Identification of Meis1 as a Target of CREB in Myeloid Leukemogenesis.

The cyclic AMP response element binding protein, CREB, is a leucine zipper transcription factor that induces the expression of genes that regulate proliferation and survival. We previously demonstrated that CREB expression was increased in bone marrow cells from patients with acute leukemia. Transgenic mice that overexpress CREB in myeloid cells develop a myeloproliferative/myelodysplastic syndrome after one year. Although CREB has been implicated in the development of leukemia, the mechanisms by which CREB overexpression leads to a malignant phenotype remain elusive. To identify CREB target genes, we performed microarray analysis with RNA from two CREB overexpressing cell lines, K562 and NFS60. Both murine (NFS60) and human (K562) myeloid leukemia cell lines were stably transfected with CREB under the control of the myeloid-specific HMRP-8 promoter. Meis1, an important transcription factor in hematopoiesis, was found to be upregulated 40-fold in CREB overexpressing cells compared to controls. In the development of granulocytes, Meis1 expression decreases as cells differentiate. Similar to CREB, Meis1 is expressed in CD34+ hematopoietic cells but not in CD34- cells. Meis1 expression has been reported to promote self-renewal of stem cells and inhibit G-CSF-dependent differentiation into granulocytes. We hypothesized that CREB overexpression may lead to persistent expression of Meis1, increased self-renewal, and a block in differentiation of primitive hematopoietic progenitor cells, thereby contributing to leukemogenesis. We first examined Meis1 expression in CREB overexpressing K562 and NFS60 cells. Significantly higher levels of Meis1 were detected in Western blot analysis with lysates from CREB overexpressing K562 cells compared to parental cells. In addition, the degree of CREB expression correlated with the degree of Meis1 expression when evaluated at the protein level. Experiments performed using the NFS60 cell line showed similar results. To investigate Meis1 expression in primary myeloid leukemia cells, bone marrow from patients with acute myeloid leukemia (AML) were analyzed for CREB and Meis1 expression. Samples from three of the four patients had elevated CREB expression. Patients whose cells had the highest levels of CREB also had highest expression of Meis1. The one patient with minimal CREB expression showed no detectible levels of Meis1. Evaluation of cell lines and primary cells using quantitative real time PCR is underway to determine potential differences in CREB and Meis1 expression at the mRNA level. Chromatin immunoprecipitation will also be performed to determine whether Meis1 is a direct target of CREB. Our data demonstrate that CREB overexpression correlates with overexpression of Meis1 in both cell lines and primary AML cells. These results suggest that CREB and Meis1 are co-regulated in AML cells and may contribute to the leukemia phenotype.