Reintroduction of C/EBPα in leukemic CD34+ stem/progenitor cells impairs self-renewal and partially restores myelopoiesis

Abstract

Abstract The CCAAT/enhancer binding protein (C/EBP) α transcription factor is indispensable for myeloid differentiation. In various myeloid leukemias, C/EBPα is mutated or functionally impaired due to decreased C/EBPα expression or phosphorylation. In order to investigate the functional consequences of decreased C/EBPα function in AML, we reintroduced C/EBPα in primary CD34+ sorted acute myeloid leukemia (AML) cells using a lentiviral approach. Self-renewal and differentiation of primary AML stem cells were studied on long-term MS5 cocultures. Activation of C/EBPα immediately led to a growth arrest in all AML cultures (N = 7), resulting in severely reduced expansion compared with control cultures. This growth arrest corresponded with enhanced myeloid differentiation as assessed by fluorescence-activated cell sorter (FACS) analysis for CD14, CD15, and CD11b. Myeloid differentiation was further confirmed by the up-regulation of neutrophil elastase and granulocyte colony-stimulating factor (G-CSF) receptor in C/EBPα transduced cells. C/EBPα-expressing AML CD34+ cells failed to generate second and third leukemic cobblestone areas (L-CAs) in serial replating experiments, while control cultures could be sequentially passaged for more than 4 times, indicating that reintroduction of C/EBPα impaired the self-renewal capacity of the leukemic CD34+ compartment. Together, our data indicate that low C/EBPα levels are necessary to maintain self-renewal and the immature character of AML stem cells.