C/EBPa is a key mediator of myeloid development. The CEBPA gene harbors point mutations in 10% of AMI cases. Also, AML1-ETO, CBFb-SMMHC, and FI13ITD inhibit CEBPA transcription, and bcr-abl and AML1-MDS1-EVI1 inhibit CEBPA translation. CEBPA gene mutations have also been detected in 8% of MDS cases and predict imminent progression to AMI. 65% of CEBPA mutations are N-terminal and lead to expression of a truncated p30 protein from an internal ATG, and 35% are inframe mutations in the leucine zipper (LZ) which prevent DNA-binding. We have made the novel finding that the C/EBPap30 and C/EBPaLZ oncoproteins inhibit apoptosis via induction of bcl-2, dependent upon functional and likely direct interaction with the p50 subunit of NF-kB. Inhibition of apoptosis may be a key step in progression of MDS to AML. To further our understanding of the role of this pathway in MDS progression, we propose the following aims:
AIM 1 : To determine whether direct interaction with NF-kappaB p50 is critical for induction of bcl-2 by C/EBPa.
AIM 2 : To identify the critical residues within C/EBPa and NF-kappaB p50 which mediate their biochemical and functional interaction.
AIM 3 : To develop murine models of C/EBPa associated MDS and to determine the role of interaction with NFkappaB and the LZ in these processes.
AIM 4 : To use microarray analysis to identify genetic targets of C/EBPa in murine MDS and AML cells dependent upon its interaction with NF-kappaB and those dependent upon DNA-binding. Co-immunoprecipitation will be used to assess biochemical interaction, promoter studies, ChIP, expression of a p50 inhibitor, and use of NF-kappaB knockout cell line lines will evaluate functional interactions. Retroviral transduction of C/EBPap30 or C/EBPaLZ in p15(-/-) marrow, alone or with activated N-Ras, will be employed to develop models of MDS (at early time points) and AML, (as the mice age). Variants of C/EBPap30 and C/EBPaLZ which do not bind NF-kB will be evaluated similarly for comparison. Bcl-2 induction and susceptibility to spontaneous and chemotherapy induced apoptosis will be evaluated using murine MDS and AML cells, and their mRNA will be subjected to microarray analysis to identify direct C/EBPa genetic targets and those reflecting activation of genes indirectly, via tethering to NF-kappaB. These data will be useful for interpreting expression data from human MDS patients and may uncover a common pathway leading to inhibition of apoptosis and progression to AML.
