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

• 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
• 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

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.

项目概要/摘要 多发性骨髓瘤是一种侵袭性血液恶性肿瘤，尽管最近取得了进展，但仍然无法治愈。 这种恶性浆细胞疾病从根本上与异常的蛋白质稳态有关，定义为 由于免疫球蛋白合成的极高负担。蛋白酶体抑制剂（PI），广泛使用的一线药物 骨髓瘤的治疗被认为可以通过增加未折叠蛋白来直接利用这种异常 压力导致细胞死亡。然而，这一机制尚未得到充分证明，需要进一步深入了解 PI 诱导的 细胞死亡可能会导致更有效的组合策略。此外，PI耐药是临床上的一个主要问题。 骨髓瘤的问题，需要新的策略来克服这种情况。在这里，我们假设 治疗后浆细胞蛋白质组的重塑对于 PI 反应和耐药性至关重要。我们 特别提出蛋白质组重塑是通过蛋白质稳态途径的重新布线来介导的，涉及 分子伴侣、VCP/p97 复合物和泛素-蛋白酶体系统，以及通过改变 选择性剪接景观，由剪接机制的翻译后修饰介导。到 为了探索这个假设，我们将利用新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、初级样本分析、RNA 测序、 和质谱方法。该提案的总体目标是 1) 开发新的治疗策略 与 PI 结合或在 PI 难治性环境中，2) 描述一种新的、系统的方法 探索蛋白质稳态网络的结构。重要的是，我们的初步结果挑战了现有的 与 PI 功效相关的范式。在目标 1 中，我们解决了与未折叠蛋白质相关的矛盾发现 响应、p97 降解机制和 PI 之间的相互作用以及诱导型的相关性 HSP-家庭伴侣。我们将利用我们可用的新颖药理学，包括活性位点 和 p97 的变构抑制剂和 HSP70 的变构抑制剂，与功能遗传学相结合 CRISPR 干扰，定义中央蛋白质稳态节点的作用，定义 PI 反应和 反抗。此外，我们将利用我们在脉冲 SILAC 蛋白质组学方面的独特专业知识来确定特定的 存在临床相关耐药性时 p97 机制和蛋白酶体的底物 修改。为了实现目标 2，我们使用无偏质谱法的初步研究表明 PI处理后剪接体显着磷酸化。我们首先旨在描述这种关系的特征 PI 后特定的选择性剪接事件和蛋白质组重塑之间的关系。然后我们的目标是扩展我们的 有希望的初步数据证明了剪接抑制剂作为一种新的抗骨髓瘤疗法的功效。 总的来说，这里的研究将对描绘 PI 介导的令人惊讶的广泛范围产生直接影响。 浆细胞中的作用，验证剪接抑制的新治疗策略，并揭示新机制 剖析蛋白质稳态网络和选择性剪接的方法可能远远超出骨髓瘤范围。