Elevated Expression of C/EBPα/ε and c-myb Is Required for Myeloid Differentiation in CML.

The BCR/ABL fusion protein transforms hematopoietic stem cells and causes chronic myeloid leukemia (CML). However, the mechanism leading to the expansion of mature myeloid cells in CML remains obscure. To investigate this mechanism, we compared the gene expression profile of CML neutrophils with that of normal neutrophils by microarray analysis. The genes upregulated in CML neutrophils include critical transcription factors that regulate granulocytic differentiation and proliferation, as well as genes encoding neutrophil granule proteins (Table 1). The protein levels of these upregulated genes were also elevated in the CML neutrophils. To investigate whether BCR/ABL regulates the expression of these upregulated genes, primary CML neutrophils were exposed to 1 μM imatinib (a BCR/ABL tyrosine kinase inhibitor) for 12–16 hours. C/EBPα protein expression was downregulated in primary CML neutrophils after treatment with imatinib, compared to control cultures. We further explored the function of these upregulated genes in KCL22 cells, which are derived from CML blastic crisis. To investigate the effect of C/EBPα or C/EBPε on myeloid differentiation, we established KCL22 cells in which C/EBPα or C/EBPε expression was inducible (KCL22/α or KCL22/ε, respectively). Overexpression of either C/EBP protein resulted in morphological changes, such as reduction of the nuclear to cytoplasmic ratio, more condensed nuclear chromatin, and segmented nuclei, as well as the expression of differentiation specific markers including the G-CSF receptor and macrophage adhesion molecule-1 (Mac-1). These data indicate that C/EBPα /ε expression is sufficient to induce myeloid differentiation in BCR/ABL positive CML cells. We examined whether BCR/ABL regulates the protein expression of c-Myb. The expression of c-Myb in KCL22 cells treated with 1 μM imatinib was downregulated and growth in these cells decreased in parallel. Knock-down of c-Myb expression with siRNA inhibited the growth of KCL22 cells and these data indicate that c-Myb expression is important for the growth of CML cells. To test the effect of c-Myb on differentiation of KCL22 cells, we knocked down c-Myb expression with siRNA in KCL22/α or KCL22/ε cells after induction of C/EBP protein. The knock-down of c-Myb in KCL22α /ε cells resulted in downregulation of differentiation specific genes, including G-CSF receptor and neutophil elastase. Morphologically, the fraction of KCL22α /ε cells with segmented nuclei decreased when c-Myb expression was knocked down. Our results suggest that elevated expression of C/EBPα /ε and c-Myb play a key role in proliferation and differentiation in CML cells.