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调节细胞增殖、分化和死亡的一系列基因的表达来触发白血病细胞凋亡。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。本PI的长期目标是了解细胞凋亡的分子途径，并开发使白血病细胞对药物诱导的细胞凋亡敏感的新策略。PI的实验室先前已经证明了在一对人白血病姐妹T细胞系CEM-C7-14和CEM-C1-15中转录抑制因子E4 BP 4的上调与糖皮质激素（GC）诱导的细胞凋亡之间的相关性，所述细胞系分别对GC诱发的细胞凋亡敏感或难治。Bcl-2家族的促凋亡成员（特别是Bim）响应于化疗剂的上调触发白血病细胞的凋亡。在之前的SC 3资助期间，PI的小组已经产生了E4 BP 4过表达和敲低的CEM细胞，以证明E4 BP 4促进GC介导的Bim上调。PI建议扩展这些研究，并使用一系列白血病细胞系评价E4 BP 4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。具体目标1建议使用启动子-报告基因测定、EMSA和ChIP测定来描绘E4 BP 4介导响应GC和其他抗白血病药物的Bim转录水平的途径。已知拮抗E4 BP 4作用的脯氨酸和富酸（PAR）家族蛋白具有干性性质，并且初步研究表明它们的表达与E4 BP 4的表达相反。在特定目标2中，E4 BP 4过表达和敲低细胞系将用于评估E4 BP 4和PAR家族基因的相对表达协同调节白血病细胞分化及其对抗白血病药物的应答的假设。将通过细胞表面标志物的流式细胞术分析来鉴定LSC。另一个基因，Bcl 11b，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性，其下调可以诱导E4 BP 4的表达。特异性目的3将确定抗白血病药物是否改变Bcl 11b表达，以及Bcl 11b是否是E4 BP 4和/或Bim表达的上游调节剂。将产生Bcl 11b敲低和过表达系，以评价Bcl 11b表达与E4 BP 4和Bim转录物和蛋白水平之间的关系。与公共卫生的相关性：对化疗药物的异常或不充分的细胞凋亡是化疗耐药性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，以便开发更有效的治疗策略。

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.