Role of Different C/EBPα Mutations in AML Transformation.

Abstract

Acute Myeloid Leukemia (AML) is characterized by an abnormal hematopoietic differentiation and uncontrolled cell proliferation. Mutations in several transcription factors (TFs) have been implicated in the development of leukemia. One of these TFs is CCAAT/enhancer-binding protein-α (C/EBPα). In normal hematopoiesis, C/EBPα plays a central role to coordinate myeloid differentiation and growth arrest. C/EBPα is mutated in approximately 9% of AML; these mutations take place either in C or N terminal domains of the protein, although there are several familial cases of AML where both types of mutations have been found. We use C and/or N terminal C/EBPα mutations from one case of sporadic AML to investigate the role of each mutation in leukemic transformation (Smith et al., 2004, N Engl J Med 351, 2403–2407). Human lineage negative (Lin-) umbilical cord blood were transduced with lentiviral vectors carrying the wild type C/EBPα (WT), N terminal mutated C/EBPα (N-ter) or N and C terminal mutated (NC-ter) C/EBPα cloned from this sporadic case of AML. We observed differences in proliferation of transduced Lin- in vitro: WT C/EBPα expression resulted in G0 cell cycle arrest causing a progressive extinction of the transduced cells overtime; N-ter cells showed a higher proliferative advantage over untransduced cells. The NC-ter CEBPα cells like untransduced cells kept their levels throughout culture. Furthermore, when induced into myeloid differentiation in vitro, WT C/EBPα cells were mainly inducing fully mature granulocytes whereas N-ter C/EBPα was not able to induce terminal granulocytic differentiation; in contrast NC-ter C/EBPα did not increase myeloid differentiation. Additionally, their ability to form Colony Forming Units (CFUs) in primary, secondary and tertiary replating was also tested: WT transduced cells gave rise to few primary CFUs; contrary, N and NC-ter could generate both primary and secondary CFUs, but only NC-ter cells were able to produce CFUs in tertiary replating, indicating its ability to maintain undifferentiated hematopoietic progenitors in vitro. These results were confirmed using Long-Term Culture Initiating Cells (LTC-IC) where the NC-ter mutated cells showed the highest LTC-IC after 5 weeks. Finally, in vivo transplantation in NOD/SCID/β2mnull indicated that NC-ter mutated cells engraft better than WT and N-ter 8 week post- transplant. Serial transplantation experiments are underway to evaluate their self-renewal capacity. Our results confirmed some known functions of WT C/EBPα in human hematopoiesis, such as inducing myeloid differentiation and cell cycle arrest. On the other hand, we showed new functions for the C/EBPα mutants. The N-ter C/EBPα mutation caused an increase in cell proliferation and blockage of terminal granulocytic differentiation, whereas the NC-ter C/EBPα mutation increased the self-renewal capacity of progenitor/stem cells without having an influence on myeloid differentiation. This work provides further insight into the mechanisms by which different C/EBPα mutations induce AML.