A Novel Subgroup of Poor Prognostic AML with Low CEBPA Expression, CEBPA Promoter Hypermethylation and DNMT3b Overexpression.

Abstract

The CCAAT/Enhancer Binding Protein a (CEBPA) is a 37kDa transcription factor essential for granulocytic differentiation. The C-terminal part is composed of a basic region that binds to DNA and a leucine zipper that mediates dimerization (bZIP). Recent studies reported N and C-terminal mutations in approximately 5 – 10% of patients with acute myeloid leukemia (AML). In case a C-terminal mutation is observed in one CEBPA allele, the other allele most frequently harbors an N-terminal mutation. N-terminal mutations cause the expression of a 30kDa protein, whereas C-terminal mutations usually represent in-frame insertions or deletions in the bZIP-domain resulting in deficient DNA-binding of CEBPA. Gene array analysis carried out on 285 AML cases, revealed a group of AML patients with a unique gene expression profile, which contained most of AML cases with biallelic, CEBPA mutations (Valk et al. N Engl J Med. 2004, 350: pp1617–28). Interestingly, among this cluster 50% of the cases did not harbor any CEBPA mutation, while presenting exactly the same gene expression profile. Instead of carrying CEBPA mutations, these particular cases did not express CEBPA mRNA at all. Moreover, the patients with low CEBPA expression showed a poor response to anti leukemic treatment, whereas the CEBPA mutant AML cases respond favorably to therapy. To study the mechanism by which CEBPA expression might have been down regulated, methylation-specific PCR was applied using the bisulfite method. This analysis revealed that in most of the cases with low CEBPA mRNA levels, the CEBPA promoter was silenced by hypermethylation. None of the other AML cases that we analyzed showed CEBPA hyper methylation. To verify whether the hypermethylation correlated with the expression levels of any of the distinct DNA-methyl transferase (DNMTs), the expression of these genes was studies in all 285 AML cases. Intriguingly, AML blasts in which CEBPA was methylated showed significant overexpression of the DNA-methyl transferase DNMT3B, whereas the expression this gene was low in the cases with CEBPA mutations. The fact that AML cases with mutated CEBPA show the same gene expression profile as the cases that harbored methylated CEBPA, indicate that the same pathways are affected within those two subclasses of AML. Intriguingly, the majority of the genes within this profile are strongly down regulated, suggesting that they may represent CEBPA target genes whose expression is negatively affected by the absence of wt-CEBPA or the presence of mutated CEBPA. The gene that was most significantly down regulated in AML cases with either CEBPA mutations or CEBPA methylation is alpha-catenin (CTNNA1) (10 fold downregulation), a gene that we previously identified as a common virus integration site in retrovirally induced myeloid leukemias in mice (Erkeland et al., J Virol. 2004; 78, pp1971–80). We suggest that down regulation of CTNNA1 in CEBPA defective AML may be a critical step in leukemic transformation. Here we report the identification of a novel subgroup of AML with poor-prognosis, which overexpress DNMT3B and in which CEBPA expression is turned off, frequently as the result of hypermethylation of the CEBPA promoter.