Glucocorticoid-induced leukemia apoptosis: Molecular mechanisms and development of new therapeutic concepts

Research project P 14482 Glucocorticoid-Induced Leukemia Apoptosis Reinhard KOFLER 09.10.2000 Over 60% of adults and about one third of children with malignant lymphoproliferative disorders succumb to their disease in spite of intensive chemotherapy, irradiation and bone marrow transplantation. The survivors suffer from an increased risk of therapy-related secondary tumors and other sequela of treatment. Glucocorticoids (GC) specifically induce cell cycle arrest and programmed cell death (apoptosis) in the majority of these malignant lymphoid cells. However, although GC, as a physiologic hormone with extremely low acute toxicity and mutagenic potential, represents an almost ideal cancer therapy drug, it cannot be used on its own because of almost invariable development of GC resistance. This, all currently accepted protocols combine GC with chemotherapeutic drugs (and, in certain instances, irradiation or bone marrow transplantation) with teir severe side effects. This proposal is based upon the concept that a better understanding of the molecular basis of GC-induced cell death and resistance against it might facilitate development of treatment modalities that increase GC efficiency and/or prevent GC resistance without cancerogenic chemotherapeutic drugs. In partivular, we propose three specific aims: 1. to further improve our molecular understanding of GC-induced apoptosis, we attempt to compare DANN chip analyses derived expression profiles from untreated and in vitro GC-treated acute lymphoblastic leukemia (ALL) sells with each other and with the profiles that we have already obtained from CCRF-CEM human ALL cells undergoing GC-induced apoptosis. We expect these comparisons to reveal "candidate genes" and related signaling pathways responsible for GC-induced G1 arrest and/or cell death 2. We intend to investigate the role of these "candidate genes" by testing their function in GC-induced apoptosis of leukemia. 3. We finally propose to test (in established cell lines, in ex vivo cells, and in a mouse model) a variety of novel combinatorial treatment regimens that are based on recent observations made in our and other laboratories as well as on the data that can be expected from the experiments outlined in Specific Aims 1 and 2. In conclusion, we propose the development and preclinical assessment of new treatment strategies for malignant lymphopropose disorders, in particular ALL, based on an increased understanding of GC-induced leukemia cell death.

Glucocorticoid (GC) and its analogues induce cell death (apoptosis) in cells of the lymphoid lineage and are therefore used in essentially all therapy regimens of lymphoid malignancies including childhood acute lymphoblastic leukemia (ALL). However, although GC, as a physiologic hormone with extremely low acute toxicity and mutagenic potential, represents an almost ideal cancer therapy drug, it cannot be used on its own because of almost invariable development of GC resistance. Thus, all current treatment protocols combine GC with chemotherapeutic drugs (and, in certain instances, irradiation or bone marrow transplantation) with their attendant severe side effects. To address this problerm we proposed 3 Specific Aims: (1) to identify "candidate genes" responsible for GC-induced apoptosis via comparative DNA microarray-based expression profiling, (2) to investigate the role of these "candidate genes" by testing their function in GC-induced apoptosis of leukemia cells using various transient and stable gene expression systems, and (3) to exploit the generated knowledge to derive novel combinatorial treatment concepts and test them in pre-clinical settings. Regarding Specific Aim 1, we have generated a number of relevant biologic systems (GC-sensitive and resistant ALL cell lines and ex vivo cells from patients) and subjected them to microarray analyses thereby generating a list of candidate genes including the GC receptor (GR) itself, genes for apoptosis/survival decisions, like the BH3-only molecule Bim, but also genes controlling metabolism (in particular glycolysis) and other critical cellular events. Concerning their functional role (Specific Aim 2), we have shown that GC repression of c-myc and cyclin D3 is responsible for GC-induced cell cycle arrest but not for apoptosis. We further found that the GR auto-induction occurs in GC-sensitive but not in GC-resistant ALL cell lines. Mimicry of GR-auto-induction by GR transgenesis restored GC sensitivity suggesting a critical role of this auto-regulatory loop for cell death induction. With respect to a first translational attempt of our findings into therapeutic concepts (Specific Aim 3), we found super-additive apoptosis induction by a combination of GC with low dose of the glycolysis inhibitor 2-deoxyglucose (2-DG). This combination is particularly attractive because 2-DG is already in clinical use for other indications.