CEBPA Gene Mutations Interfere with Expressions of NKG2D Ligands Ulbps in AML Cells and Modulate Their Susceptibility to NK-Mediated Lysis

Introduction:CEBPA gene encodes CCAAT/enhancer-binding protein-alpha (C/EBPα), a crucial granulocytic differentiation factor and tumor suppressor in hematologic and many non-hematologic malignancies. We previously reported a donor-derived relapse of AML patient after allogeneic hematopoietic stem cell transplantation with multiple mutations in CEBPA gene: a N-terminal frameshift mutation (247dupC causing overproduction of truncated 30-KDa isoform, lacking the TAD1 domain, p30), a N-terminal germ-line mutation (584_589dup disrupting the TAD2 domain of protein, NM2), and a C-terminal mutation (914_916dup disrupting the bZIP domain, CM) (Blood 2011; 117: 5257-5260). Although studies from multiple laboratories have contributed immensely to our understanding that how different CEBPA mutations disturb C/EBPα functions including granulopoiesis and leukemic transformation in AML, whether C/EBPα might regulate immunosurveillance remains unknown.

Methods: AML cell line cells infected with lentivirus to over-express of wild type C/EBPα as well as 3 types of C/EBPα mutants were co-cultured with NK92MI cells and detected cytotoxic lysis through FCM. NK92MI cells were stained with CD107a to detect degranulation.We performed gene expression microarray profiling analysis in AML cell line cells with over-expression of wild type C/EBPα and mutants . Flag tagged wide type C/EBPα was over-expressed in 293T cells and ChIP with anti-Flag antibody followed by sequencing assay was performed to explore candidate gene binding sites of C/EBPα. Finally, independent ChIP-qPCR of candidate sequences were performed to further verify the transcription factor binding sites of C/EBPα.

Results: ULBPs expressed on the surface of tumor or infected cells are important ligands of NK cell receptor NKG2D. Our gene expression microarray profiling analysis showed that wild type C/EBPα could up-regulate the expression of ULBP2/5/6 in AML cell line cells. Consistent with the results of gene expression microarray profiling analysis, over-expression of wild type C/EBPα and a N-terminal germ-line mutant (NM2) can up-regulate ULBP2/5/6 expression in NB4 cells, whose endogenous expression of ULBPs was low. Meanwhile, the sensitivities of NB4 cells to the cytotoxicity of NK92MI cells were also increased by over-expression of wide type C/EBPα and NM2 mutant. In contrast, leukemia-associated somatic mutations, C/EBPα p30 and C-terminal mutant (CM), were disabled to up-regulate ULBPs expression. In dual-luciferase reporter assay, the ratio of level of firefly luciferase and renilla luciferase significantly increased when co-transduced report plasmid with wide type C/EBPα expressing plasmid compared with vector control, indicating that C/EBPα could up-regulate the transcription of ULBP2 as a transcription factor. Through ChIP-seq assay we identified 12 peaks nearby ULBP genes in chromosome 6. We further performed ChIP-qPCR to target the sequences acting as enhancers of ULBP genes, which located +7kb upstream of transcription start site of ULBP2 gene, +11kb upstream of ULBP5 gene, -9kb downstream of ULBP6 gene and -40kb downstream of ULBP1 gene. Wide type C/EBPα showed higher binding affinity to the ULBP2/5 enhancers with more than 50 folds' enrichment and to the ULBP6/1 enhancers 9 folds' enrichment compared with IgG control. The N-terminal germ-line mutant (NM2) conserved part of the binding affinity, but the enrich fold was lower than wide type. As expected, leukemia-associated C-terminal mutant (CM) totally lost its binding ability to both sequences due to the damage of DNA binding domain. Althoughleukemia-associated truncated 30-KDa isoform partly conserved its binding ability to these DNA sequences, the mutant lost the function of regulating ULBPs expression.

Conclusions: C/EBPα played an important role in innate immunosurveillance of AML. C/EBPα could bind to the promoter and potential enhancers of ULBP genes as a transcription factor, up-regulate expression of ULBPs and eventually induce AML cells to be recognized and killed by NK cells. Mutations in TAD2 domain did not affect this regulation function, while mutations in TAD1 and bZIP domain lost the specific ability. Leukemia cells with N-terminal frameshift mutations (p30), or C-terminal mutations could escape from surveillance of NK cells and may play pivotal roles in leukemia relapse.