Activation of Proto-Oncogenes by Chromosomal Translocation

染色体易位激活原癌基因

• 批准号: 8350088
• 负责人: Peter Aplan
• 金额: $ 44.45万
• 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/8350088
• 关键词: Age-Months; Animal Model; Animals; Biological Assay; Bone Marrow; Cells; Chromosomal translocation; Chronic Lymphocytic Leukemia; Development; Disease; Embryo; Erythroid; Fertility; Fishes; Gene Cluster; Gene Expression; Gene Expression Profiling; Generations; Genes; Genetically Engineered Mouse; Genotype; HOXA9 gene; Helper-Inducer T-Lymphocyte; Hematologic Neoplasms; Hematopoietic; Hematopoietic stem cells; Histones; Human; Incidence; Inflammatory; Large T Antigen; Lesion; Life; Lymphoblastic Leukemia; Malignant - descriptor; Malignant lymphoid neoplasm; Manuscripts; Metabolism; MicroRNAs; Mus; Mutation; Myeloid Leukemia; NUP98 gene; Oncogenes; Oncogenic; Pathway interactions; Patients; Peripheral; Phenotype; Pregnancy; Premalignant; Preparation; Process; Proto-Oncogenes; Publishing; Recurrence; Regulatory Element; Seminoma; Sequence Homologs; Series; Simian virus 40; Spermatids; Spermatocytes; Stem cells; T cell differentiation; T-Cell Lymphoma; T-Lymphocyte; TOP1 gene; Takifugu; Testicular Germ Cell Tumor; Testicular Neoplasms; Testicular Seminoma; Testing; Thymus Gland; Time; Torafugu; Transgenes; Transgenic Mice; Transgenic Organisms; Translocation Breakpoint; Viral Tumor Antigens; Zebrafish; abstracting; chromatin immunoprecipitation; cohort; comparative genomics; cytokine; egg; fusion gene; gene cloning; leukemia; leukemia/lymphoma; novel; offspring; overexpression; pluripotency; promoter; pup; research study; self-renewal; teleost fish; tumor

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 NHD13 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. In order to better understand the leukemogenicity of NUP98 fused to non-HOX genes, we generated mice that express either a NUP98-RAP1GDS (NRG) or NUP98-PHF23 (NP23) fusion in hematopoietic cells using Vav regulatory elements to direct expression in the hematopoietic compartment. NRG mice were generated and transmitted the transgene in expected Mendellian ratio, and were documented to express the transgene. However, we could discern no hematopoietic abnormalities in these mice. 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. Interestingly, the leukemic phenotype is very broad, including T and B cell leukemias, myeloid leukemias, and erythroid leukemias. We have begun a series of studies, including chromatin immunoprecipitation and gene expression profiling to identify targets of the NP23 fusion. These experiments show that the leukemias are clonal, and frequently activate a cluser of genes within the Hoxa locus, including Hoxa7,9,10,11, and Meis1. In addition, we have identified novel genes, including Gm525, that are overexpressed in the NP23 leukemias. Overexpression of the Hoxa cluster genes was associated with the prsence of active histone marks (H3K4Me3), and the absence of inactive histone marks (H3K27Me2). An abstract describing these findings has been presented, and a manuscript is in preparation. Recent studies have demonstrated that HOXA9 is important for hematopoietic stem cell self-renewal and is one of the most differentially expressed genes in patients with AML and MDS. Indeed, Hoxa cluster genes, especially Hoxa7/9/10, were among the most differentially expressed genes in the NHD13 and CALM-AF10 mice described in previous projects, suggesting that Hoxa9 is an important target for leukemic transformation. Therefore, we have generated mice that express Hoxa9 in hematopoietic cells, using Vav regulatory elements to enable us to compare these mice to the NHD13 mice. Some of the potential founders did not transmit the transgene, despite having >30 pups genotyped, suggesting that the transgene may have been embryonic lethal in these mice. We have euthanized mice with timed pregnancies, and were able to document embryos that were transgenic, further supporting the possibility that the transgene was embryonic lethal in some founders. Two founders were able to transmit the transgene; however, they only expressed levels of the Hoxa9 transgene that were only slightly higher than wild-type controls. Despite this low level of expression, the Hoxa9 mice have developed a leukemic phenotype. At approximately 12 months of age, some of the Hoxa9 mice have developed a precursor T cell lymphoblastic leukemia/lymphoma (pre-T LBL). These pre-T LBLs are clonal and are typically accompanied by spontaneous Notch1 mutations. We are currently characterizing additional tumors, and assaying the Hoxa9 mice for evidence of disordered T cell differentiation prior to the onset of the pre-T LBL. We noted that Lin28b, a gene involved in microRNA metabolism and the 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, 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. A manuscript describing these features is currently under review. We tested the ability of SCL, LMO1, and/or SV40 Large T antigen (TAg) to cause leukemia in zebrafish. The rationale for studying zebrafish is that they have a short generation time, high fecundity, small size, large, visible eggs, and visible, ex vivo development; development of T-lymphoid leukemia in fish would also allow comparative genomic approaches to determine common abnormalities and pathways among humans, mice, and fish. We used an Lck promoter from a related teleost fish (Fugu rubripes) to drive expression of genes in the developing fish thymus, and established transgenic lines for SCL, LMO1, and TAg. After 3 years of study, there was no evidence that any of these lines developed lymphoid malignancies. However, the SCL, LMO1, and TAg lines have decreased survival and a markedly increased incidence of seminoma (testicular germ cell tumor). Two different Lck-TAg lines were followed, and showed a cumulative incidence of seminoma of 12 or 16% by 36 months of life. Transgenic SCL and LMO1 fish are also predisposed to seminoma, with a cumulative incidence of 25 and 14 % by 36 months of life. The seminomas showed variable contribution of spermatocyte, spermatid, and spermatogonial components, and expression of genes that have been used as markers for human (AP2a, OCT4) or fish (Sox9a, Vas, Wt1) testicular tumors. We were puzzled by these findings, as we anticipated that the Lck promoter would direct expression exclusively in the thymus. However, the Fugu Lck promoter was promiscuous, and we detected TAg expression in seminomas and testes. A manuscript describing these findings was recently published.

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-HOXA99和NHD13融合已显示出具有属性的属性在Genetiner in Genetiner in Genetinelication in Genetinelication在Genetinelication in Genetinelication inenetiner inenetine inenetiner inenetiner inenet in netrication inenet inenet in netrication enet netrication in netrication inenet inenet inenet inenet inenet inenet inenet in net nhd13融合。工程鼠标。然而，当从转基因小鼠中的VAV启动子表达时，NUP98-TOP1融合充其量是微弱的致癌性。 为了更好地了解NUP98与非Hox基因的白血病性，我们产生了表达NUP98-RAP1GDS（NRG）或NUP98-PHF23（NP23）融合的小鼠，在造血细胞中使用VAV调节元素中的表达在造血细胞中，以直接在造血细胞中表达表达车厢。生成NRG小鼠并以预期的门德利比率传输转基因，并记录以表达转基因。但是，我们不能辨别这些小鼠的造血异常。 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。有趣的是，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。 我们已经开始进行一系列研究，包括染色质免疫沉淀和基因表达分析，以鉴定NP23融合的靶标。这些实验表明，白血病是克隆的，并且经常激活Hoxa基因座中的基因簇，包括Hoxa7,9,10,11和Meis1。此外，我们已经确定了NP23白血病中过表达的GM525在内的新基因。 HOXA簇基因的过表达与活性组蛋白标记的实践（H3K4ME3）和不存在非活性组蛋白标记（H3K27ME2）有关。已经提出了描述这些发现的摘要，并且正在准备手稿。 最近的研究表明，HOXA9对于造血细胞自我更新至关重要，并且是AML和MDS患者中最差异表达的基因之一。实际上，HOXA簇基因，尤其是Hoxa7/9/10，是NHD13中最差异表达的基因之一，而先前项目中描述的Calm-AF-10小鼠是Hoxa9是白血病转化的重要靶标。因此，我们使用VAV调节元素生成了在造血细胞中表达HOXA9的小鼠，使我们能够将这些小鼠与NHD13小鼠进行比较。尽管有30个幼崽的基因分型，但一些潜在的创始人并未传输转基因，这表明转基因在这些小鼠中可能是胚胎致死的。 我们已经对小鼠定时怀孕了，并能够记录转基因的胚胎，进一步支持了转基因在某些创始人中是胚胎致死的可能性。两个创始人能够传输转基因。但是，他们仅表达仅略高于野生型对照的HOXA9转基因。尽管表达较低，但Hoxa9小鼠还是发展了白血病表型。在大约12个月大的时候，一些HOXA9小鼠已经发展了前体T细胞淋巴细胞白血病/淋巴瘤（T PRE-T LBL）。这些前T LBL是克隆的，通常伴有自发的Notch1突变。我们目前正在表征其他肿瘤，并分析HOXA9小鼠，以证明T前LBL发作之前的T细胞分化无序。 我们注意到，NHD13小鼠Lin28b是一种参与microRNA代谢和干细胞多能性的基因。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转基因小鼠会形成一种侵略性，克隆，致命的外周T细胞淋巴瘤，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。目前正在审查描述这些功能的手稿。 我们测试了SCL，LMO1和/或SV40大T抗原（TAG）在斑马鱼中引起白血病的能力。研究斑马鱼的理由是，它们的生成时间很短，繁殖力高，大小，大，可见的鸡蛋和可见的，离体发育；鱼类中T淋巴白血病的发展还将允许比较基因组方法来确定人类，小鼠和鱼类之间的常见异常和途径。 我们使用了来自相关硬骨鱼（Fugu Rubripes）的LCK启动子来驱动发展中心的胸腺中基因的表达，并为SCL，LMO1和TAG建立了转基因线。经过3年的研究，没有证据表明这些线中的任何一种都会出现淋巴恶性肿瘤。但是，SCL，LMO1和TAG系的存活率降低，明显增加了精子瘤的发病率（睾丸生殖细胞肿瘤）。 遵循两条不同的LCK-TAG系，并在36个月的寿命到36个月的时间内累积精液瘤的累积发生率为12或16％。转基因SCL和LMO1鱼也易于精液瘤，到36个月的生命，累积发生率为25和14％。精液显示精子细胞，精子和精子成分的贡献以及已用作人（AP2A，OCT4）或FISH（SOX9A，VAS，VAS，WT1）睾丸肿瘤的基因的表达。我们对这些发现感到困惑，因为我们预计LCK启动子将仅引导在胸腺中表达。但是，FUGU LCK启动子是混杂的，我们在seminomas和睾丸中检测到了TAG表达。最近发表了描述这些发现的手稿。