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）：各种血小板性疾病会导致威胁生命的出血。在发展血小板增长因素方面已经做出了巨大的努力。 巨核细胞和血小板发育的主要细胞因子是血小板蛋白（TPO）。通过其受体MPL信号传导的TPO在造血干/祖细胞（HSPC）膨胀中也起着重要作用。 TPO与MPL结合诱导MPL/ Janus激酶2（JAK2）复合物的构象变化，该复合物导致JAK2的激活并触发了一系列信号事件。 FDA最近批准了两种刺激剂：AMG531（Romiplostim），TPO肽模拟物和Eltrombopag（SB497115），一种小分子MPL激动剂。我们在合作研究中表明，Eltrombopagag-家族化合物SB559457刺激了人骨髓（BM）Megakaryopoieis。引人注目的是，与人MPL的跨膜（TM）结构结合的SB559457和Eltrombopag，可牢固地激活MAPK和MTOR/S6K途径，但仅激活STAT5。受体TM和juxtammbrane（JM）的1螺旋构象对于正确激活JAK2和下游信号级联对于级联至关重要。因此，我们假设MPL/JAK2复合物在TPO/AMG531或Eltrombopag刺激时会采用不同的方向/构象。 Eltrombopag连接导致次优MPL构象部分激活JAK2/STAT5途径，这可能会导致巨核细胞和HSPC的不同细胞结局。特定目标1：剖析依赖于方向的MPL信号传导，由TPO，AMG531和Eltrombopag在细胞系中诱导。因此，解决MPL细胞内结构域的结构的尝试失败了，因此我们将向MPL TM/JM螺旋引入丙氨酸插入突变，以剖析TPO，AMG或Eltrombopopag诱导的方向依赖性MPL信号传导。特定目标2：在原代BM巨核细胞培养物中由TPO，AMG531和Eltrombopag诱导的信号传导途径。在鉴定出差异影响TPO的MPL突变体之后，AMG531与细胞系中的Eltrombopag功能相比，我们将在功能上验证一级BM培养物中不同MPL激动剂诱导的细胞增殖效应和信号传导途径。特定目标3：在BM重建的表达人MPL的小鼠模型中，TPO，AMG531和Eltrombopag诱导的依赖方向 - 依赖性信号传导和造血。我们将测试从生理环境中的最后目标中获得的结果。我们将向与野生型或突变的人类MPL重构的小鼠管理TPO，AMG531或Eltrombopag，并比较其支持Megakaryopoiesis和HSPC扩展的能力，以及长期的效能和安全性和安全性和安全性和安全性。此处提出的研究旨在对不同类别的血小板生长因子如何激活MPL/JAK2介导的信号传导，调节巨核细胞和HSPC扩展。这些研究可能会导致更好的治疗策略，以利用不同的血小板促进药物来治疗自身免疫性和医源性血小板减少症。 公共卫生相关性：各种血小板性疾病，例如特发性血小板性紫癜和化学疗法诱发的癌症患者的血小板减少症，可能导致威胁生命的出血，从而对患者施加很大的风险。这里提出的研究旨在实现机械理解，以了解不同类别的血小板生长因子如何引起信号转导并调节造血的多个方面。 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.