Signaling Mechanisms of Different Classes of Thrombopoietin Receptor Agonists

不同类别血小板生成素受体激动剂的信号传导机制

• 批准号: 7875957
• 负责人: Wei Tong
• 金额: $ 20.56万
• 依托单位: CHILDREN'S HOSP OF PHILADELPHIA
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2012-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7875957
• 关键词: 70-kDa Ribosomal Protein S6 Kinases; AMG531; Acute Myelocytic Leukemia; Adopted; Adverse effects; Affect; Agonist; Alanine; Antibodies; Attenuated; Autoimmune Process; Binding; Blast Cell Proliferation; Blood Platelets; Bone Marrow; Bone Marrow Transplantation; CD34 gene; Cancer Patient; Cell Line; Cell Survival; Cells; Clinical Trials; Commit; Complex; Cytokine Receptors; Data; Development; Disease; Dysmyelopoietic Syndromes; Event; Extracellular Domain; Family; Generations; Growth; Growth Factor; Hematopoiesis; Hematopoietic; Hematopoietic stem cells; Hemorrhage; Histology; Human; In Vitro; Insertion Mutation; Janus kinase 2; Lead; Life; Ligands; Ligation; Long-Term Effects; MAP Kinase Gene; Mediating; Megakaryocytes; Megakaryocytopoieses; Modeling; Molecular Conformation; Mus; Mutagenesis; Mutation; Myeloproliferative disease; Outcome; Pathway interactions; Patients; Peptides; Pharmaceutical Preparations; Physiological; Play; Proliferating; Proteins; Purpura; Recombinants; Reporting; Research; Risk; Role; Safety; Scanning; Signal Pathway; Signal Transduction; Stem cells; Structure; Test Result; Therapeutic; Thrombocytopenia; Thrombopoiesis; Thrombopoietin; Transmembrane Domain; United States Food and Drug Administration; bone marrow reticulin fibrosis; chemotherapy; cytokine; human FRAP1 protein; human MPL protein; in vivo; interest; mimetics; mouse model; mutant; patient population; public health relevance; receptor; reconstitution; small molecule; stem

DESCRIPTION (provided by applicant): Various thrombocytopoietic disorders can cause life-threatening hemorrhage. There have been intense efforts in developing thrombopoietic growth factors. The primary cytokine for megakaryocyte and platelet development is thrombopoietin (Tpo). Tpo, signaling through its receptor Mpl, also plays important roles in hematopoietic stem/progenitor cell (HSPC) expansion. Tpo binding to Mpl induces a conformational change in the Mpl/ Janus kinase 2 (JAK2) complex, which leads to the activation of JAK2 and triggers a cascade of signaling events. The FDA recently approved two thrombopoiesis- stimulating agents: AMG531 (Romiplostim), a Tpo peptide mimetic, and Eltrombopag (SB497115), a small molecule Mpl agonist. We show in collaborative studies that Eltrombopag- family compound, SB559457, stimulate human bone marrow (BM) megakaryopoieis. Strikingly, SB559457 and Eltrombopag, which bind to the transmembrane (TM) domain of human Mpl, robustly activate MAPK and mTOR/S6K pathways, but only minimally activate Stat5. The 1-helical conformation of the receptor TM and juxtamembrane (JM) is critical for the proper activation of JAK2 and downstream signaling cascade. We therefore hypothesize that the Mpl/JAK2 complex adopts different orientation/ conformations when stimulated by Tpo/AMG531 or Eltrombopag. Eltrombopag ligation results in a suboptimal Mpl conformation that partially activates JAK2/Stat5 pathway, which may result in different cellular outcomes in megakaryocytes and HSPCs. Specific Aim 1: Dissect orientation- dependent Mpl signaling induced by Tpo, AMG531, and Eltrombopag in cell lines. Attempts to solve the structure of Mpl intracellular domain have failed, we therefore will introduce alanine insertion mutations to the Mpl TM/JM helix to dissect the orientation-dependent Mpl signaling induced by Tpo, AMG or Eltrombopag. Specific Aim 2: Dissect signaling pathways induced by Tpo, AMG531, and Eltrombopag in primary BM megakaryocyte culture. After identifying the Mpl mutants that differentially affect Tpo, AMG531 versus Eltrombopag function in cell lines, we will functionally validate the cell proliferative effects and signaling pathways induced by different Mpl agonists in primary BM cultures. Specific Aim 3: Dissect orientation- dependent signaling and hematopoiesis induced by Tpo, AMG531, and Eltrombopag in BM reconstituted mouse models expressing human Mpl. We will test the results gained from last aims in physiological settings. We will administrate Tpo, AMG531, or Eltrombopag to mice that are reconstituted with wild type or mutant human Mpl, and compare their abilities to support megakaryopoiesis and HSPC expansion, as well as the long-term efficacy and safety profiles. The research proposed here aims to achieve a mechanistic understanding of how different classes of thrombopoietic growth factors activate Mpl/JAK2- mediated signaling, and regulate megakaryocytes and HSPC expansion. These studies could potentially lead to better therapeutic strategies in utilizing different thrombopoietic promoting drugs in the treatment of autoimmune and iatrogenic thrombocytopenia. PUBLIC HEALTH RELEVANCE: Various thrombocytopoietic disorders, such as idiopathic thrombopoietic purpura and chemotherapy- induced thrombocytopenia in cancer patients, can cause life-threatening bleeding, thereby imposing great risk to the patients. The research proposed here aims to achieve a mechanistic understanding of how different classes of thrombopoietic growth factors elicit signaling transduction and regulate multiple aspects of hematopoiesis. Since the efficacy and safety of the long-term therapy with these drugs rely on their action on both lineage committed megakaryocytes and multi-potential stem/progenitor cells, these studies could potentially lead to better therapeutic strategies in utilizing different thrombopoietic promoting drugs in the treatment of autoimmune and iatrogenic thrombocytopenia.

描述（由申请人提供）：各种血小板生成障碍可导致危及生命的出血。在开发血小板生成生长因子方面已经进行了大量的努力。 巨核细胞和血小板发育的主要细胞因子是血小板生成素（Tpo）。通过其受体Mpl进行信号传导的Tpo在造血干/祖细胞（HSPC）扩增中也起重要作用。Tpo与Mpl的结合诱导Mpl/ Janus激酶2（JAK 2）复合物的构象变化，这导致JAK 2的活化并触发信号传导事件的级联。FDA最近批准了两种血小板生成刺激剂：AMG 531（Romiplostim），一种Tpo肽模拟物，和Elt（SB 497115），一种小分子Mpl激动剂.我们在合作研究中发现，艾曲泊帕家族化合物SB 559457可刺激人骨髓（BM）巨核细胞生成。引人注目的是，SB 559457和艾曲泊帕结合人Mpl的跨膜（TM）结构域，强烈激活MAPK和mTOR/S6 K途径，但仅最低限度地激活Stat 5。受体TM和质膜（JM）的1-螺旋构象对于JAK 2和下游信号级联的适当激活是关键的。因此，我们假设Mpl/JAK 2复合物在受到Tpo/AMG 531或艾曲泊帕刺激时采用不同的取向/构象。艾曲泊帕连接导致次优Mpl构象，部分激活JAK 2/Stat 5通路，这可能导致巨核细胞和HSPC中不同的细胞结局。具体目的1：剖析Tpo、AMG 531和艾曲泊帕在细胞系中诱导的方向依赖性Mpl信号传导。解决Mpl胞内结构域的结构的尝试失败了，因此我们将在Mpl TM/JM螺旋中引入丙氨酸插入突变以剖析由Tpo、AMG或艾曲泊帕诱导的取向依赖性Mpl信号传导。具体目的2：在原代BM巨核细胞培养物中分析Tpo、AMG 531和艾曲泊帕诱导的信号传导通路。在鉴定了差异影响细胞系中Tpo、AMG 531与艾曲泊帕功能的Mpl突变体后，我们将在原代BM培养物中功能性验证不同Mpl激动剂诱导的细胞增殖效应和信号传导途径。具体目标3：在表达人Mpl的BM重建小鼠模型中解剖Tpo、AMG 531和艾曲泊帕诱导的方向依赖性信号传导和造血。我们将在生理环境中测试最后一次目标所获得的结果。我们将对用野生型或突变型人Mpl重建的小鼠给予Tpo、AMG 531或艾曲泊帕，并比较其支持巨核细胞生成和HSPC扩增的能力以及长期疗效和安全性特征。本文提出的研究旨在从机制上理解不同类型的血小板生成生长因子如何激活Mpl/JAK 2介导的信号传导，并调节巨核细胞和HSPC扩增。这些研究可能会导致更好的治疗策略，利用不同的促血小板生成药物治疗自身免疫性和医源性血小板减少症。 公共卫生关系：各种血小板生成障碍，如癌症患者中的特发性血小板生成性紫癜和化疗诱导的血小板减少症，可导致危及生命的出血，从而给患者带来极大的风险。本文提出的研究旨在从机制上理解不同类型的血小板生成生长因子如何引发信号转导并调节造血的多个方面。由于这些药物的长期治疗的疗效和安全性依赖于它们对谱系定向巨核细胞和多潜能干/祖细胞的作用，因此这些研究可能会在利用不同的促血小板生成药物治疗自身免疫性和医源性血小板减少症中产生更好的治疗策略。