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）是一种血液恶性肿瘤，其特征是造血干细胞功能障碍，导致低血细胞计数。尽管MDS可以转变为急性白血病，但MDS的大部分死亡是由于血细胞减少所致。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，阻碍了有效治疗的发展。我们证明骨髓抑制性细胞因子 TGF-β 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-β受体-I激酶的负调节因子）在MDS中显着减少，并通过Smad2介导的TGF-β信号过度激活导致造血无效。我们还确定 SMAD7 是 microRNA-21 的直接靶向，而 microRNA-21 的表达在 MDS 干细胞中增加。最重要的是，抑制 miR-21 可以减弱 MDS 中过度激活的 TGF-β 信号传导，并刺激体内和体外的造血作用。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组成型激活的机制。我们已经证明 STAT3 在 MDS 干细胞中选择性过表达，并且在其他模型中也被证明可以调节 miR-21 的表达。我们将结合生化和功能方法来确定 STAT3 表达/激活在 MDS 中 miR-21 上调和 TGF-β 信号传导刺激中的作用。我们还将确定 miR-21 是否由于原代 MDS 样本中的基因扩增或表观遗传改变而过度表达。目标 2 将确定 miR-21 过表达对 MDS 无效造血的功能作用，并确定其下游效应器。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 SMAD7 之外，miR-21 还可以靶向许多其他潜在致病基因。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 在介导红细胞发育不良中的作用。最后，目标 3 将确定新型临床相关 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功效。化学稳定的 miR-21 反义抑制剂和临床相关的 STAT3 抑制剂乙胺嘧啶将在体外和体内测试其逆转 MDS 中异常增加的 TGF-β 信号传导的功效。 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的功效。原代 MDS 异种移植物也将用于这些研究。这些抑制剂的功效还将在体外对大量原代 MDS 骨髓祖细胞进行评估，并将与患者特征相关联，以确定可能受益于基于 miR-21 通路抑制的治疗的 MDS 子集。

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.