The CEBPA gene is mutated in 10% of acute myeloid leukemia (AML) cases, and wild-type CEBPA levels are reduced in additional AMLs, e.g. as a result of AML1-ETO or flt3ITD mediated inhibition of transcription or bcr-abl mediated inhibition of translation. One consequence of diminished C/EBPα activity or expression is likely inhibition of myeloid differentiation. We now find, via analysis of data from two published microarray studies, that CEBPA and Bcl-2 RNA levels correlate highly in low risk human AMLs (P = 0.003 and 0.001), suggesting that inhibition of apoptosis via induction of bcl-2 by C/EBPα or its mutant variants also contributes to transformation. Lack of correlation in other risk groups may reflect the presence of additional mutations that inhibit apoptosis. Among the low-risk patients, those with AML1-ETO had the lowest, CBFβ-SMMHC intermediate, and PML-RARα the highest levels of CEBPA (P < 0.05). Patients with CEBPA mutations were mainly in the intermediate-risk, normal karyotype group, and within this group the average CEBPA level was higher than those having wild-type alleles (P = 0.0002), suggesting a selective advantage to enable dominant-inhibition of remaining wild-type C/EBPs and/or induction of bcl-2. The two categories of C/EBPα mutant alleles associated with AML are C/EBPα p30, lacking an N-terminal transactivation domain, and C/EBPα LZ, carrying in-frame mutations in the leucine zipper (LZ) that prevent DNA-binding. Each of this oncoproteins induced bcl-2 in hematopoietic cell lines, and wild-type C/EBPα also induced bcl-2 when transduced into normal murine myeloid progenitors or in the splenocytes of H2K-C/EBPalpha]-Eμ transgenic mice. Remarkably, C/EBPα LZ oncoproteins activated the bcl-2 P2 promoter despite lack of DNA-binding, and C/EBPα p30 also activated the promoter. C/EBPα and the C/EBPα oncoproteins cooperated with NF-κB p50, but not p65, to induce bcl-2 promoter transcription. Endogenous C/EBPα preferentially coimmunoprecipitated with p50, versus p65, in myeloid cell extracts. Mutation of residues 297–302 in the C/EBPα basic region (BR) prevented induction of endogenous bcl-2 or the bcl-2 promoter and interaction with p50 but not p65 when co-expressed in 293T cells, whereas C/EBPα p30 or a C/EBPα LZ oncoprotein retain the ability to co-ip with p50. These findings offer an explanation for preferential in-frame rather than out-of-frame mutation of the LZ with sparing of the BR in the C/EBPα LZ oncoproteins that constitute one-third of CEBPA mutations in AML. C/EBPα or its mutant variants may tether to a subset of NF-κB targets genes, including Bcl-2, via p50 to facilitate gene activation. Targeting interaction between the C/EBPα basic region and NF-κB p50 may contribute to the therapy of AMLs expressing either mutant or residual wild-type C/EBPα , by increasing susceptibility to apoptosis. Targetting this interaction may also be relevant to the therapy of other malignancies expressing C/EBPs, not only via down-regulation of bcl-2 but also by preventing their cooperative activation of genes mediating inflammation and angiogenesis, which have recently been shown to contribute to the pathogenesis of several solid tumors in animal models.

Author notes

Corresponding author