CREB Regulates Early Myelopoiesis and Myeloid Engraftment.

Abstract 1452

The cAMP-responsive element binding protein (CREB) is a nuclear transcription factor that regulates genes that control cell proliferation, differentiation, and survival. CREB overexpression leads to increased proliferation and survival of myeloid cells. Transgenic (Tg) mice overexpressing CREB under the control of the myeloid specific promoter hMRP8 develop myeloproliferative disease (MPD) but not leukemia. We hypothesized that transplantation of hematopoietic stem cells from CREB transgenic mice into lethally irradiated recipient wild type mice would lead to enhanced myelopoiesis and myeloid engraftment.

The goal of our study was to determine if proliferative stress through transplantation would result in increased myeloid engraftment and progression of CREB overexpressing cells from MPD to leukemia. Steady state analyses were performed on CREB Tg mice, including flow cytometry to resolve common myeloid progenitors (CMP), granulocyte macrophage progenitors (GMP), and megakaryocyte erythroid progenitors (MEP), as well as cell cycle analysis to determine baseline proliferative state. In vitro and in vivo models that exposed CREB-expressing cells to proliferative stress were used. In the former case, long-term bone marrow cultures (LTBMC) were established on an adherent layer of stromal cells prepared from wild type (WT) bone marrow (BM) with media specific for myeloid cell growth. BM cells (2 × 10⁶) from CREB Tg mice or WT controls were seeded onto the stroma and evaluated at 4 and 8 weeks for myeloid cell proliferation. In vivo studies were conducted by transplanting (2.5 × 10⁶) BM cells from CREB Tg mice into lethally irradiated recipients that were sacrificed at 4 weeks. Cells harvested from LTBMC or transplant recipients were analyzed by flow cytometry to evaluate cell lineage and proliferation or were plated in methylcellulose and assessed for colony formation. In addition, kinetic analyses were performed on these populations.

At baseline, CREB Tg mice have an increased percentage of early progenitors (1.8% vs. 1.2%, p=0.0001) with increased absolute numbers of CMP (17,683 cells vs. 11,650 cells, p=0.0001) at 12 weeks of age compared to WT controls. CREB Tg mice also have increased number of cells in S phase at baseline (26% vs. 20%, p=0.0022) due to upregulation of cyclins A and D. LTBMCs seeded with BM cells from CREB Tg mice had greater numbers of myeloid cells at 4 weeks compared to cultures established with WT marrow (4.5 × 10⁶ cells/mL and 1.3 × 10⁶ cells/mL respectively, p = 0.0135). Consistent with these data, mice transplanted with CREB Tg BM had a significantly higher percentage of donor myeloid cells at 4 weeks, detected using cell surface markers Gr-1+Mac-1+ (67% vs. 40%, p=0.0061). These mice also had a higher percentage of more differentiated Mac-1+ myeloid cells (11% vs. 0%, p=0.0014) and a higher number of myeloid cells in BM colony assays compared to recipients of WT marrow (69% vs. 13%, p<0.0001). At 4 weeks post-transplant, the histology of the spleen and liver from mice transplanted with CREB Tg marrow demonstrated replacement of the lymphocytes in the white pulp with macrophages, as well as extramedullary hematopoiesis in the liver that was not observed in WT controls. Our results provide evidence that CREB overexpression enhances myelopoiesis and short-term myeloid engraftment, but is not sufficient for transformation to AML. Therefore, CREB plays a critical role in normal hematopoietic dynamics and myeloid progenitor cell kinetics.