Regulation of protein ubiquitination in hematopoietic cytokine signaling

造血细胞因子信号传导中蛋白质泛素化的调节

• 批准号: 9310835
• 负责人: Wei Tong
• 金额: $ 56.7万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-18 至 2021-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9310835
• 关键词: Acute Myelocytic Leukemia; Adaptor Signaling Protein; Address; Alleles; Attenuated; Automobile Driving; Biochemical; Blood Cells; Bone Marrow Transplantation; CBL Protein; CBL gene; Cell Count; Cell Line; Cell Proliferation; Cells; Chronic; Clonal Hematopoietic Stem Cell; Collaborations; Cytokine Receptors; Cytokine Signaling; Data; Development; Disease; Disease remission; Engineering; Exhibits; FDA approved; Family; Frequencies; Genetic; Goals; Granulocyte-Macrophage Colony-Stimulating Factor Receptors; Growth; Half-Life; Hematopoiesis; Hematopoietic; Hematopoietic Neoplasms; Hematopoietic stem cells; Homeostasis; Human; Hypersensitivity; Interleukin-3; Janus kinase; Janus kinase 2; Juvenile Myelomonocytic Leukemia; Kinetics; Knock-in Mouse; Knock-out; Knockout Mice; MPL gene; Malignant - descriptor; Mediating; Modeling; Molecular; Molecular Conformation; Mus; Mutation; Myelogenous; Myeloproliferative disease; Patients; Pharmacology; Phenocopy; Phosphorylation; Phosphotransferases; Play; Protein Tyrosine Kinase; Proteins; Proteomics; Regulation; Role; Series; Signal Pathway; Signal Transduction; Site; Stem cell transplant; Stem cells; Testing; Therapeutic; Tyrosine; Ubiquitin; Ubiquitination; Ursidae Family; Viral; actionable mutation; cell growth; cytokine; gain of function; human stem cells; in vivo; induced pluripotent stem cell; insight; kinase inhibitor; loss of function mutation; mouse model; mutant; novel; novel therapeutics; outcome forecast; overexpression; patient population; progenitor; receptor; therapeutic target; treatment strategy; ubiquitin-protein ligase

Abstract Hematopoietic stem and progenitor cell (HSPC) homeostasis is regulated by intricate cytokine receptor and tyrosine kinase signaling pathways. Janus Kinase 2 (JAK2) is the key tyrosine kinase in the signaling pathway of an array of hematopoietic receptors, including thrombopoietin (TPO) receptor (MPL) in hematopoietic stem cells (HSCs) and granulocyte macrophage colony-stimulating factor receptor (GM-CSFR) in myeloid progenitors. While JAK2 plays an essential role in hematopoietic development, uncontrolled JAK2 signaling results in hematopoietic malignancies. Gain-of-function JAK2 mutations such as V617F are found in large populations of patients with myeloproliferative neoplasms (MPNs), a clonal HSC disease. Genetically targeting JAK2 abrogates MPN in mice; the JAK2V617F mutation is the driving mutation found in the HSCs of human MPN patients. However, current FDA-approved JAK kinase inhibitors have low curative potential, indicating the need for a better understanding of the regulation of JAK2 for efficient targeting. A number of E3 ubiquitin ligases for JAK2 have been proposed, but none of them, when lost in vivo, increases JAK2 protein level in HSPCs, expands HSC pool, or enhances multiple lineage hematopoiesis. Thus, the E3 ligase(s) regulating JAK2 turnover remain elusive. Through a series of biochemical and functional studies, we found that JAK2 stability is regulated by the CBL family E3 ubiquitin ligases, c-CBL and CBL-b, via the adaptor protein LNK (also called SH2B3). C-Cbl-/- mice phenocopy Lnk-/- mice, exhibiting an augmented HSPC pool with superior transplantability and hypersensitivity to cytokines. Importantly, CBL loss-of-function mutations have been found in a wide range of myeloid malignancies with the most frequent occurrence in chronic and juvenile myelomonocytic leukemia (CMML and JMML), both of which bear poor prognosis. Here, we propose to define the molecular basis underlying the regulation of JAK2 stability and signaling through this novel signaling axis, CBL/LNK/JAK2, and exploit it for therapeutic strategies in treating myeloid neoplasms. The following specific aims will be addressed: 1. Investigate mechanisms by which CBL regulates JAK2 ubiquitination, stability and signaling in hematopoietic cell lines and HSPCs from Cbl deficient and Cbl E3 ligase inactive mouse models. 2. Determine the influence of CBL on the stability of constitutively active JAK2 mutants and mutant JAK2- mediated MPN development. 3. Determine the role of CBL in regulating JAK2 level and signaling in primary human progenitors and explore therapeutic potential of JAK inhibition in treating murine and human myeloid malignancies with CBL mutations. Collectively, our data point to a novel direct role of CBL-mediated JAK2 ubiquitination and degradation in cytokine signaling and hematopoiesis. The proposed studies will likely provide mechanistic insights into the regulation of wild type JAK2 in normal HSPCs as well as JAK2 mutants in MPNs. In addition, our studies may reveal novel therapeutic strategies for the treatment of CBLmut myeloid malignancies with poor prognosis.

摘要 造血干细胞和祖细胞（HSPC）的稳态是由复杂的细胞因子受体和 酪氨酸激酶信号通路。Janus激酶2（JAK 2）是信号通路中的关键酪氨酸激酶 一系列造血受体，包括造血干细胞中的血小板生成素（TPO）受体（MPL 细胞（HSC）和粒细胞巨噬细胞集落刺激因子受体（GM-CSFR） 祖先虽然JAK 2在造血发育中起着重要作用，但不受控制的JAK 2信号传导 导致造血系统恶性肿瘤。JAK 2功能获得性突变，如V617 F，在大部分人中发现。 骨髓增生性肿瘤（MPN）患者群体，一种克隆性HSC疾病。基因靶向 JAK 2消除小鼠中的MPN; JAK 2 V617 F突变是在人MPN的HSC中发现的驱动突变 患者然而，目前FDA批准的JAK激酶抑制剂具有较低的治愈潜力，这表明需要 为了更好地理解JAK 2的调节以实现有效靶向。许多E3泛素连接酶， 已经提出了JAK 2，但是当在体内丢失时，它们中没有一个增加HSPC中的JAK 2蛋白水平， 扩增HSC库或增强多谱系造血。因此，调节JAK 2的E3连接酶 营业额仍然难以预测。通过一系列的生化和功能研究，我们发现JAK 2的稳定性是 通过衔接蛋白LNK（也称为CBL家族E3遍在蛋白连接酶c-CBL和CBL-b）调节 SH2B3）。C-Cbl-/-小鼠与Lnk-/-小鼠表型相似，表现出HSPC池增加，具有上级 可移植性和对细胞因子的超敏性。重要的是，已经发现CBL功能丧失突变， 在广泛的骨髓恶性肿瘤中，最常见于慢性和青少年 骨髓单核细胞白血病（CMML和JMML），这两种疾病预后差。在这里，我们建议定义 JAK 2稳定性调节和通过这种新型信号传导轴的信号传导的分子基础， CBL/LNK/JAK 2的表达，并将其用于治疗髓系肿瘤的治疗策略。以下具体 目标将被处理：1。研究CBL调节JAK 2遍在化、稳定性和功能的机制 在来自Cbl缺陷型和Cbl E3连接酶失活小鼠模型的造血细胞系和HSPC中的信号传导。2. 确定CBL对组成型活性JAK 2突变体和突变JAK 2-突变体的稳定性的影响。 介导的MPN发育。3.确定CBL在调节原发性肝癌中JAK 2水平和信号传导中的作用。 人祖细胞和探索JAK抑制在治疗鼠和人骨髓中治疗潜力 CBL突变的恶性肿瘤。总的来说，我们的数据表明CBL介导的JAK 2 细胞因子信号转导和造血中的泛素化和降解。拟议的研究可能会 为正常HSPC中野生型JAK 2的调节以及HSPC中JAK 2突变体的调节提供了机制见解。 MPN。此外，我们的研究可能揭示治疗CBLmut髓样白血病的新的治疗策略。 预后不良的恶性肿瘤。