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.
Apoptosis is a form of physiological cell death essential for maintaining normal health, and for preventing diseases such as cancer. Induction of apoptosis by chemotherapeutic agents is widely used to treat cancers including leukemias; however, the mechanism of drug-induced apoptosis is poorly understood. Resistance to drug-induced apoptosis is a serious impediment to successful treatment of cancer. Apoptosis is triggered by altered expression or function of multiple genes, one of them being E4BP4, a transcriptional repressor implicated as a central player in lymphoid development, proliferation and differentiation and known to be regulated by glucocorticoids and other chemotherapeutic agents. This project proposes to understand how E4BP4 and related genes may trigger apoptosis of leukemic cells, and how the gene regulatory pathway may be manipulated to prevent or overcome drug resistance.
