Functional and Molecular Dissection of Mutant Calreticulin in Myeloproliferative Neoplasms

项目总结 尽管钙网蛋白(CALR)突变导致骨髓增生性肿瘤(MPN)的机制 目前，在将这种知识转化为创新的治疗方法方面存在着根本的差距 战略。长期目标是推进CALR突变MPN的治疗，特别是开发新的 具有疾病修改活性和治疗潜力的治疗方法。此应用程序的总体目标是 利用我们通过了解改变的生化特性和独特的分子获得的洞察力 突变的CALR驱动的MPN的依赖性，以确定新的治疗脆弱性，包括在上下文中 CALR/ASXL1共突变。中心假设是CALR突变的造血干细胞(HSC) 具有独特的性质，这是突变体CALR致癌机制的结果，它 我们之前已经澄清过了。我们假设CALR突变的HSC具有的特定属性， 包括蛋白质动态平衡的改变和对关键细胞通路的不同依赖(例如N-糖基化 和蛋白质分泌)以求生存。我们进一步假设，共同操作的遗传事件(例如伴随的 ASXL1突变)改变CALR突变体HSC的染色质状态，以推动MPN的疾病进展。这个 这项拟议研究的基本原理是，一旦我们开发出新的治疗策略来针对独特的 利用CALR突变的HSC的特性，我们将能够优先靶向患者的CALR突变的MPN细胞。这 有可能改变CALR突变的MPN的自然历史，包括在ASXL1共突变的背景下， 这对CALR突变的MPN有负面的预后影响。在强劲的初步数据指引下， 假设将通过追求三个具体目标来检验：1)确定蛋白质的动态平衡和对 CALR突变体HSC中蛋白酶体的抑制；2)决定突变体CALR驱动的分子脆弱性 3)体内检测突变体ASXL1对CALR突变体MPN的影响。在第一个目标下，一个变种人 CALR敲打蛋白(KI)小鼠模型能很好地概括人CALR突变MPN的特征 用来测量CALR-突变型HSC的蛋白质合成和蛋白酶体活性，并确定CALR- 突变型HSC对体内蛋白酶体抑制具有不同的敏感性。在第二个目标下，关键细胞 我们发现突变的表达CALR的造血细胞生存所独有的途径 在体外全基因组CRISPR基因敲除筛选中，将使用功能遗传和 CALRKI突变小鼠的药理学研究。在第三个目标下，突变体ASXL1对组蛋白的影响 CALR突变体HSC的修饰、染色质状态和转录组将使用突变体来确定 ASXL1KI小鼠。这种方法是创新的，通过应用新的小鼠模型，在体外和体内 CRISPR/Cas9基因编辑、化学筛选和基于质谱仪(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CALR突变的新的治疗漏洞 MPN.最终，这些知识有可能对这种疾病的治疗产生革命性的影响。

PROJECT SUMMARY Although the mechanism by which calreticulin (CALR) mutations cause myeloproliferative neoplasms (MPN) has been elucidated, there is currently a fundamental gap in translating this knowledge into innovative therapeutic strategies. The long-term goal is to advance the treatment of CALR-mutant MPN, in particular to develop new therapies with disease-modifying activity and curative potential. The overall objective in this application is to exploit the insights we have gained from understanding the altered biochemical properties and unique molecular dependencies of mutant CALR-driven MPN, to identify novel therapeutic vulnerabilities, including in the context of CALR/ASXL1 co-mutation. The central hypothesis is that CALR-mutant hematopoietic stem cells (HSC) have unique properties, which arise as a consequence of the mechanism of oncogenicity of mutant CALR, which we have previously elucidated. The specific properties, which we hypothesize that CALR-mutant HSC possess, include altered protein homeostasis and a differential dependency on key cellular pathways (e.g. N-glycosylation and protein secretion) for survival. We further hypothesize that co-operating genetic events (e.g. concomitant ASXL1 mutation) alter the chromatin state of CALR-mutant HSC to drive disease progression in MPN. The rationale for the proposed research is that, once we develop novel therapeutic strategies to target the unique properties of CALR-mutant HSC, we will be able to preferentially target CALR-mutant MPN cells in patients. This has the potential to alter the natural history of CALR-mutant MPN, including in the context of ASXL1 co-mutation, which confers a negative prognostic impact on CALR-mutant MPN. Guided by strong preliminary data, the hypothesis will be tested by pursuing three specific aims: 1) Determine protein homeostasis and sensitivity to proteasome inhibition in Calr-mutant HSC; 2) Determine the molecular vulnerabilities of mutant CALR-driven MPN; and 3) Determine the impact of mutant Asxl1 on Calr-mutant MPN in vivo. Under the first aim, a mutant CALR knockin (KI) mouse model that closely recapitulates the features of human CALR-mutant MPN will be employed to measure protein synthesis and proteasome activity in Calr-mutant HSC and to determine if Calr- mutant HSC are differentially sensitive to in vivo proteasome inhibition. Under the second aim, key cellular pathways we have found to be uniquely required for the survival of mutant CALR-expressing hematopoietic cells in an in vitro whole genome CRISPR knockout screen, will be inhibited using functional genetic and pharmacological approaches in mutant CALR KI mice. Under the third aim, the impact of mutant Asxl1 on histone modifications, chromatin state and the transcriptome of Calr-mutant HSC, will be determined using a mutant Asxl1 KI mouse. The approach is innovative through the application of novel murine models, in vitro and in vivo CRISPR/Cas9 gene editing, chemical screening and mass spectrometry (MS)-based quantitative proteomics. The proposed research is significant because it will uncover novel therapeutic vulnerabilities in CALR-mutant MPN. Ultimately, such knowledge has the potential to be transformative in the treatment of this disease.