Fig 2: CEBPA decreases CGI methylation at target gene promoters.(A) Summary of CEBPA mutations in total 5976 AML patients according to the TCGA database and other literatures. (B) Reanalysis of DNA methylation in 194 sequenced AML patients by dividing the TCGA cohort into four groups, including CEBPA nonmutation (i.e., WT), single N-terminal mutations (i.e., TADmut), single C-terminal mutations (i.e., bZIPmut), and double mutations (i.e., both TADmut/bZIPmut). The patient numbers for each group are shown. Asterisks denote statistical significance with two-tailed unpaired Student’s t test. (C) In HL-60 cells, Flag-tagged wild-type CEBPA or N321D mutant protein was ectopically expressed, followed by Western blot to detect overexpressed proteins (top). In these cells, genomic DNA was isolated and then subjected to LC-MS/MS analysis to quantify global 5mC levels. See Materials and Methods for more details. (D) Stable HL-60 cells with knockdown of endogenous CEBPA and reinduction of lentiviral vectors expressing empty vector (VEC) or Flag-tagged wild-type CEBPA or the N321D mutant to a comparable level as endogenous CEBPA protein. In these stable cells, the occupancy of Flag-CEBPA at the promoter regions of indicated target genes was determined by ChIP-qPCR. See Materials and Methods for more details. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was included as a negative control. (E) In stable HL-60 cells with CEBPA knockdown and put-back as described in (E), 5mC levels at CEBPA binding sites at the promoter regions of indicated CEBPA target genes were determined by BS-PCR. See Materials and Methods for more details. 5mC level was calculated as the percentage of methylated cytosines. (F) In stable HL-60 cells in (E), the mRNA expression of indicated CEBPA target genes was determined by quantitative reverse transcription (qRT-PCR). Average values of triplicated results with SD are shown. Asterisks denote statistical significance with two-tailed unpaired Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001 for the indicated comparison.

Fig 3: CEBPAN321D mutation sensitizes leukemia cells to DNMTis.(A) K562 and HL-60 stable cells with knockdown of endogenous CEBPA and reinduction of lentiviral vectors expressing empty vector (VEC) or Flag-tagged wild-type CEBPA or N321D mutant to a comparable level as endogenous CEBPA protein. Moreover, DNMT3A was deleted by CRISPR-Cas9–mediated genome editing technique in these stable cells. For each group, 2 × 106 cells were initially grown in 3 ml of culture medium without or with 5-Aza (1 µM) or DAC (1 µM), and the cell numbers were monitored during the period of 4 days by cell counting. DMSO, dimethyl sulfoxide. (B) Strategy to generate K562 CDX mouse model. See Materials and Methods for more details. (C) Numbers of mWBC and K562 cells in peripheral blood samples of xenografted mice after DAC therapy (n = 9 for each group). The data were collected on the date of sacrifice when an individual animal had lost =25% of body weight, and the results are representative of two independent experiments. (D) Representative data for Giemsa staining in peripheral blood from xenografted mice after DAC therapy (blue arrow, mWBC; red arrow, K562). (E) Kaplan-Meier survival curves and the log-rank test in xenografted mice with K562 overexpressing wild-type CEBPA or CEBPAN321D with DAC therapy (n = 9 to 10 and n = 16 per group for PBS and DAC treatment, respectively). Average values of triplicated results with SD are shown. Asterisks denote statistical significance with two-tailed unpaired Student’s t test. *P < 0.05, **P < 0.01, and ***P < 0.001 for the indicated comparison.

Fig 4: AML-derived C-terminal mutations in CEBPA are associated with DNA hypermethylation of PRC2 targets.(A) Summary of gene sets enriched in the list of CEBPA C-terminal mutation–associated hypermethylated genes (CEBPAC-mut) from the TCGA dataset. Gene sets were obtained from the Molecular Signatures Database (MSigDB). Hypergeometric test P values are indicated, and gene numbers are also illustrated. (B) Enrichment plots from GSEA showing significant enrichment of gene sets associated with CEBPA C-terminal mutations in TCGA AML patients. P values were determined using a hypergeometric test. (C) Venn diagram showing overlap of high CpG promoter genes and CEBPAC-mut genes that are hypermethylated PRC2 target genes in TCGA AML patients. Overlap of these gene sets was measured by a hypergeometric test. (D) Venn diagram showing overlap of the CEBPAC-mut genes from the TCGA dataset and genes that are significantly down-regulated in AML. Overlap of these gene sets was measured by a hypergeometric test. (E) Pathway enrichment of CEBPAC-mut genes that are hypermethylated PRC2 target genes derived from the TCGA dataset. cAMP, adenosine 3',5'-monophosphate; MAPK, mitogen-activated protein kinase; NES, normalized enrichment score.

Fig 5: CEBPA physically interacts with DNMT3A.(A) Heatmap and Venn diagram showing different DNA methylation in 194 sequenced AML patients from the TCGA cohort, including mutations that are mostly linked to DNA hypermethylation (WT1, IDH2, and CEBPA) and hypomethylation (DNMT3A). (B) Schematic representation of the mammalian two-hybrid system and dual-luciferase reporter system used to search for CEBPA-interacting DNA-modifying enzymes in HEK293K cells. Average values of triplicated results with SD are shown. (C) Myc-tagged DNMT proteins, including DNMT1, DNMT3A, DNMT3B, and DNMT3L, were coexpressed with Flag-CEBPA in HEK293T. Protein-protein interaction was examined by IP-Western using the indicated antibodies. (D) Schematic representation of two isoforms of DNMT3A. (E) HA-tagged DNMT3A or DNMT3A2 was coexpressed with Flag-CEBPA in HEK293T cells. Protein-protein interaction was examined by IP-Western using the indicated antibodies. (F) Endogenous DNMT3A protein was purified by IP with an anti-DNMT3A antibody from HL-60 cells with or without knockout (KO) of DNMT3A, followed by Western blot to detect CEBPA. WT, wild type.