Role of TET2 in normal hematopoiesis and pathogenesis of myeloid malignancies

描述(申请人提供)：TET2基因突变和/或缺失在包括CMML、MDS、MPN和AML在内的多种形式的髓系恶性肿瘤中频率很高。大多数TET2突变导致无意义/移码，提示功能丧失。因此，TET2被认为是一种可能的肿瘤抑制基因，与髓系恶性肿瘤的发病机制密切相关。小发夹RNA介导的小鼠造血干/祖细胞(HSC/HPC)中TET2的缺失改变了HSC/HPC的细胞分化，并增加了培养中HSC/HPC的比例，提示TET2在调节正常造血中起重要作用。本项目的目的是确定TET2在体内的生理功能以及在髓系恶性肿瘤的发病机制中的作用。我们建立了几个以TET2为靶点的小鼠模型。TET2基因缺失的小鼠在3个月龄时表现出类似于CMML的表型，其中约30%的小鼠在10个月龄前由于进展为红系/髓系白血病样表型而死亡。因此，这些TET2缺失的小鼠使我们能够建立髓系恶性肿瘤患者的模型。在竞争性重建实验中，TET2-/-HSCs的造血重建能力增强。在移植TET2-/-而不是对照BM细胞后，7个受体中有2个表现出与TET2-/-小鼠相似的CMML表型。因此，我们假设TET2通过调节HSC的行为在骨髓生成中发挥肿瘤抑制作用。目的1：通过对TET2-/-和TET2+/-小鼠的表型鉴定，证明TET2在骨髓生成中是一种肿瘤抑制基因。我们将确定与小鼠TET2功能丧失相关的血液学表型。在TET2基因缺失的小鼠中，任何类型的髓系恶性肿瘤的发生频率都很高，这将增加有说服力的证据，支持TET2作为骨髓生成中的肿瘤抑制基因。目的2：明确TET2功能缺失导致小鼠髓系恶性肿瘤的细胞机制。我们将通过连续移植HSC和具有造血特异性TET2失活的小鼠模型来检查TET2缺失是否诱导HSC自主的表型。目的：明确TET2在骨髓生成中发挥肿瘤抑制作用的分子机制。我们将确定TET2和TET2相互作用蛋白的潜在遗传靶点，这将使我们能够揭示TET2的调控网络，并为揭示TET2调节造血和发挥其肿瘤抑制功能的机制铺平道路。这些研究的完成将极大地提高我们对TET2在正常造血和髓系恶性肿瘤发病机制中的作用的理解。这些信息可能导致识别治疗髓系恶性肿瘤患者的新的分子靶点。TET2小鼠模型还提供了一种生物学背景，可以在其中测试和开发药物和其他疗法。

DESCRIPTION (provided by applicant): TET2 gene is mutated and/or deleted with high frequencies in multiple forms of myeloid malignancies including CMML, MDS, MPN and AML. The majority of the TET2 mutations lead to nonsense/frameshift, suggesting loss of function. Therefore, TET2 has been speculated to be a putative tumor suppressor gene that is strongly implicated in the pathogenesis of myeloid malignancies. Small hairpin RNA-mediated depletion of Tet2 in murine hematopoietic stem/progenitor cells (HSC/HPC) alters their cell differentiation and increase the proportion of HSC/HPC in culture, suggesting that Tet2 is important for regulating normal hematopoiesis. The objective of this project is to define the physiological function of Tet2 in vivo and in the pathogenesis of myeloid malignancies. We generated several Tet2-targeted murine models. Tet2- null mice displayed a phenotype resembling CMML at 3 months of age, and ~30% of these mice died by 10 months of age due to the progression to an erythroid/myeloid leukemia-like phenotype. Therefore, these Tet2-null mice allow us to model patients with myeloid malignancy. In a competitive reconstitution assay, Tet2-/- HSCs had an increased hematopoietic repopulating capacity. After transplantation of Tet2-/-, but not control BM cells, two of the seven recipients exhibited CMML phenotype similar to that of Tet2-/- mice. We, therefore, hypothesize that Tet2 acts as a tumor suppressor in myelopoiesis by regulating the behavior of HSC. Three specific aims are proposed: Aim 1: To prove Tet2 acts as a tumor suppressor gene in myelopoiesis by characterizing the phenotype of Tet2-/- and Tet2+/- mice. We will determine the hematological phenotype associated with the loss of Tet2 function in mice. The development of any type of myeloid malignancies at an elevated frequency in Tet2-null mice will add persuasive evidence to support Tet2 as a tumor suppressor gene in myelopoiesis. Aim 2: To define the cellular mechanisms by which loss of Tet2 function in mice leads to myeloid malignancies. We will examine if deletion of Tet2 induces a phenotype that is HSC autonomous by using serial transplantation of HSCs and mouse models with hematopoiesis-specific Tet2 inactivation. Aim 3: To define the molecular mechanisms by which Tet2 exerts its tumor suppressor function in myelopoiesis. We will identify potential genetic targets of Tet2 and Tet2-interacting proteins, which will allow us to unveil the regulatory network of Tet2 and pave a way for uncovering the mechanism by which Tet2 regulates hematopoiesis and exerts its tumor suppressor function. The completion of these studies will greatly improve our understanding of the role of Tet2 in normal hematopoiesis and pathogenesis of myeloid malignancies. This information could lead to the identification of novel molecular targets for the treatment of patients with myeloid malignancies. The Tet2 murine models also offer a biological context in which drugs and other therapies can be tested and developed.