Epigenetic regulation by microRNA of MDS pathogenesis

描述（由申请人提供）：骨髓增生异常综合征（MDS）是一种无法治愈的干细胞疾病，通常进展为骨髓性白血病。一种异常的表观遗传调节在这种疾病的发病、进展和进化中起着至关重要的作用。迄今为止，MDS的有效治疗被证明是难以捉摸的，支持性治疗用于改善症状，而造血干细胞移植是唯一可用的治疗选择。通过抑制特定靶基因的表达，MicroRNAs最近被认为与血液恶性肿瘤有关。在这种情况下，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，并重塑血液系统恶性肿瘤的表观遗传格局。为了通过其活性更好地了解参与干细胞生物学和MDS发病机制的关键途径，我们提出以下具体目标：我们在造血室中培养了有条件地表达这种新发现的致癌microRNA的转基因小鼠，其10 - 11易位基因2 （TET2）和全局5-羟甲基胞嘧啶的水平低于对照细胞。生物信息学分析一致认为TET2是该microRNA的潜在靶点，在我们的MDS患者大队列数据集中，其表达与TET2水平直接抗相关，这使我们假设TET2是其关键靶点。本研究旨在阐明TET2异位表达对该microRNA诱导的造血表型在体内和体外的影响。我们期望我们的研究将为靶向TET2治疗血液系统恶性肿瘤的治疗潜力提供理论依据。2. 在MDS临床前模型中测试抑制microRNA的治疗潜力。我们已经证明了这种microRNA的异常表达与MDS的低生存率之间的直接相关性。我们的初步研究结果表明，在体外阻断该microRNA可降低小鼠原发性白血病细胞和人类白血病细胞系的白血病发生性，并伴有TET2的升高，对正常小鼠造血的损伤最小。在这个目的中，我们建议评估在人类原发性白血病样本中抑制microRNA的安全性和有效性。我们将把这些发现与正在进行的骨髓增生异常综合征小鼠模型临床前试验的数据结合起来，并最终探索启动有效根除骨髓增生异常综合征的正式临床试验的可能性。3. 为了阐明microRNA-TET2通路调控的关键靶基因的机制：为了进一步了解造血过程中microRNA抑制TET2蛋白的后果，我们将研究异常的microRNA-TET2串扰对TET2蛋白可能靶点的影响。我们将重点分析这些基因在体内和体外对转基因小鼠小鼠造血干细胞的遗传操作，观察microRNA对MDS发病机制的后续影响。这些拟议的研究不仅将确定microRNA作为一种有效的原癌基因，而且还将确定microRNA- tet2调控网络中的畸变是血液系统恶性肿瘤中最常见的事件之一，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：Drs。David E. Avigan（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，Paul S. Frenette博士（干细胞利基）与Arthur Skoultchi博士（血液学表观遗传重编程）密切支持我们的研究项目。

DESCRIPTION (provided by applicant): 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 hematopoiesis. 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).