Glucocorticoid apoptosis in childhood lymphoblastic leukemia

Glucocorticoid (GC) - induced apoptosis is a central component in the therapy of acute lymphoblastic leukemia (ALL), and defining its molecular basis and that of GC resistance is crucial for therapeutic improvements. Previously, our laboratory and others have delineated numerous GC-regulated candidate genes in experimental systems (leukemia lines and mouse thymocytes). Very recently, we have exploited a whole genome comparative expression profiling screen to identify genes regulated in lymphoblasts from children with ALL under GC mono- therapy. This study has essentially ruled out several model-based hypotheses and identified a small number of new candidates that now need to be investigated with regard to their functional role in apoptosis induction. For this purpose, we have developed lentiviral expression systems that allow conditional (i.e., tetracycline-regulated) gene over-expression and gene knock-down by RNA interference (RNAi) with small hairpin RNAs. These systems were designed to be "Gateway compatible" (i.e., allowing quick subcloning by highly efficient recombinatorial processes) and, hence, enable functional analyses of several candidate genes within a reasonable time. Regarding biologic systems for these analyses, we have established childhood ALL derived cell lines with constitutive expression of tetracycline-responsive transactivators and/or transrepressors. Since lentiviral constructs have a high transduction efficiency and do not require cell cycle progression for integration into the host genome, the corresponding systems may also be suitable for studies in primary lymphoblasts from patients. Combining these systems, we propose to delineate the functional role of the most promising GC-regulated candidate genes derived from the above mentioned screen both individually or in combinations of two to several genes. In conclusion, the proposed research may considerably improve our understanding of the clinically important anti-leukemic effects of GC.

Glucocorticoid (GC) - induced apoptosis is a central component in the therapy of acute lymphoblastic leukemia (ALL), and defining its molecular basis and that of GC resistance is crucial for therapeutic improvements. Our laboratory and others have delineated, and are still identifying, GC-regulated candidate genes in experimental systems (leukemia cell lines and mouse thymocytes) and, more importantly, in lymphoblasts from children with ALL under GC mono-therapy. Central aim of this project was to investigate the functional role of the most promising candidate genes in apoptosis induction. For this purpose, we developed lentiviral expression systems for conditional (i.e., tetracycline-regulated) gene over-expression and/or gene knock-down by RNA interference with small hairpin RNAs for NR3C1/GR (the GC receptor), PFKFB2 (2 splice variants), ZBTB16, SLA, SOCS1, SNF1LK/SIK1, TSC22D3/GILZ and RGS18 (a candidate gene for GC resistance identified in this project). Using these constructs, we analyzed the functional role of the corresponding genes in childhood ALL derived cell lines (CCRF-CEM, NALM6, PreB697). In one instance (SIK1) a corresponding knock-out mouse was generated in collaboration with J. Penninger in Vienna. With the exception of the GR, which turned out to be a crucial determinator for sensitivity and kinetics of GC-induced apoptosis, none of these candidates appeared to be an essential player in the anti-leukemic GC effects. Thus, at the current state, regulation of components of the large BCL2 family (a family of apoptosis regulating proteins that we investigated mainly in a parallel project), seems to be the most critical event for cell death induction. Some of the `top candidates` seem to play a modulatory role, such as the PFKFB2 splice variant 15B that sensitized cells for apoptosis induction, or ZBTB16 that had a protective effect. Other GC-regulated genes may account for other GC effects such as SLA that might be a critical player in the immunosuppressive action of GC. The constructs, cell lines and knock-out mice generated in the course of this project will be extremely useful for corresponding analyses.