Synthetic glucocorticoids (GCs) such as dexamethasone and prednisone are the mainstay of acute lymphoblastic leukemia (ALL) therapy. However, in spite of extensive use of these compounds in treating lymphoid malignancies, the molecular events that lead to cell death upon GC exposure are only poorly understood. Elucidation of these events is crucial to decipher the mechanisms of resistance to GC seen commonly at the time of recurrent disease as well as in older patients diagnosed with ALL and to design therapy targeted to overcome GC resistance. We investigated the molecular events leading to cell death in a panel of ALL cell lines with varying sensitivity to dexamethasone induced cell death. Dexamethasone induced cell death in sensitive cell lines was caspase dependent, preceded by mitochondrial membrane depolarization and cytochrome C release. Since the mitochondrial integrity is controlled by the members of the BCL2 family of proteins, we evaluated the expression of various pro- and anti-apoptotic BCL2 family proteins in ALL cell lines treated with dexamethasone and found a consistent upregulation of Bim, a BH3-only proapoptotic BCL2 family protein. This was accompanied by accumulation of non-phosphorylated FOXO3a, a member of the forkhead transcription factors known to be involved in transcriptional regulation of Bim expression. This accumulation of non-phosphorylated FOXO3a was associated with increased reporter activity in a FOXO-responsive luciferase construct, increased luciferase reporter activity in the Bim promoter containing the canonical foxo response element, and increased occupancy of FOXO3a in the Bim promoter as demonstrated by chromatin immunoprecipitation. However, shRNA mediated partial knock-down of FOXO3a did not rescue ALL cells from dexamethasone induced cell death. Indeed, this partial reduction in FOXO3a levels resulted in sensitization of ALL cells to dexamethasone induced cell death. Similar to the parental or control transfected cells, dexamethasone induced cell death in ALL cells with reduced FOXO3a expression could be inhibited by the pan-caspase inhibitor z-VAD-fmk. Further, the partial reduction in FOXO3a expression did not interfere with Bim up-regulation in response to GC exposure. In addition to modulating apoptosis by transcriptional regulation of molecules like Bim, FOXO family members are also involved in regulating cellular reactive oxygen species (ROS), by transcriptional regulation of molecules like manganese-superxode dismutase involved in ROS scavenging pathways. We therefore measured ROS by dihydroethidium staining in control and FOXO3 shRNA transfected ALL cell lines in the presence or absence of dexamethasone exposure. We found no difference in the ROS levels in the non-treated cells. However, upon exposure to dexamethasone, cells with lower FOXO3a levels demonstrated higher accumulation of ROS when compared to the control cell lines. We conclude that accumulation of non-phosphorylated, active FOXO3a in response to GC exposure mediates both the pro-survival and proapoptotic response in ALL cells and that selective abrogation of the antioxidant, pro-survival functions of FOXO3a might provide a feasible strategy to sensitize ALL cells to GC mediated cell death.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.