C/EBPα or Its Leukemic Mutants Directly Activate nfkb1 Transcription and Displace HDAC1 or HDAC3 From Chromatin-Associated NF-κB p50 to Induce Anti-Apoptotic Genes.

Abstract

Abstract 76Mutated variants of C/EBPα are expressed in approximately 15% of AML cases. We have previously shown that C/EBPα and its AML mutants interact directly with NF-κB p50 through the basic region. Through binding to a DNA κB site they activate anti-apoptotic genes including BCL2 and FLIP, inhibiting both the extrinsic and intrinsic apoptotic pathways. These effects do not depend on DNA binding but rather on the integrity of the basic region, which is rarely altered in C/EBPα AML oncoproteins. Interaction with p50 is necessary for the C/EBPα induction of bcl-2 and FLIP but not of myeloid differentiation genes. In addition, NF-κB p50 binds and activates the CEBPA gene in myeloid cells. We now report that C/EBPα or its AML mutants, including a leukemia-derived leucine zipper mutant that cannot bind DNA directly (C/EBPαLZ), bind in vivo to the nfkb1 (p50) promoter. Using Ba/F3 cell lines expressing an estrogen receptor fusion of C/EBPα or the C/EBPαLZ mutant we demonstrate induction of p50 mRNA within 7 hrs after the addition of estradiol. A basic region mutant (C/EBPαBR) that cannot bind the p50 protein did not activate nfkb1 transcription. Importantly, the translation inhibitor cycloheximide did not prevent nfkb1 induction by C/EBPα, suggesting direct regulation of nfkb1 transcription by C/EBPα. Remarkably, C/EBPα did not induce p65 RNA expression. A minimal 200 bp nfkb1 promoter, containing κB but not C/EBP binding sites, is trans-activated by C/EBPα 9 fold, or 33 fold by the C/EBPαLZ AML oncoprotein. The variant C/EBPαBR fails to trans-activate the nfkb1 promoter. Together, these data indicate that the gene encoding NF-κB p50 is a direct target of C/EBPα. NF-κB p65 is held in the cytoplasm IκBα and enters the nucleus to bind DNA predominantly as p65:p50 heterodimers upon cell stimulation. In contrast, p50 homodimers are present in the nucleus bound to chromatin in resting cells and inhibit transcription due to the lack of a trans-activation domain and the recruitment of co-repressors and histone deacetylases (HDACs). Therefore, the induction of p50 by C/EBPα would be expected to result in diminished expression of target genes. However, our data indicates that C/EBPα and p50 synergistically activate anti-apoptotic genes. Remarkably, ChIP analysis demonstrated that C/EBPα induction from the metallothionein promoter in Ba/F3 cells diminishes the occupation of the endogenous BCL2 and FLIP promoters by HDAC1 but not HDAC3, although induction of C/EBPα expression did not alter the cellular content of either HDAC. In contrast, occupancy of Bcl-3, another p50 transcriptional cofactor, on these promoters did not change. In transiently transfected 293T cells we confirmed that p50 binds to HDAC1 in a co-immunoprecipitation reaction. Remarkably, interaction between p50 and HDAC1 was disrupted in the presence of co-expressed C/EBPα, C/EBPβ, or the AML mutants C/EBPαLZ, or C/EBPαp30. In a striking correlation with our previous findings, the C/EBPαBR basic region mutant of C/EBPα that does not bind p50 did not interrupt this interaction. As an important control, we confirmed that HDAC1 does not interact directly with C/EBPα or its mutants, indicating that C/EBPα competes with HDAC1 for interaction with p50. Interestingly, the AML-derived C/EBPαLZ or C/EBPαp30 oncoproteins, but not wild-type C/EBPα, interrupt the interaction between HDAC3 and p50. This suggests a greater capacity for these leukemia mutants to de-repress NF-κB target genes compared with C/EBPα itself. In summary, C/EBPα cooperates with NF-κB p50 to induce several anti-apoptotic genes, thereby providing a survival advantage to the leukemic blasts. C/EBPα and p50 reciprocally regulate each other's expression at the transcriptional level, establishing a positive feedback relationship. Moreover, C/EBPα or its AML mutants displaces HDAC1, and the C/EBPα oncoproteins displace HDAC3, from NF-κB p50 homodimers bound to anti-apoptotic genes, contributing to NF-κB dysregulation in leukemia. Our data underscore the concept that the C/EBPα:NF-κB p50 complex is a potential novel therapeutic target in AML, and the C/EBPβ:p50 complex may functional similarly in lymphomas and in other malignancies where C/EBPβ rather than C/EBPα expression predominates. Disclosures:No relevant conflicts of interest to declare.