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

TET1在MLL重排白血病中的作用及作用机制

• 批准号: 8696304
• 负责人: Jianjun Chen
• 金额: $ 32.42万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-04-01 至 2019-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8696304
• 关键词: Acute Myelocytic Leukemia; Acute leukemia; Address; Apoptosis; Binding; Bone Marrow Transplantation; Breast; Cells; ChIP-seq; Chimeric Proteins; Chromosomal Rearrangement; Chromosomes, Human, Pair 11; Code; Complement; Coupled; DNA; DNA Modification Process; DNA Sequence Rearrangement; Data; Development; Disease Resistance; Down-Regulation; Epigenetic Process; Gene Expression Profiling; Gene Expression Regulation; Gene Family; Gene Targeting; Generations; Genes; Genetic; Genome; Genomic DNA; Goals; Growth; HOXA9 gene; Hematopathology; Hematopoietic; Hematopoietic Neoplasms; Human; Human Chromosomes; In Vitro; Infant; Knock-out; Lead; Leukemic Cell; Light; MEIS1 gene; MLL gene; MLLT3 gene; MLLT4 gene; Maintenance; Malignant Neoplasms; Mediating; MicroRNAs; Modeling; Modification; Molecular; Mus; Myeloproliferative disease; Oncogenic; PBX3 gene; Pathogenesis; Patients; Play; Promoter Regions; Prostate; Protein Binding; Protein translocation; Proteins; Reagent; Regulation; Reporting; Research Design; Research Personnel; Resolution; Role; Sampling; Signal Pathway; Small Interfering RNA; Solid; Solid Neoplasm; Stem cells; System; Therapeutic; Transcript; Transcription Coactivator; Transplantation; Tumor Suppressor Genes; Tumor Suppressor Proteins; Validation; Xeno; base; cell transformation; demethylation; embryonic stem cell; histone modification; in vivo; insight; interdisciplinary approach; interdisciplinary collaboration; interest; leukemia; leukemic stem cell; leukemogenesis; loss of function mutation; member; mouse model; novel; novel therapeutic intervention; novel therapeutics; outcome forecast; public health relevance; self-renewal; small hairpin RNA; stem; success; therapeutic target; therapy resistant; transcriptome sequencing; zygote

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).

描述（由申请人提供）：最近通过将 5-甲基胞嘧啶 (5mC) 转化为 5-羟甲基胞嘧啶 (5hmC)，十一个易位 (Tet) 蛋白（包括 Tet1/2/3）已被鉴定为表观遗传调控的关键调节因子，导致主动或被动的 DNA 去甲基化。尽管 TET1 是 TET 基因家族的创始成员，首次被鉴定为急性髓系白血病 (AML) 中混合谱系白血病 (MLL) 基因的融合伴侣，但其在白血病中的病理作用尚不清楚。与各种实体瘤中报道的所有三个 TET 基因的下调和潜在的肿瘤抑制作用以及造血系统恶性肿瘤中 TET2 频繁的功能丧失突变相反，我们最近发现 TET1（但不是 TET2 /3）在 MLL 重排的 AML 中显着上调； MLL 融合体与 TET1 的启动子区域结合并直接促进其表达，这与 5hmC 水平的增加有关。通过小发夹 RNA (shRNA) 或基因敲除来消除 Tet1 表达可显着抑制 MLL-AF9 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TET1 表达可显着降低人MLL-AF9 白血病细胞的活力/生长并增加细胞凋亡。此外，我们还发现了 MLL 融合的几个重要的下游直接靶点，例如 HOXA9、MEIS1 和 PBX3，这些靶点已被证明对于 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 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的可行治疗靶点-重排白血病。具体目标： 1) 确定 Tet1 在 MLL 重排白血病的发生和维持以及相关白血病干细胞 (LSC) 自我更新中的重要作用； 2) 鉴定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敲除/敲低模型与MLL融合骨髓移植模型相结合，以确定Tet1在MLL重排AML的所有五种主要亚型的发育和维持以及相关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 TET1 是否是治疗 MLL 重排 AML 的潜在治疗方法（目标 1）。 2）我们将进行Chip-Seq、单碱基分辨率5hmC/5mC-Seq和RNA-Seq来鉴定Tet1的关键潜在靶点，然后对一组选定的Tet1靶点在MLL重排中进行验证和功能研究白血病，以阐明潜在的分子机制（目标 2）。