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: 8100217
• 负责人: Wei Tong
• 金额: $ 24.68万
• 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 2013-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8100217
• 关键词: 1-Phosphatidylinositol 3-Kinase; 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; 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。在开发血小板生长因子方面已经有了很大的努力。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）。Tpo通过其受体Mpl发出信号，在造血干细胞/祖细胞（HSPC）扩增中也起重要作用。Tpo结合Mpl诱导Mpl/ Janus kinase 2 （JAK2）复合物的构象变化，导致JAK2的激活并触发一系列信号事件。FDA最近批准了两种血栓生成刺激剂：AMG531 (Romiplostim)，一种Tpo肽模拟物，和Eltrombopag (SB497115)，一种小分子Mpl激动剂。我们在合作研究中发现，Eltrombopag家族化合物SB559457可以刺激人骨髓（BM）巨核生成物。引人注目的是，SB559457和Eltrombopag结合到人类Mpl的跨膜（TM）结构域，可以强烈激活MAPK和mTOR/S6K通路，但只能最低限度地激活Stat5。受体TM和近膜（JM）的1螺旋构象对JAK2的正常激活和下游信号级联至关重要。因此，我们假设Mpl/JAK2复合物在Tpo/AMG531或Eltrombopag的刺激下呈现不同的取向/构象。Eltrombopag连接导致次优Mpl构象，部分激活JAK2/Stat5通路，这可能导致巨核细胞和HSPCs的不同细胞结果。特异性目的1：解剖Tpo、AMG531和Eltrombopag在细胞系中诱导的定向依赖的Mpl信号。试图解决Mpl胞内结构域的结构失败了，因此我们将引入Mpl TM/JM螺旋的丙氨酸插入突变，以解剖由Tpo， AMG或Eltrombopag诱导的方向依赖性Mpl信号传导。特异性目的2：解剖Tpo、AMG531和Eltrombopag在原代BM巨核细胞培养中诱导的信号通路。在鉴定了不同Mpl突变体对细胞系中Tpo、AMG531和Eltrombopag功能的影响后，我们将在原代BM培养中功能性地验证不同Mpl激动剂诱导的细胞增殖效应和信号通路。特异性目的3：解剖Tpo、AMG531和Eltrombopag在表达人Mpl的BM重组小鼠模型中诱导的定向依赖性信号传导和造血。我们将在生理环境中测试从上次目标中获得的结果。我们将给药Tpo、AMG531或Eltrombopag给药于用野生型或突变型人Mpl重组的小鼠，比较它们支持巨核生成和HSPC扩增的能力，以及长期疗效和安全性。本文提出的研究旨在了解不同类型的血小板生成生长因子如何激活Mpl/JAK2介导的信号，并调节巨核细胞和HSPC的扩增。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药物治疗自身免疫性和医源性血小板减少症。