Genetic Analysis of T Cell Leukemogenesis

描述（由申请人提供）： 一个碱性螺旋-环-螺旋（bHLH）转录因子家族和两个含LIM结构域的转录衔接子LIM-only蛋白1（LMO 1）和2（LMO 2）的异常表达构成了T细胞急性淋巴细胞白血病（T-ALL）中最常见的功能获得性异常。这可以由染色体易位或失调的单或双等位基因表达引起。这些基因的蛋白质产物有助于形成一个共同的DNA结合复合物，该复合物还包含LIM结构域结合蛋白Ldb 1和两个Ldb 1相互作用蛋白，单链DNA结合蛋白-2（SSBP 2）和-3（SSBP 3）。这种或相关的复合物通过激活和抑制特定靶基因组的转录来介导这些核蛋白的致癌作用。虽然bHLH和LMO蛋白对T-ALL发展的重要性已得到充分证实，但这种多蛋白复合物如何促成恶性淋巴母细胞的表型以及其组分蛋白之间的相互作用是否可以靶向用于治疗目的尚不清楚。我们已经确定，SSBPs保护Ldb 1和LMO蛋白的泛素化和蛋白酶体的破坏，并通过一个单独的机制，促进Ldb 1二聚化。我们假设，降低Ldb 1表达或抑制其与自身、SSBP或LMO蛋白的相互作用将对白血病细胞的存活、增殖和/或功能产生不利影响。这将在三个具体目标中得到检验。在第一个目标中，通过RNA干扰降低Ldb 1表达的效果将在来自两个T-ALL转基因小鼠模型的白血病细胞的活力、数量和侵袭性上进行测试。在第二个目的中，将通过使用分别对应于其N-末端二聚化结构域、中心Ldb 1/Chip保守结构域和C-末端LIM相互作用结构域的显性干扰Ldb 1衍生多肽来研究抑制Ldb 1与其自身、SSBP和LMO蛋白相互作用的效果。在第三个目标中，将在细胞移植研究中测试降低或灭活Ldb 1表达对体内白血病起始活性（白血病干细胞功能）的影响。这些研究将为Ldb 1在白血病细胞生物学中的功能提供新的见解，并为T-ALL和其他以bHLH和LMO蛋白异常表达为特征的恶性肿瘤开发新的靶向治疗。

DESCRIPTION (provided by applicant): PROJECT SUMMARY Aberrant expression of a family of basic helix-loop-helix (bHLH) transcription factors and two LIM domain-containing transcriptional adaptors, LIM-only protein 1 (LMO1) and 2 (LMO2), constitute the most frequent gain-of-function abnormalities in T-cell acute lymphoblastic leukemia (T-ALL). This can result from either chromosomal translocation or dysregulated mono- or bi-allelic expression. The protein products of these genes contribute to a common DNA-binding complex that also contains the LIM domain-binding protein Ldb1 and two Ldb1-interacting proteins, single stranded DNA-binding protein-2 (SSBP2) and -3 (SSBP3). This or a related complex mediates the oncogenic actions of these nuclear proteins by activating and repressing the transcription of specific sets of target genes. Although the importance of both bHLH and LMO proteins to the development of T-ALL is well established, how this multi-protein complex contributes to the phenotype of the malignant lymphoblast and whether interactions between its component proteins can be targeted for therapeutic purpose are not known. We have determined that the SSBPs protect Ldb1 and LMO proteins from ubiquitylation and proteasomal destruction and, through a separate mechanism, promote Ldb1 dimerization. We hypothesize that reducing Ldb1 expression or inhibiting its interaction with itself, SSBPs, or LMO proteins will adversely affect leukemia cell survival, proliferation, and/or function. This wil be tested in three specific aims. In the first aim, the effects of reducing Ldb1 expression by RNA interference will be tested on the viability, number, and invasiveness of leukemia cells derived from two transgenic mouse models of T-ALL. In the second aim, the effects of inhibiting Ldb1 interaction with itself, SSBPs, and LMO proteins will be investigated through use of dominant interfering Ldb1-derived polypeptides corresponding to its N-terminal dimerization domain, central Ldb1/Chip conserved domain, and C- terminal LIM interaction domain, respectively. In the third aim, the effects of reducing or inactivating Ldb1 expression will be tested on leukemia-initiating activity (leukemia stem cell function) in vivo in cell transplantation studies. These studies should provide new insights into Ldb1 function in leukemia cell biology and lead to the development of novel targeted therapies for T-ALL and other malignancies characterized by abnormal expression of bHLH and LMO proteins.