Functional and Molecular Dissection of Mutant Calreticulin in Myeloproliferative Neoplasms

项目摘要 在理解钙网蛋白（CALR），一种内质网， (ER)伴侣蛋白，导致骨髓增生性肿瘤（MPN）。这一差距的持续存在 这是一个重要的问题，因为尽管MPN中CALR突变的频率很高（40%）， 目前没有特异性靶向MPN中CALR突变细胞的治疗策略。此外，虽然 最近FDA批准的JAK 2抑制剂可以为MPN患者提供姑息性益处，包括那些 携带CALR突变，JAK 2抑制不优先靶向MPN克隆，因此不 对这些疾病有治疗潜力。长期目标是了解 突变CALR诱导MPN，以便利用这些见解获得治疗效果。在这方面的总体目标 应用是确定突变CALR如何转化造血细胞以产生MPN。中央 假设是突变CALR，由于其突变特异性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的相互作用， 诱导JAK 2-STAT信号通路激活，以驱动克隆造血的发展， MPN表型。这项研究的基本原理是，一旦我们了解了突变的CALR是如何 诱导MPN，将有可能确定可以利用的CALR特异性分子依赖性， 在治疗上开发新的治疗方法。在强有力的初步数据的指导下，该假设将 通过追求三个具体目标进行测试：1）剖析对MPL和突变CALR C- 2）确定突变型CALR表达对造血功能的影响; 3）确定差异结合突变CALR的关键蛋白。在第一个目标下， 基于诱变的结构-功能分析和亚细胞定位研究的组合（已经 在申请人手中被证实是可行的），将被应用于几个良好建立的体外模型 系统，以进一步剖析MPL和突变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突变造血干细胞的机制 (HSC)将使用突变体的小鼠模型来确定成为克隆显性并在体内诱导MPN的可能性。 CALR驱动的MPN，已经在手。在第三个目标下，与该领域的一位领导人合作， 蛋白质组学，新的蛋白质结合突变CALR的相互作用将被确定和所需的， 将利用功能基因组学验证转化能力。该方法是创新的， 新型鼠模型的应用，体外和体内CRISPR/Cas9基因组编辑和质谱 （MS）为基础的定量蛋白质组学。这项拟议中的研究意义重大，因为它将揭示 突变CALR驱动的MPN的发病机制。最终，这些知识 潜在的信息为这种疾病的治愈性治疗策略的发展。

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.