The leukemic fusion gene AML1-MDS1-EVI1 suppresses CEBPA in acute myeloid leukemia by activation of Calreticulin

Abstract

The leukemic fusion gene AML1-MDS1-EVI1 (AME) encodes a chimeric transcription factor that results from the t(3,21)(q26;q22) translocation seen in patients with acute myeloid leukemia, with therapy-related myelodysplastic syndrome, or with chronic myeloid leukemia in blast crisis. The myeloid transcription factor CEBPA is crucial for normal granulopoiesis. Here, we found that conditional expression of AME suppresses CEBPA protein by 90.8% and DNA-binding activity by 93.9%. In contrast, CEBPA mRNA levels remained unchanged. In addition, we detected no differences in CEBPA mRNA levels in leukemic blasts of patients carrying the AME translocation (n = 8) compared to acute myeloid leukemia patients with a normal karyotype (n = 9). CEBPA protein and binding activity, however, were reduced significantly (100% and 92.1%, respectively) in AME patient samples. Furthermore, we observed that calreticulin (CRT), a putative inhibitor of CEBPA translation, was strongly activated after induction of AME in the cell-line system (14.8-fold) and in AME patient samples (12.2-fold). Moreover, inhibition of CRT by small interfering RNA powerfully restored CEBPA levels. These results identify CEBPA as a key target of the leukemic fusion protein AME and suggest that modulation of CEBPA by CRT may represent a mechanism involved in the differentiation block in AME leukemias.

Fig. 1.

Conditional expression of AME in U937 leukemic cells. (A) U937 cells were analyzed before (day 0) and 1, 2, and 3 days after withdrawal of tetracycline by real-time PCR analyses for AME and CEBPA expression. Mean values and SD (error bars) are depicted. (B) Western blot analyses at the same time points as in A. CV1 cells transiently transfected with an AME expression construct served as positive control (left lane). The membrane was incubated further with antibodies against CEBPA, CEBPB, CEBPE, G-CSF receptor, and β-actin. (C) CEBPA-binding activity to a CEBP site as present in the G-CSF receptor promoter was assessed by electrophoretic mobility shift assays at the time points indicated after withdrawal of tetracycline. Kasumi-1 cells served as a CEBPA negative control. S, shifted CEBPA protein; SS, supershifted CEBPA-protein complex. (D) CEBPA-binding activity measured with the TransAM assay. A CEBP wild-type (wt) or a CEBP mutated oligonucleotide (mut) were added to nuclear extracts from day 0 as a control. Mean values and SD (error bars) are depicted. Nuclear extracts from Kasumi-1 cells served as a negative control.

Fig. 2.

CEBPA protein is specifically suppressed in AME patients. Eight patient samples carrying the AME translocation and nine AML patients with a normal karyotype were analyzed. (A) Real-time PCR analysis of CEBPA levels from AME patients and AML patients with a normal karyotype. Mean and SD (error bars) are shown. (B) Western blot analyses from lysates of three AME patient samples and of three representative AML patients with a normal karyotype. The same membrane was incubated with an antibody against β-actin for control (Lower). (C) CEBPA-binding activity was measured by using the TransAM assay. Mean and SD (error bars) are shown.

Fig. 3.

CRT expression and activity are induced after conditional expression of AME in U937 cells and AME patient samples. (A) Measurement of CRT mRNA by real-time PCR. Mean and SD (error bars) are shown. (B) CRT proteins were assessed by Western blot analysis. HeLa cells served as positive control. (C) CRT activity of U937 AME cells after withdrawal of tetracycline was assessed by UV cross-linking. The assay demonstrates the direct interaction of CRT protein to a CRT-binding site within the CEBPA mRNA. Coomassie blue (C. blue) staining is given as a loading control. (D) CRT activity by UV cross-linking. Two representative AME patient samples (lanes 1 and 2) are compared to two AML patient samples with a normal karyotype (lanes 3 and 4). Coomassie blue staining is depicted as a control. (E) U937 cells were transfected with GFP expression plasmid or GFP and CRT expression plasmids. Forty-eight hours after transfection, the expression of CEBPA was examined by immunostaining for CEBPA (Top). Levels of CEBPA expression were determined in 100 cells transfected with CRT and GFP and in 100 cells transfected with GFP alone (Bottom). DAPI, 4′,6-diamidino-2-phenylindole.

Fig. 4.

Inhibition of CRT expression by siRNA restores CEBPA-protein expression. AME protein was induced in U937 cells by withdrawal of tetracycline, and siRNA designed to knock-down CRT or mock siRNA was transfected by electroporation. As further control, U937T cells were transfected with siRNA. (A) Real-time PCR analysis of CRT levels 48 h after transfection. Mean and SD (error bars) are shown. (B) Western blot analysis of CRT, CEBPA, CEBPB, and β-actin.