Therapeutic targeting of MicroRNA-21 in Myelodysplastic syndromes

描述(申请人提供)：骨髓增生异常综合征(MDS)是一种恶性血液病，以造血干细胞功能障碍为特征，导致血细胞减少。尽管MDS可以转化为急性白血病，但MDS的大部分死亡是由于细胞减少症。由于对导致干细胞发育异常和分化的分子途径的了解有限，有效治疗方法的开发一直受到阻碍。我们证明骨髓抑制因子转化生长因子-�刺激的信号通路在骨髓增生异常综合征的干细胞中过度激活。我们进一步确定转化生长因子-�受体-I激酶的负调控因子Smad7在MDS中显著减少，并通过过度激活Smad2介导的转化生长因子-�信号而导致无效的造血。我们还确定Smad7直接被在MDS干细胞中表达增加的microRNA-21靶向。最重要的是，抑制miR-21可以减弱MDS中过度激活的转化生长因子-�信号，并刺激体内和体外的造血。因此，我们假设miR-21表达增加是导致MDS造血改变的关键分子改变。因此，目标1将研究MDS中miR-21的结构性激活机制。我们已经证明，STAT3在MDS干细胞中选择性地过表达，并在其他模型中被证明调节miR-21的表达。我们将使用生化和功能相结合的方法来确定STAT3的表达/激活在MDS中上调miR-21和刺激转化生长因子-�信号转导中的作用。我们还将确定miR-21在初级MDS样本中是否由于基因扩增或表观遗传学改变而过度表达。目的2确定miR-21过表达在MDS无效造血中的功能作用，并确定其下游效应因子。贫血是MDS的主要临床改变，我们将利用体外人红细胞生成模型来研究miR-21过表达对红细胞分化和增殖的作用。除了Smad7，miR-21还可以靶向许多其他潜在的致病基因。因此，我们将通过靶向在红细胞膜形成中起重要作用的GTP酶交换因子DOCK4来确定miR-21在介导红细胞异型增生中的作用。最后，Aim 3将确定新型临床相关的miR-21抑制剂在体外原发MDS样本和体内骨髓衰竭小鼠模型中的疗效。化学稳定的miR-21反义抑制剂和临床相关的STAT3抑制剂乙胺将在体外和体内测试它们在逆转MDS中异常增加的转化生长因子-�信号方面的有效性。白蛋白/转化生长因子转基因小鼠模型将被用来确定这些抑制剂在体内逆转细胞因子介导的骨髓衰竭的效果。主要的MDS异种移植物也将用于这些研究。这些抑制剂的有效性也将在体外对大量的MDS原代骨髓祖细胞进行评估，并将与患者的特征相关联，以确定那些可能受益于基于抑制miR-21途径的疗法的MDS亚型。

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.