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: 10210618
• 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/10210618
• 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/antagonist; 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; 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/antagonist; 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）的机制具有 被阐明了，目前存在将这些知识转化为创新治疗的根本差距 策略。长期目标是进步对Calr突变MPN的治疗，特别是开发新的 疾病改良活性和治疗潜力的疗法。该应用程序的总体目标是 利用我们了解改变的生化特性和独特的分子而获得的见解 突变体CALR驱动的MPN的依赖性，以识别新的治疗脆弱性，包括在上下文中 Calr/asxl1共同致辞。中心假设是Calr突变造血干细胞（HSC） 具有独特的特性，这是由于突变calr致癌的机理而产生的， 我们以前已经阐明了。我们假设Calr突变的HSC拥有的特定特性， 包括改变的蛋白质稳态和对关键细胞途径的差异依赖性（例如N-糖基化 和蛋白质分泌）生存。我们进一步假设合作的遗传事件（例如 ASXL1突变）改变了Calr突变HSC的染色质状态，以驱动MPN中的疾病进展。这 拟议研究的理由是，一旦我们制定了新颖的治疗策略，以针对独特 Calr突变HSC的性质，我们将能够优先针对患者的Calr突变物MPN细胞。这 有可能改变Calr突变MPN的自然历史，包括在ASXL1共同享受的背景下 这赋予了对Calr突变MPN负面的预后影响。在强大的初步数据的指导下 假设将通过追求三个特定目的来检验：1）确定蛋白质稳态和对 Calr突变HSC中的蛋白酶体抑制作用； 2）确定突变体Calr驱动的分子漏洞 mpn; 3）确定突变体ASXL1对体内Calr突变MPN的影响。在第一个目标下，突变体 Calr敲蛋白（Ki）小鼠模型紧密概括了人类Calr突变MPN的特征 用于测量Calr突变HSC中蛋白质合成和蛋白酶体活性，并确定CALR- 突变HSC对体内蛋白酶体抑制差异敏感。在第二个目标下，关键细胞 我们发现途径是表达突变体CALR造血细胞存活所必需的 在体外整个基因组CRISPR敲除筛查中，将使用功能遗传和 突变体Calr Ki小鼠的药理方法。在第三个目标下，突变体ASXL1对组蛋白的影响 将使用突变体确定修饰，染色质状态和CALR突变体HSC的转录组 asxl1 ki鼠标。通过应用新型鼠模型，体外和体内的新型鼠模型，该方法具有创新 CRISPR/CAS9基因编辑，化学筛选和质谱（MS）基于定量蛋白质组学。 拟议的研究很重要，因为它将发现Calr突变的新型治疗脆弱性 MPN。最终，这种知识有可能在治疗这种疾病方面具有变革性。