The Role of LMO2 in the Pathogenesis of T-cell Leukemia

LMO2 在 T 细胞白血病发病机制中的作用

• 批准号: 9762024
• 负责人: Utpal P Dave
• 金额: $ 34.97万
• 依托单位: INDIANA UNIV-PURDUE UNIV AT INDIANAPOLIS
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-09-15 至 2021-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9762024
• 关键词: Acute T Cell Leukemia; Address; Antigens; Attenuated; BHLH Protein; Binding; Binding Proteins; Cells; ChIP-seq; Complex; Data; Development; Disease; Distal; Distant; Elements; Enhancers; Epigenetic Process; Erythroid; Fibrinogen; GATA1 gene; Gene Expression Profile; Genes; Genetic; Genetic Transcription; Grant; Hematopoietic stem cells; Homodimerization; Human; Knock-out; LIM Domain; LYL1 gene; Leukemic Cell; Link; MYCN gene; Malignant Neoplasms; Mediating; Modeling; Molecular; Mus; Mutagenesis; Mutation; Nature; Oncogenes; Pathogenesis; Protein Binding Domain; Proteins; Regulatory Element; Resistance; Rhombotin 2; Role; Site; Stem cells; T-Cell Development; T-Cell Leukemia; T-Lymphocyte; TAL1 gene; Target Populations; Testing; Transcriptional Regulation; Transgenic Mice; Ubiquitin; Up-Regulation; clinically significant; design; experimental study; gene therapy; genetic approach; improved; leukemia; mutant; novel therapeutics; progenitor; promoter; protein complex; self-renewal; stem-like cell; thymocyte

PROJECT SUMMARY LIM domain only-2 (LMO2) is one of the most frequently deregulated oncogenes in sporadic and gene therapy- induced human acute T-cell lymphoblastic leukemia (T-ALL). Lmo2 encodes an 18 kDa protein that binds class II basic helix-loop-helix transcription factors (i.e. bHLH: TAL1 or LYL1) and GATA factors (GATA1-2) and LIM domain binding protein 1 (LDB1) in a large multi-subunit complex at promoters and enhancers of hematopoietic stem and progenitor cells (HSPCs). LDB1 binds to LMO2 via its LIM interaction domain (LID) and its homodimerization links near and distant E box-GATA sites in erythroid progenitors. The nature of the LMO2-associated protein complex in T-ALL and LMO2's transcriptional targets in T-ALL are not well characterized. We addressed these questions through a mouse genetic approach. We generated CD2-Lmo2 transgenic mice and defined two distinct subtypes of highly penetrant T-ALLs: one class of leukemias had familiar Notch1 mutation and Notch1-target gene upregulation and the second class closely modeled Early T- cell Precursor (ETP-) ALL, a highly treatment-resistant subtype. ETP-ALLs are derived from developing T-cell progenitors, express antigens of other lineages, have a transcriptional profile that resembles hematopoietic stem cells (HSCs), and express LMO2, HHEX, MYCN, MEF2C, and LYL1 oncogenes. All these features are recapitulated in CD2-Lmo2 transgenic mice including the ETP-ALL transcriptional signature, which is upregulated along with Lmo2-induced hematopoietic stem cell- (HSC) like features of differentiation arrest, relative quiescence, and enhanced self-renewal. In preliminary data, LDB1 was concordantly expressed and co-purified with LMO2 protein in ETP-ALL cells. Using conditional deletion in thymocytes, where Ldb1 is dispensable for T-cell development, we discovered that Ldb1 was required for Lmo2-induced T-ALL; through mutagenesis studies, we found specific residues within the LID of LDB1 that are required for LMO2 binding; most strikingly, LDB1 mutant proteins that could not bind LMO2 were unstable. In fact, LDB1 and LMO2 proteins stabilize each other. Additionally, to address the concordant upregulation of LMO2 with specific oncogenes in ETP-ALL, we performed LDB1 ChIP-seq analysis and discovered occupancy of LMO2 and LDB1 at many genes in the ETP-ALL HSC-like signature including LYL1 and HHEX. We also discovered that Hhex is a required transcriptional target for Lmo2-induced T-ALL. In summary, our preliminary data on Lmo2 and T- ALL establishes an essential protein partner, Ldb1, and an essential target, Hhex, in a highly treatment- resistant leukemia, ETP-ALL. In this proposal, we will test the hypothesis that Ldb1 deletion attenuates T-ALL development by reducing the pre-leukemic target population in CD2-Lmo2 transgenic mice. We will also investigate whether Ldb1 deletion causes destabilization of Lmo2 protein by ubiquitin-mediated proteasomal degradation and disrupts transcriptional regulation of key targets such as Hhex.

项目概要 仅 LIM 结构域 2 (LMO2) 是散发性和基因治疗中最常见的失调癌基因之一 - 诱导人急性T细胞淋巴细胞白血病（T-ALL）。 Lmo2 编码结合类的 18 kDa 蛋白质 II 碱性螺旋-环-螺旋转录因子（即 bHLH：TAL1 或 LYL1）和 GATA 因子（GATA1-2）和 LIM 结构域结合蛋白 1 (LDB1) 位于启动子和增强子的大型多亚基复合物中 造血干细胞和祖细胞（HSPC）。 LDB1 通过其 LIM 相互作用域 (LID) 与 LMO2 结合 其同源二聚化连接了红系祖细胞中近处和远处的E盒-GATA位点。的性质 T-ALL 中的 LMO2 相关蛋白复合物和 T-ALL 中 LMO2 的转录靶点尚不明确 特点。我们通过小鼠遗传方法解决了这些问题。我们生成了CD2-Lmo2 转基因小鼠并定义了两种不同的高渗透性 T-ALL 亚型：一类白血病具有 熟悉的Notch1突变和Notch1靶基因上调以及第二类紧密建模的早期T- 细胞前体 (ETP-) ALL，一种高度耐药的亚型。 ETP-ALL 源自发育中的 T 细胞 祖细胞，表达其他谱系的抗原，具有类似于造血细胞的转录谱 干细胞 (HSC)，并表达 LMO2、HHEX、MYCN、MEF2C 和 LYL1 癌基因。所有这些功能都是 在 CD2-Lmo2 转基因小鼠中重述，包括 ETP-ALL 转录特征，即 与 Lmo2 诱导的造血干细胞 (HSC) 样分化停滞特征一起上调， 相对静止，自我更新能力增强。在初步数据中，LDB1 的表达一致，并且 与 ETP-ALL 细胞中的 LMO2 蛋白共纯化。在胸腺细胞中使用条件删除，其中 Ldb1 是 Ldb1 对于 T 细胞发育是可有可无的，我们发现 Ldb1 对于 Lmo2 诱导的 T-ALL 是必需的；通过 诱变研究中，我们在 LDB1 的 LID 内发现了 LMO2 结合所需的特定残基； 最引人注目的是，无法结合 LMO2 的 LDB1 突变蛋白是不稳定的。事实上，LDB1和LMO2 蛋白质相互稳定。此外，为了解决 LMO2 与特定物质的一致上调问题 为了检测 ETP-ALL 中的癌基因，我们进行了 LDB1 ChIP-seq 分析，发现了 LMO2 和 LDB1 的占据 ETP-ALL HSC 样特征中的许多基因，包括 LYL1 和 HHEX。我们还发现 Hhex 是 Lmo2 诱导的 T-ALL 所需的转录靶标。总之，我们关于 Lmo2 和 T 的初步数据 ALL 在高度治疗中建立了一个重要的蛋白质伴侣 Ldb1 和一个重要的靶标 Hhex 耐药性白血病，ETP-ALL。在本提案中，我们将测试 Ldb1 缺失会减弱 T-ALL 的假设 通过减少 CD2-Lmo2 转基因小鼠的白血病前目标群体来进行开发。我们也会 研究 Ldb1 缺失是否会通过泛素介导的蛋白酶体导致 Lmo2 蛋白不稳定 降解并破坏 Hhex 等关键靶标的转录调控。