Signaling Mechanisms of Different Classes of Thrombopoietin Receptor Agonists

描述(由申请人提供)：各种血小板生成性疾病可导致危及生命的出血。人们在开发促血小板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传递信号，在造血干/祖细胞(HSPC)的扩增中也起着重要作用。TPO与MPL结合可诱导MPL/Janus kinase2(JAK2)复合体的构象改变，从而导致JAK2的激活并触发一系列信号事件。FDA最近批准了两种促血小板生成药物：TPO多肽模拟物AMG531(罗米普替丁)和小分子MPL激动剂Eltrombaog(SB497115)。我们在合作研究中表明，ELTROMBOPG家族化合物SB559457能刺激人骨髓(BM)巨核细胞生成。值得注意的是，与人MPL跨膜(TM)结构域结合的SB559457和Eltrombaog强势激活MAPK和mTOR/S6K通路，但仅最低限度地激活Stat5。受体TM和邻近膜(JM)的1-螺旋构象对于JAK2的正确激活和下游信号级联是至关重要的。因此，我们假设MPL/JAK2复合体在TPO/AMG531或ELTROMBOPG刺激下采用不同的取向/构象。ELTROMBOPG结扎导致MPL构象不佳，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：分析TPO、AMG531和Eltrombopg在细胞系中诱导的定向依赖性MPL信号转导。试图解决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TPO、AMG或Eltrombopg诱导的方向依赖的MPL信号。具体目的2：分析TPO、AMG531和Eltrombopg在原代骨髓巨核细胞培养中诱导的信号转导通路。在确定了在细胞系中影响TPO、AMG531和Eltrombaog功能的MPL突变体后，我们将从功能上验证不同MPL激动剂在原代BM培养中诱导的细胞增殖效应和信号通路。具体目的3：分析TPO、AMG531和Eltrombopg在表达人MPL的BM重组小鼠模型中诱导的定向依赖性信号和造血作用。我们将在生理环境中测试从最后目标获得的结果。我们将用TPO、AMG531或Eltrombopg给与野生型或突变型人MPL重组的小鼠，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。本研究旨在从机制上了解不同类型的促血小板生长因子如何激活MPL/JAK2介导的信号转导，并调节巨核细胞和HSPC的扩增。这些研究可能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。 与公共卫生相关：各种血小板生成性疾病，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，可导致危及生命的出血，从而给患者带来极大的风险。这项研究旨在从机制上理解不同类别的促血小板生长因子是如何引发信号转导和调节多方面的造血的。由于这些药物的长期治疗的有效性和安全性依赖于它们对定向巨核细胞和多潜能干细胞/祖细胞的作用，这些研究可能会导致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的更好的治疗策略。

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.