Signaling Mechanisms of Different Classes of Thrombopoietin Receptor Agonists

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。人们在开发血小板生成生长因子方面付出了巨大的努力。 巨核细胞和血小板发育的主要细胞因子是血小板生成素（Tpo）。 Tpo 通过其受体 Mpl 发出信号，在造血干/祖细胞 (HSPC) 扩增中也发挥着重要作用。 Tpo 与 Mpl 结合会诱导 Mpl/Janus 激酶 2 (JAK2) 复合物发生构象变化，从而导致 JAK2 激活并触发一系列信号传导事件。 FDA 最近批准了两种血小板生成刺激剂：AMG531 (Romiplostim)（一种 Tpo 肽模拟物）和 Eltrombopag (SB497115)（一种小分子 Mpl 激动剂）。我们在合作研究中表明，艾曲波帕家族化合物 SB559457 可刺激人类骨髓 (BM) 巨核细胞生成。引人注目的是，SB559457 和 Eltrombopag 与人 Mpl 的跨膜 (TM) 结构域结合，可强力激活 MAPK 和 mTOR/S6K 通路，但仅最低程度地激活 Stat5。受体 TM 和近膜 (JM) 的 1 螺旋构象对于 JAK2 和下游信号级联的正确激活至关重要。因此，我们假设 Mpl/JAK2 复合物在受到 Tpo/AMG531 或艾曲波帕刺激时采用不同的方向/构象。艾曲波帕连接导致次优 Mpl 构象，部分激活 JAK2/Stat5 通路，这可能导致巨核细胞和 HSPC 中不同的细胞结果。具体目标 1：剖析 Tpo、AMG531 和 Eltrombopag 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 Eltrombopag 诱导的方向依赖性 Mpl 信号传导。具体目标 2：剖析原代 BM 巨核细胞培养物中由 Tpo、AMG531 和 Eltrombopag 诱导的信号通路。在鉴定出对细胞系中 Tpo、AMG531 与 Eltrombopag 功能有差异影响的 Mpl 突变体后，我们将在功能上验证不同 Mpl 激动剂在原代 BM 培养物中诱导的细胞增殖效应和信号通路。具体目标 3：在表达人 Mpl 的 BM 重建小鼠模型中剖析由 Tpo、AMG531 和 Eltrombopag 诱导的方向依赖性信号传导和造血作用。我们将在生理环境中测试最后一个目标所获得的结果。我们将给用野生型或突变型人类 Mpl 重建的小鼠施用 Tpo、AMG531 或艾曲波帕，并比较它们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。本文提出的研究旨在从机制上理解不同类别的血小板生成生长因子如何激活 Mpl/JAK2 介导的信号传导，并调节巨核细胞和 HSPC 扩张。这些研究可能会产生更好的治疗策略，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。 公共卫生相关性：各种血小板生成性疾病，例如癌症患者的特发性血小板生成性紫癜和化疗引起的血小板减少症，可导致危及生命的出血，从而给患者带来巨大风险。这里提出的研究旨在从机制上理解不同类别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。由于这些药物长期治疗的有效性和安全性依赖于它们对谱系定型巨核细胞和多能干/祖细胞的作用，因此这些研究可能会产生更好的治疗策略，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。

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.