FUNCTIONAL ANALYSIS OF THE T(17--19)-ALL CHIMERA E2A-HLF

异常转录调控在肿瘤发生中的重要作用 非随机疾病的频繁发生突显了 转录因子编码基因的特定突变。这样的一个 在ALL中观察到的t(17；19)突变产生了E2A-HLF融合 具有嵌合转录因子特性的蛋白质。 功能相似的嵌合体经常由其他 急性白血病和儿童肉瘤中的易位。理解 异常转录调控的作用机制 恶变是发展的必要前提 直接针对调节分子事件的新疗法 致癌作用。拟议的研究将分两次平行进行。 E2A-HLF具有生物活性相关的实验系统 对其在急性淋巴细胞白血病中的作用：阻断生长因子诱导的细胞凋亡 依赖IL-3的小鼠PRO-B细胞株FL5.12和 NIH3T3细胞的转化。特定目标1旨在 严格检验一种假设，即转录激活 关键靶基因是E2A-HLF的主要生物学机制 活动。使相关的明确定义的结构域失活的点突变 在转录调控中将被用来描绘E2A-HLF 生物活动所必需的功能。功能性 具有足够生物活性的特性将会是 通过用具有以下功能的异源结构域替换必要的结构域来定义 完成同样的功能。这项提议的第二个目的是 鉴定受E2A-HLF转录调控的靶基因 评估它们在白血病发生中的作用。的配基结合域 雌激素受体(ER)已与E2A-HLF融合，产生一种 条件性活性转录激活剂，E2A-HLF-ER。3T3和 将E2a-HLF-ER和a-HLF-ER稳定地转染人FL5.12细胞 转录非活性突变体的构建和代表性 差异分析将用于差异识别mRNAs 在诱导E2A-HLF-ER活性后表达。候选核糖核酸 它们也在t(17；19)+人类白血病中表达，将进一步 其特征是定义它们的蛋白质产品在 白血病的发生。

The prominent role of aberrant transcriptional regulation in oncogenesis is underscored by the frequent occurrence of non-random, disease specific mutations in genes encoding transcription factors. One such mutation, the t(17;19) observed in ALL, creates an E2A-HLF fusion protein with properties of a chimeric transcription factor. Functionally analogous chimeras are frequently created by other translocations in acute leukemias and childhood sarcomas. Understanding the mechanisms by which aberrant transcriptional regulation contributes to malignant transformation is an essential prerequisite to developing new therapies directly targeted at the molecular events mediating oncogenesis. The proposed studies will be performed in parallel in two experimental system in which E2A-HLF has biological activity relevant to its role in ALL: blocking apoptosis induced by growth factor withdrawal in the murine IL3-dependent pro-B cell line FL5.12 and transformation of NIH 3T3 cells. Specific Aim 1 is designed to rigorously examine the hypothesis that transcriptional activation of crucial target genes is the major mechanism for E2A-HLF biological activity. Point mutations that inactivate well-defined domains involved in transcriptional regulation will be used to delineate E2A-HLF functions that are necessary for biological activity. Functional properties which are sufficient for biological activity will then be defined by replacing necessary domains with heterologous domains capable of accomplishing the same function. The second aim of this proposal is to identify target genes transcriptionally regulated by E2A-HLF and evaluate their role in leukemogenesis. The ligand binding domain of the estrogen receptor (ER) has been fused to E2A-HLF to create a conditionally active transcriptional activator, E2A-HLF-ER. 3T3 and FL5.12 cells will be stably transfected with E2A-HLF-ER and a transcriptionally inactive mutant construct and representational difference analysis will be used to identify mRNAs differentially expressed following induction of E2A-HLF-ER activity. Candidate mRNAs which are also expressed in t(17;19)+ human leukemias will be further characterized to define the role of their protein products in leukemogenesis.