Therapeutic targeting of MicroRNA-21 in Myelodysplastic syndromes

描述（由申请人提供）：骨髓增生异常综合征（MDS）是一种恶性血液病，其特征是造血干细胞功能障碍，导致血细胞计数降低。尽管MDS可以转化为急性白血病，但MDS的大部分死亡率是由于血细胞减少。对导致干细胞发育异常生长和分化的分子途径的有限理解阻碍了有效治疗的发展。我们证明了骨髓抑制细胞因子TGF-β刺激的信号通路在MDS的干细胞中被过度激活。我们进一步确定，SMAD 7，TGF-β受体-I激酶的负调节因子，在MDS中显著降低，并通过过度激活Smad 2介导的TGF-β信号传导导致无效的造血。我们还确定SMAD 7直接被microRNA-21靶向，其在MDS干细胞中的表达增加。最重要的是，miR-21的抑制减弱了MDS中过度活化的TGF-β信号传导，并在体内和体外刺激造血。因此，我们假设miR-21的表达增加是导致MDS造血改变的关键分子改变。因此，本研究的目的1是研究miR-21在MDS中的组成性激活机制。我们已经证明STAT 3在MDS干细胞中选择性过表达，并已显示在其他模型中调节miR-21表达。我们将使用生物化学和功能方法的组合来确定STAT 3表达/激活在MDS中上调miR-21和刺激TGF-β信号传导中的作用。我们还将确定原发性MDS样本中miR-21是否由于基因扩增或表观遗传学改变而过表达。目的2探讨miR-21过表达在MDS无效造血中的作用及其下游效应因子。贫血是MDS的主要临床改变，我们将利用人红细胞生成的体外模型来检查miR-21过表达对红细胞分化和增殖的作用。除了SMAD 7，miR-21还可以靶向许多其他潜在的致病基因。因此，我们将确定miR-21通过靶向在红细胞膜形成中具有重要作用的GTdR交换因子DOCK 4在介导红细胞发育不良中的作用。最后，目标3将确定miR-21的新型临床相关抑制剂在体外原发性MDS样本和体内骨髓衰竭小鼠模型中的疗效。miR-21的化学稳定的反义抑制剂和临床相关的STAT 3抑制剂乙胺嘧啶将在体外和体内测试它们在逆转MDS中异常增加的TGF-β信号传导中的功效。alb/TGF+转基因小鼠模型将用于确定这些抑制剂在体内逆转细胞因子介导的骨髓衰竭中的功效。原发性MDS异种移植物也将用于这些研究。还将在体外对大量原代MDS骨髓祖细胞评价这些抑制剂的疗效，并将其与患者特征相关联，以鉴定可能受益于基于miR-21途径抑制的治疗的MDS亚组。

DESCRIPTION (provided by applicant): Myelodysplastic syndromes (MDS) are hematologic malignancies characterized by hematopoietic stem cell dysfunction that leads to low blood counts. Even though MDS can transform to acute leukemias, most of mortality in MDS is due to cytopenias. Development of effective treatments has been impeded by a limited understanding of the molecular pathways that lead to dysplastic growth and differentiation of stem cells. We demonstrated that the signaling pathways stimulated by myelosuppressive cytokine TGF-� are overactivated in stem cells in MDS. We further determined that SMAD7, a negative regulator of TGF-� receptor-I kinase, is markedly reduced in MDS, and leads to ineffective hematopoiesis by overactivation of Smad2 mediated TGF-� signaling. We also determined that SMAD7 is directly targeted by microRNA-21 whose expression is increased in MDS stem cells. Most importantly, inhibition of miR-21 attenuates the overactivated TGF-� signaling in MDS and stimulates hematopoiesis in vivo and in vitro. Thus, we hypothesize that increased expression of miR-21 is the critical molecular alteration that leads to hematopoietic alterations in MDS. Therefore, Aim 1 will study the mechanisms of constitutive activation of miR-21 in MDS. We have shown that STAT3 is selectively overexpressed in MDS stem cells and has been shown to regulate miR-21 expression in other models. We will determine the role of STAT3 expression/activation in upregulation of miR-21 and stimulation of TGF-� signaling in MDS using a combination of biochemical and functional approaches. We will also determine whether miR-21 is overexpressed due to gene amplification or epigenetic alterations in primary MDS samples. Aim 2 will determine the functional role of miR-21 overexpression on ineffective hematopoiesis in MDS and identify its downstream effectors. Anemia is the predominant clinical alteration in MDS and we will utilize an in vitro model of human erythropoiesis to examine the role of miR-21 overexpression on red cell differentiation and proliferation. In addition to SMAD7, miR-21 can target many other potentially pathogenic genes. Thus, we will determine the role of miR-21 in mediating red cell dysplasia via targeting of a GTPase exchange factor DOCK4 that has essential roles in red cell membrane formation. Finally, Aim 3 will determine the efficacy of novel, clinically relevant inhibitors of miR-21 in primary MDS samples in vitro and in mouse models of bone marrow failure in vivo. Chemically stabilized antisense inhibitors of miR-21 and a clinically relevant STAT3 inhibitor Pyrimethamine will be tested in vitro and in vivo for their efficacy in reversing abnormally increased TGF-� signaling in MDS. The alb/TGF+ transgenic mouse model will be used to determine the efficacy of these inhibitors in reversing cytokine mediated bone marrow failure in vivo. Primary MDS xenografts will also be used for these studies. The efficacy of these inhibitors will also be evaluated in vitro on a large number of primary MDS bone marrow progenitors and will be correlated with patient characteristics to identify subsets of MDS that will potentially benefit from therapeutics based on inhibition of miR-21 pathway.