Bcl-Xl Mitigates p53 Activity in AML1-ETO-Expressing Human Hematopoietic Cells.

Abstract 387

A subset of acute myeloid leukemia (AML) is caused by chromosomal translocation (8;21), resulting in the expression of the AML1-ETO (AE) fusion protein. We have recently shown that AE represses DNA damage repair genes, resulting in increased DNA damage and upregulation of p53. These findings suggest that AE contributes to leukemogenesis by promoting additional mutations. However, it remains undefined how AE cells survive in the context of increased p53. In the current study, we showed that Bcl-xL is upregulated upon AE expression. Bcl-xL knockdown resulted in growth disadvantage and increased apoptosis in AE cells, but not in normal CD34+ cells. On the other hand, Bcl-xL overexpression in AE cells leads to an expansion of colony-forming cells and an increase in the frequency of long-term culture-initiating cells (LTC-IC). Interestingly, similar results were obtained when a Bcl-xL mutant (Bcl-xLG138A) that is defective in BH3 binding is overexpressed in AE cells, suggesting that the additional cytoprotection is not contributed by pro-apoptotic protein binding. Instead, published evidence on the p53 sequestering function of Bcl-xL prompted us to speculate that AE expression may lead to the establishment of a new balance between pro-apoptotic signals conveyed by activated p53 and the pro-survival characteristic of Bcl-xL. Indeed, using confocal microscopy, we observed colocalization of Bcl-xL and p53 in CD34+ AE cells. In addition, we also observed an increase in p53 target gene expression in AE cells following Bcl-xL knockdown, implicating interplay between Bcl-xL levels and p53 activity. Strikingly, examination of the two critical apoptosis-inducing p53 downstream targets, Bim and Puma, revealed that, whereas Bim is upregluated in AE cells, Puma is downregulated upon AE expression. AE ablation in an established cell line induces Puma expression. A chromatin immunoprecipitation assay revealed that AE binds to the first intron of the Puma gene. These data suggest that AE directly represses Puma gene expression, thereby preventing Puma-induced dissociation of p53 from Bcl-xL. Taken together, our in vitro findings demonstrated that the interplay among p53, Bcl-xL and Puma in response to DNA damage in normal cellular physiology is disrupted in the presence of AE to ensure cell survival. To recapitulate the critical role of Bcl-xL in AE expressing cells in an in vivo xenotransplantation assay, we transduced scrambled and Bcl-xL shRNA into AE/N-RASG12D cells, followed by transplantation into the bone marrow cavity of immunodeficient mice. We found that Bcl-xL knockdown is associated with a significant reduction in the long-term engraftment capability of AE/N-RASG12D cells. In conclusion, our data indicate that AE expressing human hematopoietic cells depend on Bcl-xL for their survival and growth.