Epigenetic regulation by microRNA of MDS pathogenesis

摘要 骨髓增生异常综合征(MDS)是一种无法治愈的干细胞疾病，通常进展为髓系白血病。 一种异常的表观遗传调节在疾病的发生、发展和发展中起着至关重要的作用。 以及这种紊乱的进化。到目前为止，MDS的有效治疗被证明是难以捉摸的，并使用了支持性护理 以改善症状，而造血干细胞移植是唯一可用的治疗选择。 MicroRNAs最近被认为与血液系统的恶性肿瘤有关，因为它们抑制了 特定靶基因的表达。在此背景下，我们已经确定了一种致癌的microRNA，它可以增强 干细胞的自我更新，并重塑了血液系统恶性肿瘤的表观遗传格局。为了更好地 通过它的活性了解干细胞生物学和MDS发病机制中的关键途径 提出以下具体目标： 1.确定TET2如何直接参与致癌microRNA在造血中的功能 我们已经产生了有条件地表达这种新发现的致癌microRNA的转基因小鼠 造血室，进而表现出较低水平的10-11易位基因2(TET2) 和全局5-羟甲基胞嘧啶。生物信息学分析一致地确定了 TET2作为该microRNA的潜在靶点，其表达水平与 在我们的MDS患者的大型队列数据集中，TET2使我们假设TET2是其关键靶点。 目前的建议旨在阐明TET2的异位表达对造血的影响。 该microRNA在体内和体外诱导的表型。我们希望我们的研究将提供一个理论基础 靶向TET2治疗血液系统恶性肿瘤的可能性。 2.在MDS的临床前模型中测试microRNA抑制的治疗潜力 我们已经证明这种微小RNA的异常表达与MDS存活率低直接相关。 费率。我们的初步研究结果表明，在体外，阻断这种微小RNA可以降低白血病的致瘤性。 小鼠原代白血病细胞和人白血病细胞系中，伴随TET2的升高， 对正常小鼠造血的最小损伤。在这一目标中，我们建议评估的安全性和有效性 抑制人类原发白血病样本中的microRNA。我们将把这些发现与以下数据相结合 正在进行的骨髓增生异常综合征小鼠模型的临床前试验，并将最终探索 有可能启动有效根除骨髓增生异常综合征的正式临床试验。 3.从机制上阐明microRNA-TET2途径调控的关键靶基因： 为了进一步了解微小RNA抑制TET2蛋白在造血中的后果， 我们将研究异常的microRNA-TET2串扰对TET2蛋白的可能靶点的影响。我们 我们将重点分析这些基因在体内和体外对小鼠的遗传操作 从转基因小鼠中获取造血干细胞，观察其对MDS发病机制的后续影响 由microRNA诱导。 这些拟议的研究不仅将确定microRNA是一种强有力的原癌基因，而且还将定义 作为血液学中最常见事件之一的microRNA-TET2调节网络中的异常 恶性肿瘤，具有重要的治疗意义。 这项工作将在以下专家的支持下进行：David E.Avigan博士 (血液学/肿瘤学)，Jan Vijg(遗传学、表观遗传学和microRNA生物发生)，Julie Teruya-Feldstein (血液病理学)，和Toshio suda(干细胞)。重要的是，保罗·S·弗雷内特博士(干细胞利基)与 与Arthur Skoultchi博士(血液学表观遗传学重编程)一起支持我们的研究项目。

ABSTRACT Myelodysplastic syndrome (MDS) is an incurable stem cell disorder that often progresses to myeloid leukemia. An abnormal epigenetic modulation has been highlighted as playing a crucial part in the pathogenesis, progress, and evolution of this disorder. To date, effective therapy for MDS has proved elusive, with supportive care used to ameliorate symptoms, and hematopoietic stem cell transplantation the only available curative option. MicroRNAs have recently been implicated in hematological malignancies through their inhibition of the expression of specific target genes. In this context, we have identified an oncogenic microRNA that enhances the self-renewal of stem cells and remodels the epigenetic landscape toward hematological malignancies. To better understand through its activity the key pathways involved in stem cell biology and MDS pathogenesis, we propose the following Specific Aims: 1. Determine how TET2 directly contributes to function of an oncogenic microRNA in hematopoiesis We have generated transgenic mice conditionally expressing this newly-identified oncogenic microRNA in the hematopoietic compartment, which in turn exhibits lower levels of ten-eleven translocation gene 2 (TET2) and global 5-hydroxymethylcytosine than control cells. Bioinformatics analyses have consistently identified TET2 as a potential target of this microRNA, whose expression was directly anti-correlated with the levels of TET2 in our large-cohort data set of patients with MDS, leading us to hypothesize that TET2 is its key target. The current proposal aims to elucidate the effect of ectopic expression of TET2 on the hematopoietic phenotypes induced by this microRNA both in vivo and in vitro. We expect our studies will provide a rationale for the therapeutic potential of targeting TET2 for the treatment of hematological malignancies. 2. Test the therapeutic potential of microRNA inhibition in preclinical models of MDS We have shown a direct correlation between aberrant expression of this microRNA and poor MDS survival rates. Our preliminary findings demonstrated in vitro that blocking this microRNA reduces leukemogenicity in mouse primary leukemic cells and in human leukemia cell lines, accompanied by elevation of TET2, with minimal injury to normal murine hematopoieisis. In this aim, we propose to assess the safety and efficacy of inhibition of microRNA in human primary leukemia samples. We will integrate these findings with the data of ongoing pre-clinical trials in faithful mouse models of myelodysplastic syndrome, and will finally explore the potential to initiate formal clinical trial towards effective eradication of myelodysplastic syndrome. 3. To elucidate mechanistically the key target genes regulated by the microRNA-TET2 pathway: To further understand the consequences of repression of TET2 protein by the microRNA in hematopoiesis, we will examine the effects of aberrant microRNA-TET2 cross-talk on putative targets of TET2 protein. We will focus our analysis on the genetic manipulation of these genes, both in vivo and in vitro, in murine hematopoietic stem cells from transgenic mice, to observe the subsequent effects on MDS pathogenesis induced by microRNA. These proposed studies will not only identify microRNA as a potent proto-oncogene, but will also define aberrations in the microRNA-TET2 regulatory network as one of the most frequent events in hematological malignancies, with important therapeutic implications. This work will be conducted with the support of the following experts; Drs. David E. Avigan (Hematology/Oncology), Jan Vijg (Genetics, Epigenetics and microRNA biogenesis), Julie Teruya-Feldstein (Hemato-pathology), and Toshio Suda (Stem Cells). Importantly, Dr. Paul S. Frenette (Stem Cell niche) is closely supporting our research program along with Dr. Arthur Skoultchi (Epigenetic reprogramming in Hematology).