Epigenetic Silencing of Bcl-2, C/Ebpa and p14^ARF by the AML1/ETO Oncoprotein Contributing to Growth Arrest and Differentiation Blockage

Abstract 3617

The t(8; 21) is one of the most frequent chromosomal translocations associated with acute leukemia. The AML1(RUNX1)-ETO(MTG8) fusion transcription factor generated by the t (8; 21) translocation is believed to deregulate the expression of genes that are crucial for normal differentiation and proliferation of hematopoietic progenitors, resulting in acute myelogenous leukemia by recruiting co-repressor complexes to DNA. To investigate the role of AML1-ETO in leukemogenesis, we transfected the cloned AML1-ETO cDNA with plasmid and expressed the AML1-ETO protein in U937 myelomonocytic leukemia cells. Interestingly, we found dichotomous phenomena in these transfected leukemic cells, i.e. growth arrest versus differentiation block (detailed data?). The AML1-ETO transfected U937cells were growing significantly slower than that of empty vector-transfected cells and nontransfected cells (P<0.01). As the surface markers for myeloid differentiation, the expression of CD11b and CD14 measured by flow cytometry demonstrated that the percentage of CD11b+ cell was 4.1%-7.0% in U937-A/E1-4 cells, which was significantly lower(P<0.01) than those in U937-Mock cells (11.4%) and U937-WT cells (11.0%). Moreover, the expression of CD14 antigen was decreased by 1.5–2-fold as compared with the control cells. By focusing on the anti-apoptotic gene (Bcl-2), a key transcription factor (C/EBPA) which regulates granulocytic differentiation and a tumor suppressor gene (p14^ARF), we found that AML1-ETO- expressing cell subclones displayed low levels of these three genes in comparison with the non-transfected U937 (P<0.001). In primary bone marrow cells of acute myeloid leukemia containing t(8;21)/AML1-ETO, levels of Bcl-2, C/EBPA and p14^ARF mRNA were markedly lower (P<0.001) when compared with other acute myeloid leukemias lacking this translocation (n=10). Chromatin immunoprecipitation assays (ChIP) demonstrated that Bcl-2, C/EBPA and p14^ARF were among the direct transcriptional regulating targets of AML1-ETO. The universal binding of AML1-ETO to genomic DNA resulted in MeCP2 recruitment (P<0.01), reduction of histone H3 (P<0.0 1) or histone H4(P<0.01) acetylation and increased tri-methylation on histone H3 lysine 9 (P<0.01)as well as histone H3 lysine 27 (P<0.01), indicating that AML1-ETO induced heterochromatic silencing of Bcl-2, C/EBPA and p14^ARF. These results suggested that the aberrant transcription factor AML1-ETO epigenetically silences the function of Bcl-2, C/EBPA and p14^ARF gene by inducing repressed chromatin configurations at their promoters through histone modification. Considering that apoptosis-enhancing effect of AML1-ETO would not be favorable to the leukemogenesis, it must be compensated by some other effects to permit its leukemogenic potential.