The role and functional mechanism of TET1 in MLL-rearranged leukemia

描述(由申请人提供)：Ten-Eleven易位(Tet)蛋白(包括Tet1/2/3)最近被确定为表观遗传调控的关键调节因子，通过将5-甲基胞嘧啶(5mC)转化为5-羟甲基胞嘧啶(5hmC)，导致主动或被动的DNA去甲基化。尽管TET基因家族的创始成员TET1首次被鉴定为急性髓系白血病(AML)混合系白血病(MLL)基因的融合伙伴，但其在白血病中的病理作用尚不清楚。与已报道的三种TET基因在各种实体瘤中的下调和潜在的肿瘤抑制作用以及TET2在血液系统恶性肿瘤中频繁的功能缺失突变相反，我们最近发现TET1(而不是TET2/3)在MLL重排的AML中显著上调；MLL融合与TET1的启动子区域结合并直接促进其表达，这与5hmC的升高有关。通过小发夹RNA(ShRNAs)或基因敲除Tet1的表达可显著抑制MLL-AF9介导的细胞转化和体内白血病的发生。此外，小干扰RNA(SiRNA)寡核苷酸抑制TET1的表达显著降低了人MLL-AF9白血病细胞的存活率和生长速度，并增加了细胞的凋亡率。此外，我们还发现MLL融合的几个重要下游直接靶点，如HOXA9、Meis1和PBX3，已被证明对MLL重排AML的诱导/维持和白血病干细胞(LSCs)的自我更新至关重要，也是TET1的直接靶基因。假设：TET1通过促进一组致癌靶基因的表达和抑制一组抑癌靶基因的表达，在MLL重排白血病的发病机制中发挥重要的致癌作用，因此TET1是治疗MLL重排白血病的有效靶点。具体目的：1)确定Tet1在MLL重排白血病的发生、维持及相关白血病干细胞(LSCs)自我更新中的重要作用；2)确定MLL重排白血病细胞中Tet1的关键靶基因。研究设计：1)首先，我们将进行体外结合和转录研究，以验证TET1是所有五种主要MLL融合(即MLL-AF9、-AF6、-AF10、-ELL和-ENL)的直接靶标。接下来，我们将使用Tet1基因敲除/基因敲除模型和MLL融合骨髓移植模型来确定Tet1在MLL重排AML的所有五个主要亚型的发展和维持以及相关LSCs的自我更新中的重要作用。我们还将使用异种移植模型来确定靶向TET1是否是治疗MLL重排AML的潜在治疗方法(AIM 1)。2)我们将通过芯片序列、单碱基分辨5hmC/5mC-Seq和RNA-Seq来确定Tet1的关键潜在靶点，然后对一组在MLL重排白血病中选择的Tet1靶点进行验证和功能研究，以阐明其潜在的分子机制(目标2)。

DESCRIPTION (provided by applicant): The Ten-eleven translocation (Tet) proteins (including Tet1/2/3) have been recently identified as critical regulators in epigenetic regulation through converting 5-methylcytosine (5mC) to 5- hydroxymethylcytosine (5hmC), leading to active or passive DNA demethylation. Although TET1, the founding member of the TET gene family, was first identified as a fusion partner of the mixed lineage leukemia (MLL) gene in acute myeloid leukemia (AML), its pathological role in leukemia is unknown. In contrast to the down-regulation and potential tumor suppressor roles of all three TET genes reported in various solid tumors and the frequent loss-of-function mutations of TET2 in hematopoietic malignancies, we recently found that TET1 (but not TET2 /3) was significantly up-regulated in MLL-rearranged AML; MLL fusions bind to the promoter region of TET1 and promote its expression directly, which is associated with an increased level of 5hmC. Depletion of Tet1 expression by small hairpin RNAs (shRNAs) or genetic knockout significantly inhibits MLL-AF9-mediated cell transformation in vitro and leukemogenesis in vivo. In addition, knockdown of TET1 expression by small interfering RNA (siRNA) oligos significantly decreases viability/growth and increases apoptosis of human MLL- AF9 leukemic cells. Furthermore, we showed that several essential downstream direct targets of MLL fusions, such as HOXA9, MEIS1, and PBX3 that have been shown to be critical for the induction/maintenance and leukemia stem cells (LSCs) self-renewal of MLL-rearranged AML, are also direct target genes of TET1. Hypothesis: TET1 plays an essential oncogenic role in the pathogenesis of MLL-rearranged leukemia through promoting expression of a group of oncogenic target genes and repressing expression of a group of tumor- suppressor target genes, and thus TET1 is a viable therapeutic target in treating MLL-rearranged leukemia. Specific Aims: 1) To determine the essential role of Tet1 in both development and maintenance of MLL- rearranged leukemia as well as in the self-renewal of relevant leukemia stem cells (LSCs); 2) To identify critical target genes of Tet1 in MLL-rearranged leukemic cells. Study Design: 1) First, we will perform in vitro binding and transcriptional studies to validate that TET1 is a direct target of all five major MLL-fusions (i.e, MLL-AF9, -AF6, -AF10, -ELL and -ENL). Next, we will use Tet1 knockout/knockdown models coupled with MLL-fusion bone marrow transplantation models to determine the essential role of Tet1 in both development and maintenance of all five major subtypes of MLL-rearranged AML and in the self-renewal of relevant LSCs. We will also use xeno-transplantation models to determine whether targeting TET1 is a potential therapeutic approach to treat MLL-rearranged AML (Aim 1). 2) We will perform Chip-Seq, single-base resolution 5hmC/5mC-Seq, and RNA-Seq to identify critical potential targets of Tet1, followed by the validation and functional studis of a group of selected targets of Tet1 in MLL-rearranged leukemia in order to elucidate the underlying molecular mechanisms (Aim 2).