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

项目总结 LIM结构域仅限-2(LIM DOMAIN Only-2，LMO2)是散发性和基因治疗中最常被解除调控的癌基因之一。 诱导人急性T淋巴细胞白血病(T-ALL)。LMO2编码一个18 kDa的蛋白，与类结合 II基本螺旋-环-螺旋转录因子(即bHLH：TAL1或LYL1)和GATA因子(GATA1-2)和LIM 大分子多亚单位复合体中的结构域结合蛋白1(LDB1) 造血干祖细胞(HSPC)。LDB1通过其LIM相互作用结构域(LID)与LMO2结合 在红系祖细胞中，其同源二聚体连接着近距离和远距离的E box-GATA位点。这个世界的本质 T-ALL中Lmo2相关蛋白复合体和T-ALL中Lmo2‘S转录靶点的研究 特色化的。我们通过老鼠遗传学的方法解决了这些问题。我们生成了CD2-Lmo2 并定义了两种不同的高穿透性T-ALL亚型：一类白血病有 熟悉的Notch1突变和Notch1靶基因上调和第二类密切模拟早期T- 细胞前体(ETP-)ALL，一种高度耐治疗的亚型。ETP-ALL来源于发育中的T细胞 祖细胞表达其他血统的抗原，具有类似于造血细胞的转录特征 干细胞(HSCs)，表达LMO2、HHEX、MYCN、MEF2C和LYL1癌基因。所有这些功能都是 在CD2-Lmo2转基因小鼠中概述，包括ETP-ALL转录特征，这是 随着Lmo2诱导的造血干细胞(HSC)样分化停止的特征而上调， 相对平静，自我更新能力增强。在初步数据中，LDB1被一致表达，并且 在ETP-ALL细胞中与LMO2蛋白共纯化。在胸腺细胞中使用条件删除，其中LDB1是 对于T细胞的发育来说是必不可少的，我们发现Ldb1是Lmo2诱导的T-ALL所必需的；通过 突变研究，我们在LDB1的盖子中发现了LMO2结合所需的特定残基； 最引人注目的是，不能与LMO2结合的LDB1突变蛋白是不稳定的。事实上，LDB1和LMO2 蛋白质之间相互稳定。此外，为了解决LMO2与特定基因的一致性上调问题 在ETP-ALL的癌基因中，我们进行了LDB1芯片序列分析，发现LMO2和LDB1 在包括LYL1和HHEX在内的许多基因的ETP-ALL-ALL-HSC样签名中。我们还发现HHEX是 Lmo2诱导的T-ALL所必需的转录靶点。总而言之，我们关于Lmo2和T-的初步数据- ALL在高度治疗中建立了一个基本蛋白质伙伴LDB1和一个基本靶点HHEX- 耐药白血病，ETP-ALL。在本提案中，我们将检验LDB1缺失会减弱T-ALL的假设 在CD2-Lmo2转基因小鼠中通过减少白血病前期靶群的发展。我们还将 研究LDB1缺失是否通过泛素介导的蛋白酶体导致Lmo2蛋白的不稳定 降解并扰乱HHEX等关键靶标的转录调控。

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.