Sox4 and CREB Cooperate in Myeloid Transformation,

Abstract 3452

The cAMP Response Element Binding Protein, CREB, is a transcription factor that is critical for cell proliferation and survival in neuronal and hematopoietic cells. We previously reported that CREB is overexpressed in leukemic blasts from patients with AML. CREB overexpression is also associated with an increased risk of relapse and decreased event-free survival in AML patients. We generated transgenic mice in which CREB is expressed under the control of the myeloid specific promoter, hMRP8. In vitro, bone marrow progenitors from CREB transgenic mice have increased proliferative potential and replating ability and these mice develop myeloproliferative disease, but not AML. Therefore, CREB is not sufficient to fully transform hematopoietic cells. To identify genes that cooperate with CREB, we performed retroviral insertional mutagenesis with CREB or wildtype C57/Blk6 mice. Newborn mice were infected with the MOL4070LTR retrovirus. Leukemia was observed in both CREB transgenic and wild type mice, however, latency for disease was significantly shortened in CREB transgenic mice at 9 months vs. 14 months for wild type mice (p<0.001). Infected mice developed hepatosplenomegaly (weighing up to 10-fold more than spleens from uninfected wild type mice). The leukemic phenotype by FACs analysis showed that 75% (24/32 mice analyzed) had AML with blast cells expressing Gr-1/Mac-1. Therefore, wild type MOL4070LTR infected mice had decreased penetrance for myeloid disease (45% or 5/11 mice analyzed). To identify potential cooperating oncogenes, genomic DNA was subjected to long-mediated PCR analysis to amplify genomic sequences upstream of the virus. Viral integration sites were identified by Southern blot analysis with genomic DNA from spleens from mice with leukemia. These sequences were mapped to the mouse genome to identify the chromosomal location. Blast analysis of retroviral integration sites (RIS) was performed on the publicly available Ensembl database and compared to known common integration sites on the Retroviral Tagged Cancer Gene Database (RTCGD http://rtcgd.ncifcrf.gov/). Sequencing of integration sites identified previously known oncogenes, such as Evi1/Mds, Evi5/Gfi1, Myb/Ahi, and Ras. Common integration sites identified in multiple mice include, sox4, Evi5/Gfi1, Myb/Ahi, Cbfa2t3. The highest incidence of integration in the mutagenesis screen was in the sox4 gene. Sox4 is a transcription factor that regulates embryonic development and cell fate. Transduction of CREB transgenic mouse bone marrow progenitor cells with a sox4 retrovirus increased survival and self-renewal by 2-fold (p<0.01) in serial replating and methylcellulose colony assays compared to wild type bone marrow progenitors. CREB overexpressing bone marrow progenitor cells stained with Brdu had a 10% increase in cycling cells compared to wild type bone marrow progenitor cells transduced with sox4 retrovirus (p<0.0001). Consistent with this observation is that sox4 retroviral transduction of CREB transgenic bone marrow cells led to increased CREB and cyclin D expression (p<0.01 and p<0.001, respectively). CREB transgenic mouse bone marrow transduced with sox4 retrovirus and stained with Annexin/7AAD also showed decreased apoptosis when cultured in low serum and absence of growth factor (p<0.05). Furthermore, the expression of the sox4 gene was increased in bone marrow cells from AML patients that also overexpressed CREB. Our results indicate that sox4 and CREB cooperate and contribute to transformation of hematopoietic progenitor cells.