Bcl-2 Family Dysregulation in AML1-ETO-Expressing Human Hematopoietic Cells

A subset of acute myeloid leukemia is caused by the chromosomal translocation (8;21), resulting in the expression of the AML1-ETO (AE) fusion protein. AE expression in human CD34+ umbilical cord blood (UCB) cells causes an expansion of the hematopoietic stem and progenitor cell (HSPC) compartment, but alone does not cause leukemia. We have recently shown that AE represses DNA damage repair genes, resulting in higher mutation frequency as well as p53 activation and increased basal apoptosis. Interestingly, we found that AE expression in CD34+ UCB cells was associated with high Bcl-xL levels, while control cells showed a gradual loss of Bcl-xL protein. Furthermore, expression of AE in aged HSPC that had already downregulated Bcl-xL also led to Bcl-xL protein upregulation. In addition, Bcl-xL protein, but not mRNA, was decreased by pharmacological and RNAi-mediated inhibition of p53 in AE cells and increased by low-intensity γ-irradiation, implying that Bcl-xL turnover may be post-translationally regulated by p53 levels. Bcl-xL knock-down by short-hairpin RNA in AE cells resulted in even higher p53 activity and growth disadvantage, suggesting that AE cells depend largely upon Bcl-xL for survival in the context of p53 activation. AE cells are also more sensitive than long-term cultured UCB cells to GX15-070, a BH3-mimetic drug that binds and inhibits all anti-apoptotic Bcl-2 family proteins. The sensitivity of AE cells to Bcl-xL inhibition and GX15-070 treatment may be due to upregulation of various pro-apoptotic Bcl-2 family proteins. Surprisingly, p53-upregulated modulator of apoptosis (PUMA) mRNA levels were decreased or remained unchanged in association with AE expression, even in the context of an upregulated p53 signaling pathway. Chromatin immunoprecipitation assay revealed that AE binds to and AML1 binding site in the putative promoter of PUMA, in a region distinct from the p53 consensus binding site. Indeed, PUMA expression is increased in response to γ-irradiation in cells expressing AE. This suggests that AE may directly repress PUMA gene expression, but does not prevent p53 binding. Taken together, our data indicate that AE expression may lead to re-establishment of a new balance between the pro-apoptotic signals conveyed by the activated p53 signaling pathway and the pro-survival signals, presumably Bcl-xL upregulation and PUMA repression, within the Bcl-2 family proteins to ensure cell survival and propagation.