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

项目摘要 LIM domain only-2（LMO 2）是散发性和基因治疗中最常见的失调癌基因之一， 诱导的人急性T细胞淋巴细胞白血病（T-ALL）。Lmo 2编码一个18 kDa的蛋白质， II碱性螺旋-环-螺旋转录因子（即bHLH：TAL 1或LYL 1）和加塔因子（GATA 1 -2）和LIM 结构域结合蛋白1（LDB 1）在一个大的多亚基复合物的启动子和增强子， 造血干细胞和祖细胞（HSPC）。LDB 1通过其LIM相互作用结构域（LID）与LMO 2结合 并且其同源二聚化连接红系祖细胞中的近端和远端E盒-GATA位点。的性质 T-ALL中的LMO 2相关蛋白复合物和T-ALL中的LMO 2转录靶点并不清楚， 表征了我们通过小鼠遗传学方法解决了这些问题。我们生成了CD 2-Lmo 2 转基因小鼠，并确定了两种不同的亚型的高度渗透的T-ALL：一类白血病有 常见的Notch 1突变和Notch 1靶基因上调，第二类密切模拟早期T- 细胞前体（ETP-）ALL，一种高度耐药的亚型。ETP-ALL来源于发育中的T细胞 表达其他谱系抗原的祖细胞具有类似于造血干细胞的转录谱， 干细胞（HSC），并表达LMO 2、HHEX、MYCN、MEF 2C和LYL 1癌基因。所有这些特征都是 在CD 2-Lmo 2转基因小鼠中重现，包括ETP-ALL转录特征， 沿着Lmo 2诱导的造血干细胞（HSC）样分化停滞特征的上调， 相对平静，自我更新增强。在初步数据中，LDB 1一致表达， 在ETP-ALL细胞中与LMO 2蛋白共纯化。在胸腺细胞中使用条件性缺失，其中Ldb 1是 通过对T细胞发育的研究，我们发现Ldb 1是Lmo 2诱导的T-ALL所必需的; 诱变研究中，我们在LDB 1的LID中发现了LMO 2结合所需的特定残基; 最引人注目的是，不能结合LMO 2的LDB 1突变蛋白是不稳定的。事实上，LDB 1和LMO 2 蛋白质相互稳定。此外，为了解决LMO 2与特异性 在ETP-ALL中的癌基因，我们进行了LDB 1 ChIP-seq分析，发现LMO 2和LDB 1 在ETP-ALL HSC样特征中的许多基因上，包括LYL 1和HHEX。我们还发现Hhex Lmo 2诱导的T-ALL所需的转录靶点。总之，我们对Lmo 2和T- ALL在高度治疗中建立了一个重要的蛋白质伴侣Ldb 1和一个重要的靶点Hhex， 耐药白血病，ETP-ALL。在这个提议中，我们将检验Ldb 1缺失减弱T-ALL的假设 通过减少CD 2-Lmo 2转基因小鼠中的白血病前靶群体来促进发展。我们还将 泛素介导蛋白酶体介导Ldb 1缺失是否导致Lmo 2蛋白不稳定 降解并破坏关键靶标如Hhex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.