Functional and Molecular Dissection of Mutant Calreticulin in Myeloproliferative Neoplasms

项目摘要 在理解内质网钙网织蛋白(CALR)的突变是如何发生的 (ER)伴侣蛋白，引起骨髓增生性肿瘤(MPN)。这种鸿沟的持续存在 是一个重要的问题，因为尽管MPN中CALR突变的频率很高(40%)，但 目前还没有针对MPN中CALR突变细胞的治疗策略。此外，尽管 最近FDA批准的JAK2抑制剂可以为MPN患者提供姑息治疗的好处，包括 含有CALR突变的JAK2抑制不会优先针对MPN克隆，因此不会 对这些疾病有治愈的潜力。长期目标是了解通过什么机制 突变的CALR诱导MPN，以利用这些洞察力获得治疗收益。这个项目的总体目标是 其应用是确定突变的CALR如何将造血细胞转化为MPN。中环 假设突变的CALR，凭借其突变特有的C末端和改变的亚细胞 定位发展了新的蛋白质结合相互作用，包括与血小板生成素受体MPL的结合 诱导JAK2-STAT信号通路激活，推动克隆性造血的发展 MPN表型。这项拟议研究的基本原理是，一旦我们了解突变的CALR如何 诱导MPN，将有可能识别可利用的CALR特异性分子依赖 在治疗方面开发新的治疗方法。在强劲的初步数据的指导下，该假说将 通过追求三个特定目标进行测试：1)剖析对MPL和突变体CALR C- 致癌转化的终点；2)确定突变的CALR表达对造血的影响 在体内；以及3)确定差异结合突变CALR的关键蛋白。在第一个目标下，一个 基于突变的结构-功能分析和亚细胞定位研究的结合(已经 在申请者手中确认为可行)，将应用于几个成熟的体外模型 系统，以进一步剖析MPL和突变体CALR之间的相互作用，并确定其在 诱导MPN。在第二个目标下，允许CALR突变的造血干细胞的机制 (HSC)成为克隆性显性并在体内诱导MPN将使用突变的小鼠模型来确定 由CALR驱动的MPN，已经在手头。在第三个目标下，与该领域的一位领导者合作 蛋白质组学，将确定突变体CALR的新的蛋白质结合相互作用及其所需的 转化能力将使用功能基因组学进行验证。这种方法是创新的，通过 新的小鼠模型在体内外CRISPR/Cas9基因组编辑和质谱学中的应用 基于(MS)的定量蛋白质组学。这项拟议的研究具有重要意义，因为它将揭示 突变的CALR驱动的MPN的发病机制。归根结底，这种知识具有 有可能为这种疾病的治疗策略的发展提供信息。

Project Summary There is a fundamental gap in understanding how mutations in calreticulin (CALR), an endoplasmic reticulum (ER) chaperone protein, cause myeloproliferative neoplasms (MPN). Continued existence of this gap represents an important problem because despite the high frequency of CALR mutations in MPN (40%), there are currently no treatment strategies to specifically target CALR-mutant cells in MPN. Furthermore, although the recently FDA-approved JAK2 inhibitors can provide palliative benefit to MPN patients, including those harboring CALR mutations, JAK2 inhibition does not preferentially target the MPN clone and therefore does not have curative potential in these diseases. The long-term goal is to understand the mechanisms by which mutant CALR induces MPN in order to exploit these insights for therapeutic gain. The overall objective in this application is to determine how mutant CALR transforms hematopoietic cells to engender MPN. The central hypothesis is that mutant CALR, by virtue of its mutant-specific C-terminus and altered sub-cellular localization develops novel protein binding interactions, including with the thrombopoietin receptor, MPL that induce JAK2-STAT signaling pathway activation, to drive the development of clonal hematopoiesis and the MPN phenotype. The rationale for the proposed research is that, once we understand how mutant CALR induces MPN, it will be possible to identify CALR-specific molecular dependencies that can be exploited therapeutically to develop novel treatment approaches. Guided by strong preliminary data, the hypothesis will be tested by pursuing three specific aims: 1) Dissect the requirement for MPL and the mutant CALR C- terminus in oncogenic transformation; 2) Determine the effects of mutant CALR expression on hematopoiesis in vivo; and 3) Determine the key proteins that differentially bind mutant CALR. Under the first aim, a combination of mutagenesis-based structure-function analyses and sub-cellular localization studies (already confirmed as feasible in the applicants' hands), will be applied to several well-established in vitro model systems, to further dissect the interaction between MPL and mutant CALR and define its precise role in inducing MPN. Under the second aim, the mechanisms that allow CALR-mutant hematopoietic stem cells (HSC) to become clonally dominant and induce MPN in vivo will be determined using mouse models of mutant CALR-driven MPN, that are already on hand. Under the third aim, in collaboration with a leader-in-the-field in proteomics, the novel protein binding interactions of mutant CALR will be determined and those required for its transforming capacity will be validated using functional genomics. The approach is innovative through the application of novel murine models, in vitro and in vivo CRISPR/Cas9 genome editing and mass spectrometry (MS)-based quantitative proteomics. The proposed research is significant because it will uncover the mechanisms underlying the pathogenesis of mutant CALR-driven MPN. Ultimately, such knowledge has the potential to inform the development of curative treatment strategies for this disease.