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)的动态平衡受复杂的细胞因子受体和 酪氨酸激酶信号通路。Janus Kinase 2(JAK2)是信号通路中的关键酪氨酸激酶 一系列的造血受体，包括造血干细胞中的血小板生成素(TPO)受体(MPL) 髓系细胞与粒-巨噬细胞集落刺激因子受体 祖先。虽然JAK2在造血发育中起着重要作用，但不受控制的JAK2信号转导 会导致血液系统恶性肿瘤。大量发现功能增益JAK2突变，如V617F 骨髓增生性肿瘤(MPN)患者群体，这是一种克隆性HSC疾病。基因靶向 JAK2在小鼠中废除MPN；JAK2V617F突变是在人类MPN的HSCs中发现的驱动突变 病人。然而，目前FDA批准的JAK激酶抑制剂的治疗潜力很低，这表明需要 以更好地理解JAK2的调节，以实现有效的靶向。一些E3泛素连接酶用于 JAK2已经被提出，但当它们在体内丢失时，都不会增加HSPC中JAK2的蛋白水平， 扩大HSC库，或增强多血统造血。因此，调节JAK2的E3连接酶(S) 营业额仍然难以捉摸。通过一系列的生化和功能研究，我们发现JAK2的稳定性是 受CBL家族E3泛素连接酶c-CBL和CBL-b的调节，通过适配蛋白LNK(也称为 Sh2b3)。C-Cbl-/-小鼠表现为LNK-/-小鼠，表现出增强的HSPC池 可移植性和对细胞因子的超敏。重要的是，已经发现了CBL功能丧失突变 在广泛的髓系恶性肿瘤中，以慢性和青少年最常见 粒单核细胞白血病(CMML和JMML)，预后均较差。在这里，我们建议定义 通过这个新的信号轴调节JAK2稳定性和信号的分子基础， CBL/LNK/JAK2，并利用其作为治疗髓系肿瘤的策略。以下是具体的 目标将涉及：1.研究CBL调节JAK2泛素化、稳定性和 Cbl缺陷和Cbl E3连接酶失活小鼠模型中造血细胞系和HSPC中的信号转导。2. 确定CBL对成分活性JAK2突变体和突变体JAK2-的稳定性的影响 介导的MPN发育。3.确定CBL在调节初生稻JAK2水平和信号转导中的作用 人类祖细胞及JAK抑制对小鼠和人髓系细胞的治疗潜力 带有CBL突变的恶性肿瘤。总的来说，我们的数据指出了CBL介导的JAK2的一个新的直接作用 细胞因子信号和造血中的泛素化和降解。拟议的研究很可能 提供对野生型JAK2在正常HSPC和JAK2突变体中的调节的机械性见解 MPN。此外，我们的研究可能为治疗CBLmut髓系疾病提供新的治疗策略。 预后不良的恶性肿瘤。