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

描述（由申请人提供）：最近已确定十-十一易位（泰特）蛋白（包括Tet 1/2/3）是表观遗传调控中的关键调节因子，通过将5-甲基胞嘧啶（5 mC）转化为5-羟甲基胞嘧啶（5 hmC），导致主动或被动DNA去甲基化。虽然TET 1是泰特基因家族的创始成员，在急性髓系白血病（AML）中首次被鉴定为混合谱系白血病（MLL）基因的融合伴侣，但其在白血病中的病理作用尚不清楚。与所有三个泰特基因在各种实体瘤中报道的下调和潜在的肿瘤抑制作用以及TET 2在造血系统恶性肿瘤中频繁的功能丧失突变相反，我们最近发现TET 1 TET 2/3在MLL重排的AML中表达显著上调; MLL融合蛋白与TET 1的启动子区结合，直接促进TET 1的表达，这与5 hmC水平的增加有关。通过小发夹RNA（shRNAs）或基因敲除消除Tet 1表达显著抑制MLL-AF 9介导的体外细胞转化和体内白血病发生。此外，通过小干扰RNA（siRNA）寡核苷酸敲低TET 1表达显著降低人MLL-AF 9白血病细胞的存活率/生长并增加细胞凋亡。此外，我们发现MLL融合的几个重要下游直接靶点，如HOXA 9，MEIS 1和PBX 3，已被证明对MLL重排AML的诱导/维持和白血病干细胞（LSC）自我更新至关重要，也是TET 1的直接靶基因。假设：TET 1通过促进一组致癌靶基因的表达和抑制一组肿瘤抑制靶基因的表达在MLL重排白血病的发病机制中起重要的致癌作用，因此TET 1是治疗MLL重排白血病的可行的治疗靶点。具体目标：1）确定Tet 1在MLL重排白血病的发展和维持以及相关白血病干细胞（LSC）的自我更新中的重要作用; 2）鉴定Tet 1在MLL重排白血病细胞中的关键靶基因。研究设计：1）首先，我们将进行体外结合和转录研究以验证TET 1是所有五种主要MLL融合体（即MLL-AF 9、-AF 6、-AF 10、-ELL和-ENL）的直接靶标。接下来，我们将使用Tet 1敲除/敲低模型结合MLL融合骨髓移植模型来确定Tet 1在所有五种主要亚型的MLL重排AML的发展和维持以及相关LSC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TET 1是否是治疗ML重排AML的潜在治疗方法（目的1）。2)我们将进行Chip-Seq，单碱基分辨率5 hmC/5 mC-Seq和RNA-Seq来识别Tet 1的关键潜在靶标，然后对一组选定的Tet 1在MLL重排白血病中的靶标进行验证和功能研究，以阐明潜在的分子机制（目的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).