Activation of Proto-Oncogenes by Chromosomal Translocation

染色体易位激活原癌基因

• 批准号: 8175426
• 负责人: Peter Aplan
• 金额: $ 37.95万
• 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/8175426
• 关键词: Animal Model; Animals; Bone Marrow; Cells; Chromosomal translocation; Chronic Lymphocytic Leukemia; Complementary DNA; Development; Embryo; Erythroid; Fertility; Fishes; Gene Cluster; Gene Expression; Gene Expression Profiling; Generations; Genes; Genetically Engineered Mouse; Genotype; Goals; HOXA9 gene; Hematologic Neoplasms; Hematopoietic; Homeobox Genes; Human; Incidence; Internal Ribosome Entry Site; Knock-in Mouse; Large T Antigen; Lesion; Life; Lymphoblastic Leukemia; Malignant - descriptor; Malignant lymphoid neoplasm; Manuscripts; Messenger RNA; Mus; Myeloid Leukemia; NUP98 gene; Oncogenes; Oncogenic; Pathway interactions; Patients; Phenotype; Pregnancy; Premalignant; Process; Production; Proteins; Proto-Oncogenes; Recurrence; Regulatory Element; Reporter; Seminoma; Sequence Homologs; Series; Simian virus 40; Spermatids; Spermatocytes; Stem cells; T-Lymphocyte; Takifugu; Testicular Germ Cell Tumor; Testicular Neoplasms; Testicular Seminoma; Testing; Thymus Gland; Time; Torafugu; Transcript; Transgenes; Transgenic Mice; Transgenic Organisms; Translocation Breakpoint; Viral Tumor Antigens; Zebrafish; chromatin immunoprecipitation; cohort; comparative genomics; egg; embryonic stem cell; fusion gene; gene cloning; leukemia; leukemia/lymphoma; novel; offspring; promoter; pup; self-renewal; sialosyl-T antigen; teleost fish; vector

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 will study 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. Using Vav regulatory elements, we demonstrated that expression of a CALM-AF10 fusion predisposed mice to AML. However, the majority of patients with CALM-AF10 fusions develop not AML, but a gamma/delta pre-T lymphoblastic leukemia/lymphoma (pre-T LBL). Although we were able to show expression of CALM-AF10 mRNA in gamma/delta T-cells, it is possible that the expression level was inadequate to induce pre-T LBL, or that expression of CALM-AF10 led to AML before a pre-T LBL developed. Therefore, we plan to generate mice that express CALM-AF10 exclusively in gamma/delta T-cells. Insertion of an IRES and GFP reporter into the mouse Tcrd locus led to production of bi-cistronic transcripts encoding both Tcrd and GFP proteins, enabling the authors to track gamma/delta T-cells in vivo81. We obtained the vector used for those studies, and will replace the GFP cDNA with a CALM-AF10 cDNA. This vector will be used to target ES cells, and knock-in ES cells will be used to generate mice that express CALM-AF10 under the control of endogenous Tcrd regulatory elements. Unfortunately, we have encountered technical difficulties in generating this vector, and have been unable to generate any mice to complete this goal. Recent studies have demonstrated that HOXA9 is an important gene for 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 greater than 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. We have recently 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 is no evidence that any of these lines develop lymphoid malignancies. However, the SCL, LMO1, and TAg lines have decreased survival and a markedly increased incidence of seminoma (testicular germ cell tumor). Preliminary studies indicate the following; analysis of larger cohorts is currently in progress. Two different Lck-TAg lines have been followed, and have 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 (AP2alpha, 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 fidings has recently been submitted.

使用逆转录病毒转导的骨髓，多个实验室已证明 NUP98-HOXA9、NUP98-HHEX、NUP98-NSD1 和 NUP98-TOP1 融合体具有致白血病性，并且 NUP98-HOXA9 和 NHD13 融合体已被证明具有致白血病性。工程小鼠。然而，当 NUP98-TOP1 融合体在转基因小鼠中从 Vav 启动子表达时，其致癌性充其量是弱的。为了更好地了解与非 HOX 基因融合的 NUP98 的致白血病性，我们将研究在造血细胞中表达 NUP98-RAP1GDS (NRG) 或 NUP98-PHF23 (NP23) 融合的小鼠，使用 Vav 调节元件来指导造血细胞中的表达。造血室。生成 NRG 小鼠并以预期的孟德尔比例传播转基因，并记录其表达转基因。然而，我们在这些小鼠中没有发现造血异常。 NP23 小鼠已经诞生，我们对两位创始人的一大群后代进行了追踪。这些小鼠几乎 100% 在出生后 1 年内患上白血病。有趣的是，白血病表型非常广泛，包括T细胞和B细胞白血病、粒细胞白血病和红细胞白血病。 我们已经开始了一系列研究，包括染色质免疫沉淀和基因表达谱分析，以鉴定 NP23 融合的靶点。 使用 Vav 调控元件，我们证明 CALM-AF10 融合蛋白的表达使小鼠易患 AML。然而，大多数 CALM-AF10 融合患者不会发展为 AML，而是发展为 γ/δ 前 T 淋巴细胞白血病/淋巴瘤（前 T LBL）。尽管我们能够在γ/δ T细胞中显示CALM-AF10 mRNA的表达，但表达水平可能不足以诱导pre-T LBL，或者CALM-AF10的表达在pre-T细胞之前导致AML。 T LBL 开发。因此，我们计划培育只在 γ/δ T 细胞中表达 CALM-AF10 的小鼠。将 IRES 和 GFP 报告基因插入小鼠 Tcrd 位点，产生编码 Tcrd 和 GFP 蛋白的双顺反子转录物，使作者能够在体内追踪 γ/δ T 细胞81。我们获得了用于这些研究的载体，并将用 CALM-AF10 cDNA 替换 GFP cDNA。该载体将用于靶向 ES 细胞，敲入 ES 细胞将用于产生在内源性 Tcrd 调控元件控制下表达 CALM-AF10 的小鼠。不幸的是，我们在生成这个向量时遇到了技术困难，并且无法生成任何小鼠来完成这个目标。 最近的研究表明HOXA9是干细胞自我更新的重要基因，也是AML和MDS患者中表达差异最大的基因之一。事实上，Hoxa 簇基因，尤其是 Hoxa7/9/10，是之前项目中描述的 NHD13 和 CALM-AF10 小鼠中差异最大的基因之一，表明 Hoxa9 是白血病转化的重要靶标。因此，我们利用 Vav 调控元件培育了在造血细胞中表达 Hoxa9 的小鼠，以便我们能够将这些小鼠与 NHD13 小鼠进行比较。尽管对超过 30 只幼鼠进行了基因分型，但一些潜在的创始人并未传播转基因，这表明转基因可能对这些小鼠具有胚胎致死性。 我们对定时怀孕的小鼠实施了安乐死，并能够记录转基因胚胎，进一步支持了转基因对一些创始人胚胎致命的可能性。两位创始人能够传播转基因；然而，它们仅表达 Hoxa9 转基因水平，仅略高于野生型对照。 我们最近测试了 SCL、LMO1 和/或 SV40 大 T 抗原 (TAg) 引起斑马鱼白血病的能力。研究斑马鱼的理由是斑马鱼的世代时间短、繁殖力高、体型小、卵大、可见、离体发育可见；鱼类 T 淋巴细胞白血病的发展也将使比较基因组方法能够确定人类、小鼠和鱼类之间的常见异常和途径。 我们使用来自相关硬骨鱼（Fugu rubripes）的 Lck 启动子来驱动发育中的鱼类胸腺中的基因表达，并建立了 SCL、LMO1 和 TAg 的转基因品系。经过三年的研究，没有证据表明这些细胞系会发展为淋巴恶性肿瘤。然而，SCL、LMO1 和 TAg 系的存活率降低，精原细胞瘤（睾丸生殖细胞肿瘤）的发病率显着增加。初步研究表明：目前正在进行更大群体的分析。两种不同的 Lck-TAg 系已被追踪，到 36 个月时，精原细胞瘤的累积发病率为 12% 或 16%。转基因 SCL 和 LMO1 鱼也容易患精原细胞瘤，36 个月内的累积发病率分别为 25% 和 14%。精原细胞瘤显示出精母细胞、精细胞和精原细胞成分的不同贡献，以及已用作人类（AP2α、OCT4）或鱼类（Sox9a、Vas、Wt1）睾丸肿瘤标记物的基因的表达。我们对这些发现感到困惑，因为我们预计 Lck 启动子将仅在胸腺中直接表达。然而，Fugu Lck 启动子是混杂的，我们在精原细胞瘤和睾丸中检测到了 TAg 表达。最近提交了一份描述这些发现的手稿。