Exploiting myeloma proteome remodeling to extend proteasome inhibitor efficacy

项目总结/摘要 多发性骨髓瘤是一种侵袭性血液系统恶性肿瘤，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， 免疫球蛋白合成的高负荷。蛋白酶体抑制剂（PI），广泛使用的一线药物 骨髓瘤的治疗，被认为是直接利用这种异常，通过增加未折叠蛋白质 压力导致细胞死亡。然而，这一机制尚未得到充分证明，进一步了解PI诱导的 细胞死亡可能导致更有效的组合策略。此外，PI耐药是主要的临床 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是PI应答和抵抗的中心。我们 特别提出蛋白质组重塑是通过蛋白质抑制途径的重新布线介导的， 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 探索这一假设，我们将利用新的药理学和遗传扰动工具，细胞 和生物化学测定，体内模型，临床试验基因组数据，原始样品分析，RNA测序， 和质谱分析方法。该提案的总体目标是：1）开发新的治疗策略 与PI联合使用或在PI难治性环境中使用，以及2）描述一种新的系统性方法， 探索蛋白质稳定网络的结构。重要的是，我们的初步结果挑战了现有的 与PI疗效相关的范例。在目标1中，我们解决了与未折叠蛋白质相关的矛盾发现， 反应，p97降解机制和PI之间的相互作用，以及诱导的 HSP-家族分子伴侣。我们将利用新的药理学提供给我们，包括活性位点， 以及p97的变构抑制剂和HSP 70的变构抑制剂，与功能遗传学结合， CRISPR干扰，以定义定义PI反应的中心蛋白质稳态节点的作用， 阻力此外，我们将利用我们在脉冲SILAC蛋白质组学方面的独特专长， p97机制和蛋白酶体的底物在临床相关耐药性的存在下 修改.为了实现目标2，我们使用无偏质谱法进行的初步研究表明， PI处理后剪接体的显著磷酸化。我们首先要描述 特异性选择性剪接事件与PI后蛋白质组重塑之间的关系。我们的目标是扩大我们的 有希望的初步数据证明剪接抑制剂作为一种新的抗骨髓瘤疗法的疗效。 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。 在浆细胞中的作用，验证了剪接抑制的新治疗策略，并揭示了新的机制， 研究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。

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.