事件和蛋白质组重塑之间。然后，我们旨在扩展我们的有希望的初步数据，证明了剪接抑制剂作为一种新的抗肌瘤治疗的功效。总体而言，这里的研究将直接影响描绘出令人惊讶的PI介导的广泛范围在血浆细胞中的作用，验证剪接抑制的新型治疗策略，并揭示新的机械剖析蛋白抑制网络和替代剪接的方法可能远远超出骨髓瘤。