Role of E4BP4 in Glucocorticoid-Evoked Lymphocyte Apoptosis

描述（由申请人提供）：抗白血病药物通过改变一系列调节细胞增殖、分化和死亡的基因的表达来触发白血病细胞的凋亡。白血病干细胞（LSC）群体对凋亡的抵抗导致一些患者预后不良和疾病复发。该项目的长期目标是了解细胞凋亡的分子途径，并开发新的策略使白血病细胞对药物诱导的细胞凋亡敏感。PI实验室先前已经在一对人类白血病姐妹t细胞系CEM-C7-14和CEM-C1-15中证明了转录抑制因子E4BP4的上调与糖皮质激素（GCs）诱导的细胞凋亡之间的相关性，这两种细胞系分别对gc诱导的细胞凋亡敏感或难治性。Bcl-2家族的促凋亡成员，特别是Bim，在化疗药物的作用下上调，引发白血病细胞凋亡。在之前的SC3资助中，PI的团队已经产生了E4BP4过表达和敲低CEM细胞，以证明E4BP4促进gc介导的Bim上调。PI建议扩大这些研究，并利用一系列白血病细胞系评估E4BP4和相关基因在广谱抗白血病药物诱导的细胞凋亡中的作用。Specific Aim 1建议使用启动子报告子测定、EMSA和ChIP测定来描绘E4BP4在响应GCs和其他抗白血病药物时介导Bim转录物水平的途径。脯氨酸和富酸（PAR）家族蛋白具有抗E4BP4作用的干性，初步研究表明其表达与E4BP4相反。在Specific Aim 2中，将使用E4BP4过表达和敲低细胞系来评估E4BP4和PAR家族基因的相对表达协调调节白血病细胞分化及其对抗白血病药物的反应的假设。LSCs将通过流式细胞术分析细胞表面标记物来鉴定。另一个基因Bcl11b是一种已知的t细胞分化调节因子，最近被证明可以诱导化疗耐药，其下调被证明可以诱导E4BP4的表达。特异性Aim 3将确定抗白血病药物是否改变Bcl11b的表达，以及Bcl11b是否是E4BP4和/或Bim表达的上游调节剂。生成Bcl11b敲低和过表达系，评估Bcl11b表达与E4BP4和Bim转录物及蛋白水平的关系。与公共卫生相关：化疗药物反应中的异常或不充分的细胞凋亡是化疗耐药和癌症预后不良的主要原因。本文提出的研究将揭示控制白血病细胞增殖、分化和凋亡的复杂途径，从而开发出更有效的治疗策略。

DESCRIPTION (provided by applicant): Anti-leukemic agents trigger apoptosis of leukemic cells by altering expression of an array of genes regulating cell prolifiration, differentiation ad death. Resistance to apoptosis among leukemic stem cell (LSC) populations results in poor prognosis and disease relapse in some patients. The long- term goal of this PI is to understand the molecular pathway for apoptosis, and to develop novel strategies to sensitize leukemic cells to drug-induced apoptosis. The PI's laboratory has previously demonstrated a correlation between upregulation of the transcriptional repressor, E4BP4, and apoptosis induced by glucocorticoids (GCs) in a pair of human leukemic sister T-cell lines, CEM-C7-14 and CEM-C1-15, that are susceptible or refractory, respectively, to GC-evoked apoptosis. Upregulation of the pro-apoptotic members of the Bcl-2 family, notably, Bim, in response to chemotherapeutic agents, triggers apoptosis of leukemic cells. During previous SC3 funding, the PI's group has generated E4BP4 overexpressing and knockdown CEM cells to demonstrate that E4BP4 facilitates GC-mediated Bim upregulation. The PI proposes to expand these studies and evaluate the role of E4BP4 and related genes in apoptosis induced by a broad spectrum of anti-leukemic agents, using an array of leukemic cell lines. Specific Aim 1 proposes to use promoter-reporter assays, EMSA and ChIP assays to delineate the pathway through which E4BP4 mediates Bim transcript levels in response to GCs and other anti-leukemic agents. Proline and Acidic Rich (PAR) family proteins, known to antagonize E4BP4 action, have stemness properties, and preliminary studies suggest that their expression is opposite to that of E4BP4. In Specific Aim 2, E4BP4 overexpressing and knockdown lines will be used to evaluate the hypothesis that relative expression of E4BP4 and PAR family genes coordinately regulate leukemic cell differentiation and their response to anti-leukemic agents. LSCs will be identified by flow cytometric analysis of cell surface markers. Another gene, Bcl11b, a known regulator of T-cell differentiation, was recently shown to induce chemoresistance, and its downregulation was shown to induce E4BP4 expression. Specific Aim 3 will determine if anti-leukemic agents alter Bcl11b expression, and whether Bcl11b is an upstream modulator of E4BP4 and/or Bim expresion. Bcl11b knockdown and overexpressing lines will be generated to evaluate the relationship between Bcl11b expression and E4BP4 and Bim transcript and protein levels. Relevance to Public Health: Aberrant or inadequate apoptosis in response to chemotherapeutic agents is the primary cause of chemoresistance and poor cancer prognosis. Studies proposed here will unravel the complex pathways that govern leukemic cell proliferation, differentiation and apoptosis so that more effective therapeutic strategies may be developed.