DEK Regulates Hematopoietic Stem Engraftment and Progenitor Cell Proliferation (2011)

Abstract Abstract 1275 Hematopoiesis is regulated by cell-cell and cytokine-cell interactions on hematopoietic stem (HSCs) and progenitor (HPCs) cells. In our continuing efforts to elucidate players involved in regulation of HSC and HPC growth, we focused on DEK, an abundant and unusual protein found in multicellular organisms. DEK has two DNA binding modules and has some affinity for specific DNA sequences, but primarily recognizes and binds to superhelical and cruciform DNA and induces positive supercoiling. DEK manifests multiple cellular activities, which include transcriptional repression and activation, mRNA processing, and chromatin architectural functions. We recently demonstrated that DEK modulates global heterochromatin integrity in vivo. Interestingly, DEK, can leave the cell and act as a chemoattractant for CD8+T cells and natural killer cells. Being intrigued that a nuclear protein was able to be secreted by hematopoietic cells, and act on other hematopoietic cells, we hypothesized that DEK might play a role in HSC/HPC function and hematopoiesis. In order to determine if DEK had an effect on steady state hematopoiesis, BM and spleen cells from DEK −/− mice were compared to that of wildtype (WT) control mice for absolute numbers and cycling status of HPC. Absolute numbers of CFU-GM, BFU-E, and CFU-GEMM per femur and per spleen were increased in DEK −/− mice. These effects were consistent with significantly increased percentages of HPCs in S-Phase of the cell cycle in DEK −/− BM and spleen, suggesting that DEK acts as a negative regulator of HPC proliferation in vivo. To confirm this, recombinant human DEK was tested for effects on HPC proliferation using unseparated mouse BM and low density human CB cells. DEK, dose-dependently suppressed colony formation by mouse BM CFU-GM stimulated by either IL-3 or GM-CSF, each alone; it did not influence colony formation stimulated by M-CSF alone. However, it dose-dependently inhibited CFU-GM colony formation by either IL-3, GM-CSF, or M-CSF when these cytokines were combined with the potent co-stimulating cytokine SCF. In fact, inhibition by DEK was greater on CFU-GM stimulated by the combination of IL-3, GM-CSF or M-CSF, each in the presence of SCF, compared to CFU-GM stimulated by IL-3, GM-CSF or M-CSF each alone in terms of percent inhibition, as well as the amount of DEK required to inhibit colony formation. Similar results were noted for HPCs present in human CB. This suggests that immature subsets of HPCs are more sensitive in vitro to the suppressive effects of DEK, than are the more mature HPCs. To determine if the DEK effects were directly or indirectly manifesting on the HPCs, DEK was assessed for effects on single isolated CD34+ cord blood cells, each in a single well stimulated by EPO, GM-CSF, IL-3, and SCF. DEK significantly decreased the number of wells containing a CFU-GM-, BFU-E-, or CFU-GEM- colony, demonstrating that DEK initiates it's suppressive effect directly on HPC. Using a mouse competitive repopulating HSC assay in vivo, allows assessment of the short- and longer-term repopulating HSC, and transplantation of BM cells from primary to secondary lethally-irradiated recipients in a non-competitive assay describes the longer-term repopulating HSC, and can offer information on the self-renewal capacity of this population of HSCs. While there was no difference in the HSC repopulating capacity of DEK −/− and WT shorter-term repopulating cells (months 1 and 2 for blood chimerism), there was a significant decrease in DEK −/− compared to WT BM cell repopulation at month 4 in the blood, and month 6 in the BM. This decreased repopulation of DEK −/− compared to WT HSC was even more apparent in secondary mouse recipients suggesting that DEK played a positive role in engraftment of longer-term repopulating HSCs, and perhaps in the self-renewal capacity of mouse BM HSCs. These studies demonstrate a here-to-fore unknown role for DEK in the regulation of HPCs, HSCs and hematopoiesis. DEK could have separate effects on HPC and HSC, as suggested by the direct acting effects of DEK on single HPC. Alternatively, DEK may alone, or in addition allow HSC to favor a self-renewal, vs. a differentiation pathway to HPC. Thus, DEK has potent effects on HSCs, HPCs, and hematopoiesis, and may be of potential clinical value for enhancing HSC activity/proliferation in vivo, or in an ex-vivo situation. Disclosures: No relevant conflicts of interest to declare.