Abstract
Introduction
CEBPA gene encodes CCAAT/enhancer-binding protein-alpha (C/EBPα), a crucial granulocytic differentiation factor and tumor suppressor in hematologic and non-hematologic malignancies. CEBPA gene is mutated in approximately 5–14% of acute myeloid leukemia (AML) patients and exhibited 3 different types of mutations, germ-line N-terminal mutation, N-terminal frameshift mutation and C-terminal mutation. Although murine models provide the functional consequences of CEBPA gene mutations in human hematopoiesis, the impacts of different mutations on abrogating C/EBPα functions and those of germ-line N-terminal mutations on bone marrow microenvironment have been received less attention.
Methods
In our previous research, we have provided the first report of multiple mutations of CEBPA contributing to the transformation of donor cells to the leukemic phenotype after allogeneic hematopoietic stem cell transplantation and identified 3 different CEBPA gene mutant forms which disrupted 3 major functional domains of C/EBPα respectively (H Xiao, Blood 2011; 117: 5257-5260). The patient and his donor-sister both harbored the N-terminal germ-line mutation (584_589dup disrupting the TAD2 domain of protein). Susceptible donor hematopoietic cells evolved to overt AML by developing two somatic CEBPA mutations, the N-terminal frameshift mutation (247dupC causing overproduction of truncated 30-KDa isoform (C/EBPα- p30) lacking the TAD1 domain) and the C-terminal mutation (914_916dup disrupting the bZIP domain), in the patient's microenvironment. We used these 3 mutant forms, as well as CEBPA gene wild type to subclone into pLenti6.3-MSC vector. Human leukemia cell lines (NB4, Kasumi-1, HL60, K562) and mouse myeloid progenitor cell line (32Dcl3) were transduced by different mutant forms to assess the impacts on blockage the major functions of C/EBPα including antileukemia effect and inducing granulocyte differentiation. To further discover previously unkown tumor suppressor genes dysregulated by C/EBPα mutant forms, the gene expression profiles of NB4 cells stably transfected with CEBPA gene different mutant forms were compared with that of CEBPA gene wild type vector by using Affymetrix PrimeView human gene expression microarray analysis. On the other hand, human normal bone marrow mesenchymal stromal cell ( MSC ) was transduced by N-terminal germ-line mutation to assess the impacts on the capacity of MSC to differentiate towards the osteogenic/adipogenic lineages, migrate and protect to leukemia cells.
Results
(1) The N-terminal germ-line mutation retains the functions of inducing apoptosis in leukemic cells and granulocyte differentiation of C/EBPα.The truncated C/EBPα-p30 protein mutant, produced from the N-terminal frameshift mutation, abrogates the effect of inducing apoptosis in leukemia cells. The C-terminal mutation (914_916 dup) abrogates both the effects of inducing apoptosis in leukemic cells and of promoting G-CSF-induced differentiation of 32Dcl3 cells into mature neutrophile granulocyte. (2) Gene expression microarray profiling analysis showed that compared with CEBPA gene wild type, leukemia-associated CEBPA somatic mutations, the N-terminal frameshift mutation and the C-terminal mutation, significantly inhibited the expression of ULBP2, an innate surface ligand of the natural killer (NK) cell receptor NKG2D, ultimately contributed to resistance to NK cell-mediated cytotoxicity. (3) MSCs harboring CEBPA N-terminal germ-line mutation showed impaired differentiation potential to osteogenic lineage by downregulation the expression of osteogenic genes (BSP1 and Runx2) , however similar in differentiation potential to adipogenic lineage, migration and the protection to leukemia cells, compared with MSCs with CEBPA wild type.
Conclusions
Our data provide clues to support that the N-terminal frameshift mutation of CEBPA works as a class I mutation, while the C-terminal mutation works as both of class I and class II mutations in inducing leukemia. Furthermore, those leukemia-associated CEBPA somatic mutations abrogate the tumor suppressor function of C/EBPα by inhibiting NKG2D-mediated NK cell cytotoxicity. The N-terminal germ-line mutation retains the major functions of C/EBPα, however impaired the osteogenic differentiation potential of bone marrow stromal cells, which is important to support hematopoiesis.
Disclosures:
No relevant conflicts of interest to declare.
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