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）是一种以造血干细胞功能障碍导致低血细胞计数为特征的血液系统恶性肿瘤。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所致。由于对导致干细胞发育不良生长和分化的分子途径了解有限，阻碍了有效治疗方法的发展。我们证明了骨髓抑制细胞因子TGF-刺激的信号通路在MDS干细胞中过度激活。我们进一步确定SMAD7， TGF-受体- 1激酶的负调节因子，在MDS中显著降低，并通过Smad2介导的TGF- 1信号的过度激活导致造血无效。我们还确定SMAD7是MDS干细胞中表达增加的microRNA-21的直接靶向。最重要的是，抑制miR-21可减弱MDS中过度激活的TGF- β信号，刺激体内和体外造血。因此，我们假设miR-21的表达增加是导致MDS中造血改变的关键分子改变。因此，Aim 1将研究miR-21在MDS中的组成性激活机制。我们已经证明STAT3在MDS干细胞中选择性过表达，并在其他模型中调节miR-21的表达。我们将采用生化和功能相结合的方法来确定STAT3表达/激活在MDS中miR-21上调和TGF-信号刺激中的作用。我们还将确定在原发性MDS样本中miR-21是否由于基因扩增或表观遗传改变而过表达。Aim 2将确定miR-21过表达在MDS无效造血中的功能作用，并确定其下游效应物。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GTPase交换因子DOCK4来确定miR-21在介导红细胞发育不良中的作用，该因子在红细胞细胞膜形成中起重要作用。最后，Aim 3将确定新型的、临床相关的miR-21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。化学稳定的反义抑制剂miR-21和临床相关的STAT3抑制剂Pyrimethamine将在体外和体内测试其在逆转MDS中异常升高的TGF- β信号传导的功效。我们将使用alb/TGF+转基因小鼠模型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。原发性MDS异种移植物也将用于这些研究。这些抑制剂的疗效也将在体外对大量原发性MDS骨髓祖细胞进行评估，并将与患者特征相关联，以确定MDS亚群，这些亚群可能受益于基于抑制miR-21途径的治疗方法。

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.