Activation of Proto-Oncogenes by Chromosomal Translocation

染色体易位激活原癌基因

• 批准号: 8938420
• 负责人: Peter Aplan
• 金额: $ 54.63万
• 依托单位: DIVISION OF CLINICAL SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/8938420
• 关键词: Animal Model; Animals; Apoptotic; Area; Binding; Birth; Bone Marrow; Cell Death; Cell Line; Cells; ChIP-seq; Childhood Leukemia; Chimeric Proteins; Chromosomal translocation; Chronic; Chronic Lymphocytic Leukemia; Data; Databases; Development; Diffuse; Disease; Disulfiram; Down-Regulation; Embryo; Erythroid; Gene Cluster; Gene Expression; Genes; Genetically Engineered Mouse; HOXA9 gene; Helper-Inducer T-Lymphocyte; Hematologic Neoplasms; Hematopoietic; High-Throughput Nucleotide Sequencing; Histones; Inflammation; Inflammatory; Injection of therapeutic agent; Lesion; Life; Malignant - descriptor; Malignant Childhood Neoplasm; Manuscripts; Metabolism; MicroRNAs; Mus; Mutate; Myelogenous; Myeloid Leukemia; NUP98 gene; Nodal; Non-Hodgkin' s Lymphoma; Oncogenes; Oncogenic; Onset of illness; Patients; Peripheral; Phenotype; Point Mutation; Polysaccharides; Premalignant; Process; Proteins; Proto-Oncogenes; Publishing; Recurrence; Regulatory Element; Research; Sequence Homologs; Site; Stem Cell Research; Stem cells; Surveys; T-Cell Lymphoma; T-Lymphocyte; TOP1 gene; Testing; Thymus Gland; Time; Transgenes; Transgenic Mice; Transgenic Organisms; Translocation Breakpoint; cancer site; chromatin immunoprecipitation; cohort; cytokine; deep sequencing; egg; fusion gene; gene cloning; leukemia; mutant; novel; offspring; overexpression; pluripotency; promoter; pup; recombinase; research study; thymocyte

Using retroviral transduced bone marrow, several labs have shown that NUP98-HOXA9, NUP98-HHEX, NUP98-NSD1, and NUP98-TOP1 fusions are leukemogenic, and NUP98-HOXA9 and NUP98-HOXD13 fusions have been shown to be been shown to be leukemogenic in genetically engineered mice. However, a NUP98-TOP1 fusion was at best weakly oncogenic when expressed from a Vav promoter in transgenic mice, as was a NUP98-RAP1GDS (NRG) . These findings were published in 2014. In order to better understand the leukemogenicity of NUP98 fused to non-HOX genes, we generated mice that expressed a NUP98-PHF23 (NP23) fusion in hematopoietic cells. NP23 mice have been generated, and we have followed a large cohort of offspring from two founders. Almost 100% of these mice develop leukemia within 1 year of life; the leukemic phenotype is very broad, including T and B cell leukemias, myeloid leukemias, and erythroid leukemias. These leukemias are clonal, and frequently activate a cluster of genes within the Hoxa locus, including Hoxa7,9,10,11, and Meis1. In addition, we have identified novel genes, including BAHCC1, that are overexpressed in the NP23 leukemias; a survey of publicly available expression data revealed that BAHCC1 was overexpressed in several distinct subsets of AML. Chromatin immunoprecipitation/sequencing (ChIP-Seq) experiments demonstrated the presence of active histone marks (H3K4Me3) at the Hoxa cluster; moreover, additional ChIP-seq experiments demonstrate binding of the NP23 fusion protein at the H3K4Me3 sites, suggesting a mechanism for the leukemic transformation. Furthermore, we have identified a compound (disulfiram) that inhibits the binding of the NP23 protein to H3K4Me3 residues. Treatment of NP23 cell lines results in rapid downregulation of Hoxa cluster genes and apoptotic cell death. These studies were published in 2014. We noted that Lin28b, a gene involved in microRNA metabolism and stem cell pluripotency, was overexpressed in the NHD13 mice. To determine if this overexpression was related to malignant transformation, we used Vav regulatory elements to express Lin28b in hematopoietic cells. Lin28b transgenic mice develop an aggressive, clonal, lethal peripheral T cell lymphoma (PTCL), which is associated with release of inflammatory cytokines. The cell of origin shows immunophenotypic and gene expression features consistent with those of T follicular helper (TFH) cells. We interrogated a publicly available gene expression database, and found that Lin28b was overexpressed (mean 7.5 fold) in patients with PTCL. A manuscript describing these features was published in 2012, and we have transferred these mice to several collaborators. Given that the PTCL in these mice was associated with diffuse, systemic inflammation, we have begun pilot experiments to determine whether chronic inflammation will accelerate the disease process. Preliminary results suggests that treatment with lipo-polysaccharide (LPS) does indeed accelerate the onset of disease. Deep sequencing studies have recently identified recurrent point mutations in ASXL1. We have generated transgenic constructs to express the mutant protein in the hematopoietic cells of mice. With respect to ASXL1, despite injections of over 200 mouse eggs, and the birth of 187 pups, no transgenic mice were identified, leading to the suspicion that expression of the mutant ASXL1 protein was embryonic lethal. We have now generated several founder mice with a revised, conditional ASXL1 mutant protein. We have recently crossed these mice to mice that express the Cre recombinase in the thymus or myeloid lineage in order to activate the conditional mutant transgene. Mice that express the mutant ASXL1 in the thymus show a marked (10 fold) reduction in total thymocytes, as well as decreased circulating T cells, demonstrating a phenotypic effect of the mutant ASXL1 transgene.

使用逆转录病毒转导的骨髓，几个实验室表明NUP98-HOXA9，NUP98-HHEX，NUP98-NSD1和NUP98-TOP1融合具有白血病，NUP98-HOXA9和NUP98-HOXA9和NUP98-HOXD13融合已显示出白血病。在基因工程的小鼠中。然而，当从转基因小鼠中的VAV启动子和NUP98-RAP1GDS（NRG）中，NUP98-TOP1融合充其量是微弱的致癌性。这些发现于2014年发表。为了更好地了解NUP98与非Hox基因的白血病性，我们产生了造血细胞中表达NUP98-PHF23（NP23）融合的小鼠。已经生成了NP23小鼠，我们遵循了两个创始人的大量后代。这些小鼠中几乎有100％在生命的1年内患白血病；白血病表型非常广泛，包括T和B细胞白血病，髓样白血病和红细胞性白血病。这些白血病是克隆的，并且经常激活Hoxa基因座中的一群基因，包括Hoxa7,9,10,11和Meis1。此外，我们已经确定了包括Bahcc1在内的新型基因，这些基因在NP23白血病中过表达。对公开表达数据的调查显示，BAHCC1在AML的几个不同子集中过表达。染色质免疫沉淀/测序（CHIP-SEQ）实验表明，HOXA簇存在活性组蛋白标记（H3K4ME3）；此外，其他CHIP-SEQ实验表明NP23融合蛋白在H3K4ME3位点的结合，这表明了白血病转化的机制。此外，我们已经确定了一种抑制NP23蛋白与H3K4ME3残基的结合的化合物（二硫兰酯）。 NP23细胞系的处理导致HOXA簇基因和凋亡细胞死亡的快速下调。这些研究于2014年发表。我们注意到，LIN28B是一种参与microRNA代谢和干细胞多能性的基因，在NHD13小鼠中过表达。为了确定这种过表达是否与恶性转化有关，我们使用VAV调节元件在造血细胞中表达LIN28B。 LIN28B转基因小鼠会形成侵略性，克隆，致命的外周T细胞淋巴瘤（PTCL），这与炎症细胞因子的释放有关。原始细胞显示出与T卵泡辅助器（TFH）细胞相一致的免疫表型和基因表达特征。我们询问了公开可用的基因表达数据库，发现PTCL患者的LIN28B过表达（平均7.5倍）。描述这些功能的手稿于2012年发布，我们将这些老鼠转移给了几位合作者。鉴于这些小鼠中的PTCL与弥漫性，全身性炎症有关，我们已经开始进行试点实验，以确定慢性炎症是否会加速疾病过程。初步结果表明，脂糖糖（LPS）的治疗确实确实加速了疾病的发作。深度测序研究最近已经确定了ASXL1中的复发点突变。我们已经产生了转基因构建体以表达小鼠造血细胞中的突变蛋白。关于ASXL1，尽管注射了200多个小鼠卵，并且187只幼崽的出生，但未发现转基因小鼠，导致人们怀疑突变体ASXL1蛋白的表达是胚胎致死的。现在，我们已经生成了几只具有修订后的ASXL1突变蛋白的创始人小鼠。我们最近将这些小鼠越过了表达胸腺或髓样谱系中CRE重组酶的小鼠，以激活条件突变转基因。表达胸腺中突变体ASXL1的小鼠在总胸腺细胞中显示出明显的（10倍）降低，并减少了循环T细胞，表明突变体ASXL1转基因的表型效应。