Functional and Molecular Dissection of Mutant Calreticulin in Myeloproliferative Neoplasms

骨髓增生性肿瘤中突变钙网蛋白的功能和分子解剖

• 批准号: 10436307
• 负责人: Ann Mullally
• 金额: $ 40.2万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2026-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10436307
• 关键词: Acute Myelocytic Leukemia; Acute leukemia; Biochemical; Bioinformatics; Biology; Blood Cells; Blood coagulation; Bone Marrow; CRISPR/Cas technology; Cells; Chemicals; Chromatin; Clinic; Clustered Regularly Interspaced Short Palindromic Repeats; Data; Dependence; Development; Disease; Disease Progression; Dissection; Event; FDA approved; Genes; Genetic; Goals; Hematological Disease; Hematopoietic; Hematopoietic stem cells; Hemorrhage; Human; In Vitro; JAK2 gene; Knock-in Mouse; Knock-out; Knowledge; Mass Spectrum Analysis; Measures; Mission; Molecular; Mutate; Mutation; Myelofibrosis; Myeloproliferative disease; Natural History; Oncogenic; Outcome Study; Pathway interactions; Patients; Pharmacology; Phenotype; Production; Prognosis; Property; Proteasome Inhibition; Protein Biosynthesis; Protein Secretion; Proteomics; Public Health; Publishing; Reagent; Repression; Research; Risk; Stem cell transplant; Testing; Therapeutic; Transcriptional Activation; Translating; United States National Institutes of Health; Work; base; calreticulin; curative treatments; functional genomics; glycosylation; histone modification; human disease; in vivo; inhibitor; innovation; insight; mouse model; multicatalytic endopeptidase complex; mutant; novel; novel strategies; novel therapeutic intervention; novel therapeutics; palliative; prognostic; proteostasis; screening; single cell sequencing; thrombocytosis; transcriptome; translational impact; treatment strategy; whole genome

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.

项目摘要 尽管钙网蛋白（CALR）突变引起骨髓增生性肿瘤（MPN）的机制已经被证实， 目前，在将这些知识转化为创新的治疗方法方面存在着根本性的差距。 战略布局长期目标是推进CALR突变型MPN的治疗，特别是开发新的治疗方法。 具有改善疾病活性和治愈潜力的疗法。本申请的总体目标是 利用我们从了解改变的生化特性和独特的分子生物学特性中获得的见解， 依赖突变CALR驱动的MPN，以确定新的治疗漏洞，包括在上下文中 CALR/ASXL 1共突变。中心假设是CALR突变造血干细胞（HSC） 具有独特的性质，这是由于突变CALR的致癌机制而产生的， 我们先前已经阐明。我们假设CALR突变型HSC具有的特定性质， 包括改变的蛋白质稳态和对关键细胞途径（例如N-糖基化）的差异依赖性 和蛋白质分泌）来维持生存。我们进一步假设，协同遗传事件（例如， ASXL 1突变）改变CALR突变型HSC的染色质状态以驱动MPN中的疾病进展。的 这项研究的基本原理是，一旦我们开发出新的治疗策略， 由于CALR突变型HSC的特性，我们将能够优先靶向患者中的CALR突变型MPN细胞。这 具有改变CALR突变型MPN的自然史的潜力，包括在ASXL 1共突变的背景下， 其对CALR突变型MPN产生负面预后影响。在强有力的初步数据的指导下， 假设将通过追求三个具体目标进行测试：1）确定蛋白质稳态和对 Calr突变型HSC中的蛋白酶体抑制; 2）确定突变型CALR驱动的HSC的分子脆弱性， 3）测定突变体Asxll对Calr-突变体MPN的体内影响。在第一个目标下， CALR敲入（KI）小鼠模型，其紧密地再现了人CALR突变型MPN的特征， 用于测量Calr突变型HSC中的蛋白质合成和蛋白酶体活性，并确定Calr突变型HSC中的蛋白质合成和蛋白酶体活性。 突变HSC对体内蛋白酶体抑制敏感性不同。在第二个目标下，关键细胞 我们已经发现，表达突变CALR的造血细胞的生存所需的独特途径 在体外全基因组CRISPR敲除筛选中，将使用功能性遗传和 在突变CALR KI小鼠中的药理学方法。在第三个目标下，突变体Asxl 1对组蛋白的影响 将使用突变型HSC，测定Calr突变型HSC的修饰、染色质状态和转录组。 Asxl 1 KI小鼠。该方法是创新的，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（MS）的定量蛋白质组学。 这项研究具有重要意义，因为它将揭示CALR突变体的新的治疗漏洞。 MPN。最终，这种知识有可能在治疗这种疾病方面产生变革性的影响。