C/EBPγ deregulation results in differentiation arrest in acute myeloid leukemia

C/EBPs are a family of transcription factors that regulate growth control and differentiation of various tissues. We found that C/EBPγ is highly upregulated in a subset of acute myeloid leukemia (AML) samples characterized by C/EBPα hypermethylation/silencing. Similarly, C/EBPγ was upregulated in murine hematopoietic stem/progenitor cells lacking C/EBPα, as C/EBPα mediates C/EBPγ suppression. Studies in myeloid cells demonstrated that CEBPG overexpression blocked neutrophilic differentiation. Further, downregulation of Cebpg in murine Cebpa-deficient stem/progenitor cells or in human CEBPA-silenced AML samples restored granulocytic differentiation. In addition, treatment of these leukemias with demethylating agents restored the C/EBPα-C/EBPγ balance and upregulated the expression of myeloid differentiation markers. Our results indicate that C/EBPγ mediates the myeloid differentiation arrest induced by C/EBPα deficiency and that targeting the C/EBPα-C/EBPγ axis rescues neutrophilic differentiation in this unique subset of AMLs.

The temporal and spatial control of cell-specific transcription factors and their abundance determines to a large extent the cell fate. The hematopoietic system, due to its well characterized cell hierarchy and well defined cell types, has been extensively used to study the function of transcription factors in cell renewal and differentiation (1, 2). Balancing these 2 processes is crucial for homeostasis, and defects in lineage-specific transcription factors, often caused by translocations or mutations, have been described in human leukemia (3–5).

CCAAT/enhancer-binding proteins (C/EBPs) are a family of basic region leucine zipper DNA–binding proteins of which 6 core members have been identified: C/EBPα, C/EBPβ, C/EBPγ, C/EBPδ, C/EBPε, and C/EBPζ (5–8). In the hematopoietic system, C/EBPα plays a crucial role in the commitment of multipotent progenitor cells into the myeloid lineage, and mice deficient in C/EBPα are characterized by a specific block in the transition from common myeloid progenitors (CMP) to granulocyte-macrophage progenitors (GMP), resulting in the lack of mature and functional granulocytes (9, 10). Defects in CEBPA, such as differentiation-deficient mutations, posttranscriptional modifications, posttranslational inhibition, and epigenetic regulation, have been shown to inhibit C/EBPα function in acute myeloid leukemia (AML) (11–17). C/EBPβ plays a role in macrophage activation (18) and is required during emergency granulopoiesis following administration of cytokines, a process that involves rapid production of granulocytes (19). C/EBPε, C/EBPδ, and C/EBPζ have also been implicated at different stages of granulocytic development (20, 21), and aberrant expression of these C/EBP members has been observed in AML and secondary granule deficiency syndromes (22–25). Thus, C/EBPγ is the only C/EBP member that has not been studied in the context of myeloid differentiation or human AML.

C/EBPγ is ubiquitously expressed and was first identified by its affinity for cis-regulatory sites in the immunoglobulin heavy chain promoter and enhancer (26). Structurally, C/EBPγ is characterized by the absence of transactivation domains, although it retains the basic region and the leucine zipper domains, required for DNA binding and homo/heterodimerization, respectively (27, 28). C/EBPγ alone neither activates nor represses transactivation; however, C/EBPγ can inhibit transcriptional activation by other C/EBP members in a cell-specific manner (27, 29). C/EBPγ-KO mice show a high mortality rate within 48 hours after birth, and analysis of the lymphoid lineage demonstrated that C/EBPγ plays a critical role in the functional maturation of natural killer cells (30). In addition, overexpression of C/EBPγ in transgenic mice causes a block in definitive erythropoiesis, leading to severe anemia and fetal lethality (31).

In the present study, we identify a unique subset of AML patients with increased CEBPG expression levels and methylation/silencing of the CEBPA gene. We demonstrate that C/EBPγ is a suppressor of myeloid differentiation in these AML samples and that targeting the C/EBPα-C/EBPγ axis represents a novel therapeutic approach in this particular subtype of AML.

CEBPG is upregulated in a specific subset of AML patients with CEBPA promoter hypermethylation and silencing. In order to determine whether CEBPG is aberrantly expressed in certain cases of AML, we analyzed the gene expression profile (GEP) of 526 AML cases. This analysis revealed that CEBPG mRNA was significantly upregulated in a small subset of patients (n = 8) (Figure 1A). The 526 AML cases include a previously reported cohort of n = 285 samples (32), and as found in the original study, unsupervised clustering of the 526 cases defined a group of patients characterized by defects in CEBPA (Figure 1A). The defects in CEBPA were caused by either CEBPA biallelic mutations (n = 21) or CEBPA silencing, primarily due to hypermethylation (n = 9) (Figure 1A and refs. 17, 33). These later cases showed very low or no CEBPA mRNA levels compared with all other AML cases (Supplemental Figure 1A; supplemental material available online with this article; doi: 10.1172/JCI65102DS1), and C/EBPα protein could not be detected (17). In addition, it was reported that these leukemias highly express Trib2, a protein that degrades C/EBPα p42 protein, contributing to the absence of C/EBPα in the silenced cases (34). Interestingly, CEBPG expression levels were particularly high in 8 out of 9 cases characterized by CEBPA silencing in comparison with the other AML samples, normal bone marrow, and CD34⁺ specimens (Figure 1A). We verified these changes in CEBPG RNA levels by quantitative RT-PCR (Supplemental Figure 1B). In summary, we identified a subset of AML patients characterized by silencing of CEBPA and upregulation of CEBPG.

Figure 1

CEBPG RNA is upregulated in the absence of CEBPA in a subset of human AML and in murine C/EBPα-deficient hematopoietic stem/progenitor cells. (A) Pairwise correlations between GEPs of 526 AML samples hybridized to the Affymetrix HGU133Plus 2.0 GeneChips identifies a subset with high CEBPG and low C/EBPA levels. The rectangle amplifies a cluster that gathers samples with defects in CEBPA. The bar and histograms next to each sample indicate the following: CEBPA status (mutations are shown in red and silencing in green), CEBPA, and CEBPG expression levels. In total, 8 cases present high CEBPG levels and CEBPA silencing due to hypermethylation. The single case with a blue arrow is a patient without CEBPA mutation but with very high CEBPA mRNA expression levels and without CEBPG mRNA expression. As this case shows a very similar gene expression signature, we propose this represents another defect within the C/EBPα/C/EBPγ “pathway.” (B) Quantitative RT-PCR analysis in murine sorted LKS cells from Cebpa^Flox/Flox cre^– (WT) and Cebpa^Flox/Flox cre⁺ (KO) mice. Cebpa and Cebpg mRNA expression levels were determined as percentage of Gapdh (relative expression) and represent the average value plus SD of at least 5 mice in each group. *P = 1.1 × 10^–7; **P = 1.6 × 10^–6. (C) C/EBPα-KO LKS cells were transduced with either pMSCV empty vector or C/EBPα pMSCV expression construct. Cebpa and Cebpg mRNA expression levels were determined in GFP⁺ cells 1 days after transduction. Data are the average of 3 independent experiments expressed as percentage of GAPDH plus SD. *P = 0.00045; **P = 0.0065.

Loss of C/EBPα in vivo results in upregulation of Cebpg in early stem/progenitor cells. Since we identified a subgroup of AML patients characterized by silencing of C/EBPα and upregulation of CEBPG, we asked whether ablation of Cebpa in vivo would lead to an increase of Cebpg expression, in particular in the early hematopoietic stem/progenitor cells (lineage^–c-kit⁺ Sca-1⁺ [LKS]). For these experiments we used a well defined Cebpa conditional KO model previously developed in our laboratory (10). Cebpa^Flox/Flox Mx1Cre⁺ conditional KO mice and Cebpa^Flox/Flox Mx1Cre^– control mice (WT) were treated with polyinosinic-polycytidylic acid (pI:pC) to induce excision of the single exon Cebpa gene (10). Quantitative RT-PCR analysis revealed that KO LKS cells lost detectable expression of Cebpa (P = 1.1 × 10⁷) and that the Cebpg transcripts had increased significantly compared with control LKS (P = 1.6 × 10^–6) (Figure 1B). Further, we determined that LKS cells from Cebpa heterozygous mice (Cebpa^Flox/+ Mx1Cre⁺) did not present increased Cebpg levels compared with the Cebpa^Flox/+ Mx1Cre^– control mice upon pI:pC treatment (Supplemental Figure 1C). Reintroduction of C/EBPα into C/EBPα-KO LKS cells using retroviral infection significantly reduced Cebpg expression (P = 0.0065) (Figure 1C). Similarly, reintroduction of the C/EBPα p30 isoform also diminished the Cebpg mRNA levels (P = 0.034) (Supplemental Figure 1D). In summary, our data suggest that C/EBPα negatively regulates Cebpg expression not only during later stages of granulopoiesis, but also in murine hematopoietic stem/progenitor cells. Further, the Cebpa^Flox/Flox Mx1Cre⁺ conditional KO mice mimic the human AMLs characterized by silencing of C/EBPα and upregulation of Cebpg, providing an excellent model to study the contribution of C/EBPγ to the development of those leukemias.

C/EBPα binds to the CEBPG promoter. Since we observed that CEBPG and C/EBPA expression levels are inversely correlated in a subset of AML patient samples and in the Cebpa conditional KO mice, we questioned whether CEBPG repression is directed by C/EBPα. First, we studied C/EBPα interaction with Cebpg regulatory regions in murine 32D/G-CSF–R cells transduced with a β-estradiol–inducible (E₂) C/EBPα-ER expression construct. When stimulated with E₂ to induce nuclear translocation of C/EBPα, these cells differentiate toward neutrophils within 3–4 days of culture, even in the presence of IL-3 (data not shown). After 4 hours of treatment, we performed ChIP followed by promoter array analysis and identified 4826 chromosomal regions, sized between 310 and 7399 nucleotides (median 1084), enriched in C/EBPα-ER cells compared with ER control cells at P < 1 × 10^–5 (false discovery rate 2.3%). The transcriptional start site of 1064 unique RefSeq genes was located within 1 kb downstream of 1 or more of the 4826 fragments. Among those, we identified previously reported genes to be directly or indirectly regulated by C/EBPs, such as Mpo (35), Hp (36), C3 (36), PU.1 (37), and IL-6rα (38) (data not shown). A strong interaction with the Cebpg promoter was observed (Figure 2A), whereas almost none of the other loci in that region appeared to be enriched by ChIP. Next, ChIP was performed in 32D/G-CSF–R cells to determine whether endogenous C/EBPα could immunoprecipitate Cebpg promoter. Similarly to the C/EBPα-ER overexpressing system, we observed immunoprecipitation of Cebpg promoter by endogenous C/EBPα (Supplemental Figure 2A), but not of a control upstream region (data not shown). To further investigate the binding of C/EBPα to the CEBPG promoter, we used K562 cells stably transfected with C/EBPα-ER as a model for human granulocytic differentiation (39). We identified a putative C/EBPα-binding site in the human CEBPG proximal promoter, which was highly conserved between mouse and human (–871 bp to –762 bp, oligos A, corresponding to the region amplified by oligos no. 2 in the murine promoter, Supplemental Figure 2B). ChIP assays showed binding of C/EBPα to this region (oligos A), but not to a control upstream region (oligos B) (Supplemental Figure 2, C and D). Together, these results demonstrate that C/EBPα can bind to the proximal promoter of CEBPG and potentially downregulate CEBPG expression.

Figure 2

C/EBPα interacts with the Cebpg promoter and mediates Cebpg repression. (A) ChIP on DNA promoter microarrays (ChIP on chip). 32D/G-GSF–R-C/EBPα-ER cells or ER-expressing cells were treated with E₂ for 4 hours. The diagram shows an amplified view of the Cebpg locus in chromosome 7. Genes (including Cebpg) are indicated in green, either on the plus or on the minus strand. Specific hybridization was observed (multiple probes in blue) to the representative Cebpg promoter region (– strand), whereas almost none of the other loci in that region appeared to be enriched by ChIP. (B) C/EBPα represses CEBPG promoter–driven luciferase activity. 293T cells, which endogenously express C/EBPγ, were cotransfected with 100 ng CEBPG luciferase reporter vector (–354 to +1) and increasing amounts of a C/EBPα expression plasmid, encoding for either the p42 or p30 form. (C) Mutation of the C/EBP-binding sites in the CEBPG luciferase reporter vector does not affect C/EBPα repression. 100 ng of the mutated reporter vector were cotransfected with increasing amounts of p42 C/EBPα plasmid. (D) C/EBPα suppresses CEBPG promoter activity of a short CEBPG reporter construct. 100 ng of a reporter construct containing 3 E2F-binding sites were cotransfected with different amounts of C/EBPα expression plasmid. (E) C/EBPα reduces E2F1 transactivation of the C/EBPγ promoter. 100 ng of the short C/EBPγ reporter construct was cotransfected with 100 ng E2F1 expression construct and increasing amounts of C/EBPα expression plasmid. The y axis indicates the relative percentage of luciferase activity. Results represent the average of at least 3 independent experiments; bars indicate SD.

C/EBPα mediates CEBPG repression by affecting E2F1 transcriptional activity. In order to determine whether C/EBPα could negatively regulate CEBPG promoter, we performed luciferase reporter assays. We generated a human CEBPG promoter construct (–354 bp to +1 bp from TSS, Supplemental Figure 2B) in the pXP2 reporter vector and performed luciferase assays to analyze CEBPG promoter activity. Cotransfection of the CEBPG reporter construct with increasing amounts of a C/EBPα expression plasmid showed that C/EBPα was able to repress CEBPG promoter reporter gene activity (Figure 2B). Of note, both a p42-C/EBPα plasmid and a p30-C/EBPα plasmid inhibited the CEBPG reporter gene activity in a dose-dependent manner and with similar efficiency (Figure 2B). As a positive control for C/EBPα transactivation, we cotransfected the C/EBPα expression vector with a reporter for the gene encoding the G-CSF receptor (CSF3R), denoted here as the G-CSF–R reporter. Luciferase assays showed that C/EBPα transactivated the G-CSF–R reporter gene, indicating that negative regulation by C/EBPα is specific to the CEBPG promoter (data not shown). Based on the AML microarray (Figure 1A) C/EBPγ is only upregulated in the C/EBPα silenced leukemias, suggesting that mutated C/EBPα proteins are still capable of repressing C/EBPγ expression. Luciferase assays with the C/EBPγ reporter construct and increasing amounts of distinct mutated C/EBPα expression plasmids showed that the N-terminal mutants repress C/EBPγ luciferase activity (Supplemental Figure 2E). These results demonstrate that WT p42 and p30 C/EBPα forms as well as N-terminal mutant C/EBPα proteins are able to negatively regulate the C/EBPγ promoter.

To identify the cis-acting elements on the CEBPG promoter that respond to C/EBPα, we first compared 2 human CEBPG reporter constructs: one corresponding to –957 bp to +1 bp of the CEBPG proximal promoter and the other corresponding to a shorter version (from –354 bp to +1 bp), which is missing the first 2 potential C/EBP-binding sites (Supplemental Figure 2B). Luciferase assays with increasing amounts of C/EBPα expression vector showed similar C/EBPα-mediated repression in both C/EBPγ reporter constructs, indicating that the first 2 potential C/EBP-binding sites are not necessary for the repression (data not shown). Next, we investigated whether the other 2 potential C/EBP-binding sites present in the shorter reporter construct were necessary to repress the luciferase activity. We therefore generated a CEBPG reporter construct with mutated C/EBP-binding sites in the context of the shorter reporter. These 2 C/EBP sites appeared not to be required, since C/EBPα was still able to repress the luciferase activity (Figure 2C). Thus C/EBPα represses either through other binding sites and/or in an indirect manner, through interaction with other transcriptional regulators. In fact, we and others have previously reported that C/EBPα can act as a transcriptional repressor through E2F-binding sites (40, 41). We determined the presence of 3 E2F-binding sites in the C/EBPγ luciferase constructs (Supplemental Figure 2B). To determine whether the E2F sites were required for C/EBPα repression, we generated a luciferase construct containing solely these 3 sites (–139 bp to +1 bp from TSS) and observed that C/EBPα could still significantly repress luciferase activity (Figure 2D). Deletion or mutation of these sites (Supplemental Figure 2F) resulted in a lack of reporter activity, pointing to a critical role of E2F in transcriptional control of CEBPG (data not shown). Several mechanisms may explain how C/EBPα regulates CEBPG through these E2F-binding sites. First, we assessed whether E2F binding to the CEBPG promoter could be affected by C/EBPα. ChIP performed on K562-C/EBPαER cells at distinct time points upon E₂ treatment showed that C/EBPα binds to the proximal promoter of CEBPG without displacing E2F1 (Supplemental Figure 2G). This result is in agreement with a previous publication by Johansen et al. demonstrating that C/EBPα had no effect on E2F1 binding to a consensus E2F DNA–binding site (40). Alternatively, C/EBPα may interfere with E2F transcriptional activity and repress the CEBPG promoter. We demonstrated that E2F1 protein (but not other E2F family members) could transactivate the CEBPG promoter and that E2F1-induced reporter activity could be reduced by increasing amounts of C/EBPα expression plasmid (Figure 2E). Together, these results indicate that C/EBPα can bind to CEBPG proximal promoter and downregulate C/EBPγ expression by affecting E2F1 transcriptional activity.

Next, we investigated whether changes in chromatin structure, which regulate gene expression, would be present in the C/EBPα-KO cells versus WT. DNA methylation HpaII tiny fragment enrichment by ligation mediated PCR (HELP) arrays in lineage^–c-kit⁺ murine bone marrow cells demonstrated no differences in the Cebpg DNA methylation status from KO versus WT mice (Supplemental Figure 2H). Similarly, HELP arrays in human AML patient samples showed no differences in the CEBPG DNA methylation profile of samples with CEBPA silencing and other AML subtypes (data not shown). Further, ChIP using H3K4me3 and H3K27me3 antibodies in lineage^–c-kit⁺ cells demonstrated increased H3K4me3 enrichment in the Cebpg promoter of C/EBPα-KO compared with WT cells (Supplemental Figure 2I), whereas no difference was observed in the enrichment of the H3K27me3 mark (data not shown). Together, these experiments indicate that the active histone modification mark H3K4me3, but not changes in DNA methylation, contributes to the elevated Cebpg levels observed in the C/EBPα-KO cells.

Cebpg is expressed in murine stem/progenitor cells and downregulated in committed myeloid cells. To understand the role of C/EBPγ in the pathogenesis of AML, we first investigated the expression of this transcription factor in normal hematopoiesis and determined Cebpg expression levels in distinct hematopoietic progenitor cell populations. RT-PCR showed that Cebpg transcripts are expressed in the LKS (lineage^–c-kit⁺ Sca-1⁺) population, which is enriched in both long- and short-term HSCs. Cebpg expression is still high in the CD150^–CMP subset, but decreases significantly as cells commit to the myeloid lineage and reach the CD150⁺CMP and GMP stages (Figure 3A). Cebpg mRNA levels are also high in megakaryocyte-erythroid progenitors (MEP), independently of endoglin and CD150 markers, and in common lymphoid progenitors (CLP).

Figure 3

C/EBPγ is downregulated with neutrophilic differentiation, whereas constitutive expression of C/EBPγ blocks G-CSF–induced neutrophilic differentiation. (A) Cebpg and Cebpa RNA in murine bone marrow–sorted populations: LKS, CD150⁺CMP, CD150^–CMP, GMP, MEP endo⁺ (endoglin⁺ CD150–), MEP ⁺/⁺ (endoglin⁺ CD150⁺), MEP CD150⁺ (endoglin– CD150⁺) CLP(78). Average and SD of 3 independent experiments. Relative expression calculated as percentage of Gapdh. (B) Cebpg and Cebpa expression in 32D/G-CSF–R cells during G-CSF–induced differentiation. The y axis indicates the relative expression as percentage of Gapdh. (C) Differential counts of 2 ER and 2 C/EBPγ-ER 32D/G-CSF–R– expressing clones cultured in G-CSF with (+) or without (–) 4-hydroxytamoxifen (8 days). (D) Morphologic analysis of May-Grunwald-Giemsa–stained cytospins of 2 ER and 2 C/EBPγ-ER clones (day 8) cultured in G-CSF (G) with or without 4-hydroxytamoxifen (T). (E) C/EBPγ binds to the proximal promoter of Csf3r (encoding G-CSF–R) and Cebpe. ChIP on C/EBPγ-ER or ER cells stimulated with (+) or without (–) 4-hydroxytamoxifen. The y axis represents binding fold enrichment. (F) Quantification of CFU-GEMM, CFU-GM, CFU-M, and CFU-G in semi-solid cultures (day 12). Lineage-depleted murine bone marrow cells infected with MSCV empty vector (ϕ) or C/EBPγ MSCV (γ) were sorted into GFP^– (–) or GFP⁺ (⁺). The y axis indicates the number of colonies per 5000 input cells. Average and SD of 3 independent experiments. *P = 0.001; **P = 0.0004. (G) Differential counting of cytospun semi-solid cultures. The y axis indicates the percentage of mature neutrophils present in each condition. Average and SD of 3 independent experiments. *P = 0.028; **P = 0.031, ***P = 0.005. (H) Cell morphology on cytospins of sorted GFP⁺ infected cells. Original magnification, ×100.

As C/EBPα is a crucial factor for myelopoiesis, we analyzed Cebpa expression in parallel in the same sorted populations. We observed that Cebpa is also expressed in LKS cells, however, with further lineage commitment, the expression of Cebpa and Cebpg followed a reciprocal pattern, showing upregulation of Cebpa in CD150⁺CMP and GMP cells (Figure 3A and Supplemental Figure 3A). This inverse correlation was also seen in CD150^–CMP and MEP, as well as CLP, where Cebpg levels were increased and Cebpa reduced (Figure 3A and Supplemental Figure 3A). In summary, Cebpg is expressed at the earliest hematopoietic stem/progenitor stage and at highest levels in MEP and CLP, with relatively lower levels in myeloid cells.

C/EBPγ is downregulated in G-CSF–induced neutrophilic differentiation. To further investigate C/EBPγ expression during granulocytic differentiation, we made use of the 32D/G-CSF–R murine cell line model. 32D/G-CSF–R cells proliferate in the presence of IL-3 and can fully differentiate toward mature neutrophils upon G-CSF stimulation (42, 43). Quantitative RT-PCR and Western blot analysis demonstrated that C/EBPγ is highly expressed in 32D/G-CSF–R cells while proliferating under the control of IL-3 (day 0) (Figure 3B and Supplemental Figure 3B). This expression level rapidly decreases as cells differentiate in the presence of G-CSF (day 2 to 8). In contrast, C/EBPα expression levels are low in the presence of IL-3, but gradually increase during G-CSF–induced differentiation (Figure 3B and Supplemental Figure 3C). These data demonstrate that C/EBPγ and C/EBPα levels are inversely correlated during neutrophilic differentiation and that C/EBPγ is more abundant in immature cells, similar to the bone marrow–sorted subsets.

Overexpression of C/EBPγ induces a block of granulocytic differentiation. To investigate whether downregulation of C/EBPγ is required for granulocytic differentiation, we overexpressed C/EBPγ in the 32D/G-CSF–R system described above. We introduced a tamoxifen-inducible form of C/EBPγ (C/EBPγ-ER) into 32D/G-CSF–R cells. ER control clones (containing the estrogen receptor peptide without C/EBPγ) and C/EBPγ-ER–expressing clones were cultured in the presence of G-CSF with or without 4-hydroxytamoxifen. All clones cultured in the presence of G-CSF fully differentiated toward granulocytes; however, a complete block of differentiation was observed in the C/EBPγ-ER–expressing cells when cultured in the presence of G-CSF and 4-hydroxytamoxifen (Figure 3, C and D). Accordingly, upregulation of granulocytic markers such as myeloperoxidase (Mpo), Cebpe, and Csf3r (G-CSF–R) was observed when 32D/G-CSF–R/CEBPγ-ER cells were cultured with G-CSF, but not when 4-hydroxytamoxifen was added to the cultures (Supplemental Figure 3D). ER control clones differentiated toward neutrophils when stimulated with G-CSF+4-hydroxytamoxifen and stopped proliferating after 4–5 days of culture, whereas the C/EBPγ-ER–expressing clones remained proliferative in the same conditions, with no signs of differentiation observed at any given time point (cultures were kept till day 19) (Supplemental Figure 3E).

To investigate the mechanism by which C/EBPγ promotes a block of granulocytic differentiation, we made use of the 32D/G-CSF–R cell line expressing either C/EBPγ-ER or the ER control alone. Cells were treated for 4 hours with 4-hydroxytamoxifen and ChIP was performed using an ER-specific antibody or an IgG control antibody. Oligos were designed on the proximal promoter of putative target genes, such as Csf3r (44) and Cebpe (45), which presented potential C/EBP-binding sites and were downregulated upon 4-hydroxytamoxifen treatment. Quantitative RT-PCR showed enrichment of the Csf3r and Cebpe promoters upon 4-hydroxytamoxifen treatment in the C/EBPγ-ER–expressing cells, but not in the absence of 4-hydroxytamoxifen or the ER control cells (Figure 3E). Further, enrichment was not observed in the IgG antibody control (Figure 3E) or in deserted regions (data not shown).

Next, we investigate whether enforced C/EBPγ expression would affect neutrophilic differentiation of murine bone marrow cells. Cebpg and pMSCV empty vector control were introduced into WT lineage depleted bone marrow cells, and overexpression was verified by quantitative RT-PCR (Supplemental Figure 3F). GFP^– (noninfected) and GFP⁺ (infected) cells were cultured in semi-solid medium, and morphological analysis determined that the C/EBPγ overexpressing cells had reduced number of granulocytic-like colonies (G-CFU), whereas the ability to form other colony types was not affected (Figure 3F). Morphological analysis of cytocentrifuged cells from the semi-solid cultures demonstrated that overexpression of C/EBPγ resulted in a lack of mature neutrophils (Figure 3, G and H). Together, these experiments demonstrate that C/EBPγ overexpression induces a block in granulocytic differentiation, suggesting that downregulation of C/EBPγ is required for myeloid differentiation.

Downregulation of C/EBPγ rescues granulopoiesis in murine C/EBPα-KO stem/progenitor cells. C/EBPα-KO mice are characterized by the lack of mature neutrophils in the bone marrow and blood (9, 10). Our observations that LKS from this mouse model have high levels of Cebpg mRNA and that sustained C/EBPγ expression blocks granulocytic differentiation (Figure 1B and Figure 3, C–H) led us to hypothesize that downregulation of C/EBPγ is required for neutrophilic differentiation in vivo. To study the contribution of C/EBPγ to the block in neutrophilic differentiation, we designed several shRNA constructs specifically targeting Cebpg (and not other C/EBPs) and demonstrated their efficiency at protein and RNA levels (Figure 4A and Supplemental Figure 4, A and B). C/EBPα-KO LKS cells were infected with a lentivirus expressing either a C/EBPγ-specific shRNA (sh#1) or a nonsilencing control shRNA (NSC#1). Cebpg was significantly downregulated in sh#1-infected LKS cells in comparison with cells infected with the NSC#1 (Figure 4B). Furthermore, Cebpa expression could not be detected in these cells (data not shown). Each individual colony in semi-solid culture was picked, and morphological analysis of cytocentrifuged cells revealed that downregulation of C/EBPγ gave rise to neutrophil-containing colonies (Figure 4, C and D). Quantitative RT-PCR of these cultures showed upregulation of granulocyte-specific genes such as neutrophil elastase (Elane), Mpo, Cebpe, and G-CSF–R (Csf3r) in C/EBPγ shRNA-infected cells (Figure 4E). Serial replating experiments demonstrated that while C/EBPα-KO NSC#1-infected cells could be replated for up to 4 times, downregulation of C/EBPγ in these cells abolished their replating abilities (Supplemental Figure 4C). Further, C/EBPα-KO infected cells were also placed in liquid culture, and flow cytometric analysis revealed a decrease in immature cell-surface markers such as c-kit and Sca-1 and an increase in mature markers such as Gr-1 and Mac-1 in sh#1-infected cells in comparison with NSC#1-infected cells (Supplemental Figure 4D). Morphological analysis on Wright-Giemsa–stained cytospins showed a more differentiated phenotype in cells lacking C/EBPα and downregulation of C/EBPγ (Supplemental Figure 4E). Quantitative RT-PCR was used to verify absence of Cebpa in those liquid cultures and downregulation of Cebpg in sh#1-infected cells in comparison with NSC#1-infected cells (Supplemental Figure 4, F–G, and data not shown). Similar experiments were performed using a retroviral vector (46) coexpressing another shRNA specifically targeting Cebpg, shRNA#2, and a GFP reporter (Figure 4A and Supplemental Figure 4, A and B). Semi-solid cultures of sorted GFP⁺ infected cells showed the presence of neutrophil-containing colonies when C/EBPγ was downregulated in C/EBPα-deficient LKS cells in comparison with the NSC#2 colonies (Supplemental Figure 4, H and I). Together, these results show that downregulation of C/EBPγ levels in murine C/EBPα-KO LKS cells is sufficient to restore neutrophilic differentiation in vitro. In addition, serial replating experiments suggest that downregulation of C/EBPγ restricts the increased self-renewal properties of C/EBPα-KO cells (10).

Figure 4

Downregulation of C/EBPγ in murine C/EBPα-KO LKS cells restores neutrophilic differentiation in cell culture. (A) Western blot analysis on 293T cells transfected with or without C/EBPγ-HA in combination with shRNA-expressing constructs. sh, shRNA targeting C/EBPγ. Blots were stained with HA and HSP90 (loading control) antibodies. (B) LKS cells from pI:pC-treated Cebpa^Flox/Flox cre⁺ (KO) mice were transduced with either NSC#1 or C/EBPγ shRNA#1 (sh#1). Cebpg mRNA expression was determined 2 days after infection. The y axis indicates relative Cebpg levels expressed as percentage of Gapdh plus SD in 2 independent mice, P = 0.0042. (C) C/EBPα-KO LKS cells were infected with NSC#1 or C/EBPγ shRNA#1 (sh#1) and seeded in semi-solid medium in the presence of puromycin. 12 days after culture, individual colonies were picked up and cell morphology was analyzed. Immature colonies contained blast and myeloblast; macrophage colonies contained mainly monocytes and macrophages; mix colonies contained immature cells, monocyte-macrophages, and neutrophils; and neutrophilic colonies had only neutrophils. Original magnification, ×40. (D) Percentage of the distinct colony types determined by cell morphology analysis in individual colonies from NSC#1- or sh#1-infected C/EBPα-KO LKS cells 12 days after culture. 3 independent experiments are shown (LKS#1, 2, and 3). (E) Quantitative RT-PCR was performed in NSC#1- and sh#1-infected C/EBPα-KO LKS cells 12 days after semi-solid culture (cultures shown in D). Expression of Cebpg, neutrophil elastase (Ela2), Mpo, Cepbe, and G-CSF–R (Csf3r) was determined in 2 independent mice. The y axis indicates relative expression in 1 representative mouse as percentage of Gapdh plus SD.

We next studied the effects of knocking down C/EBPγ in the absence of C/EBPα in vivo. For these experiments we made use of the shRNA retroviral vector containing a GFP reporter. C/EBPα-KO LKS cells were isolated and infected with retrovirus containing either shRNA#2, targeting C/EBPγ, or a control scrambled shRNA (NSC#2) and transplanted into lethally irradiated recipient mice (Figure 5A). Figure 5B shows flow cytometry data from 2 representative mice, transplanted with either sh#2- or NSC#2-infected cells. Plots were gated on Ly5.2⁺ cells (C/EBPα-KO cells), and further divided into GFP^– (noninfected) and GFP⁺ (infected) cells. We observed that mice transplanted with sh#2 had a high level of Gr-1/Mac-1⁺ cells (neutrophils) in the GFP⁺ fraction, whereas an absence of Gr-1/Mac-1⁺ cells was observed in the sh#2 GFP^– fraction or in the NSC#2 transplanted mice (Figure 5, B and C, and Supplemental Figure 5A) (n = 8 for NSC#2 and n = 11 for C/EBPγ shRNA#2). Of note, 9 out of 11 mice transplanted with C/EBPγ shRNA#2 contain granulocytes in the GFP⁺ fraction, whereas 0 out of 8 control mice did. In Supplemental Figure 5A, we measured the percentage of Gr-1/Mac-1⁺ cells present in blood from reconstituted mice 6 weeks after transplantation. At this time point, Ly5.2⁺ blood cells were sorted into GFP^– and GFP⁺ cells, and genomic DNA was extracted. Complete excision of Cebpa^Flox/Flox alleles was observed by PCR of genomic DNA in all transplanted animals and, accordingly, a PCR-specific excision band was detected (Supplemental Figure 5B). Further, GFP^– and GFP⁺ cells from NSC#2 and sh#2 transplanted mice gave rise to B220/IgM⁺ cells, indicating that downregulation of C/EBPγ in the absence of C/EBPα does not affect the production of B-lymphocytes (Supplemental Figure 6A). Animals were sacrificed 6–10 weeks after transplantation, and the absence of Cebpa was determined by RT-PCR in peripheral blood, total bone marrow, and LKS cells (Supplemental Figure 6, B and C, and data not shown). In summary, these transplantation experiments demonstrate that downregulation of C/EBPγ can rescue granulopoiesis in C/EBPα-KO mice, demonstrating that upregulation of C/EBPγ contributes to the myeloid differentiation arrest.

Figure 5

Downregulation of C/EBPγ in murine C/EBPα-KO LKS cells restores neutrophilic differentiation in vivo. (A) Schematic representation of the bone marrow transplantation experiment. (B) Sorted LKS cells from C/EBPα-KO mice were infected with either NSC#2 or C/EBPγ shRNA#2 (sh#2) and transplanted into lethally irradiated mice. 6 weeks after transplantation, recipient mice were bled and flow cytometry analysis was performed. Upper histogram panels are gated on Ly5.2⁺ cells (donor cells) and further divided in GFP^– (noninfected) and GFP⁺ (infected cells). Lower dot plot images show Gr-1/Mac-1 expression and indicate the percentage of double-positive cells. See also Supplemental Figure 5. (C) Cell morphology of peripheral blood mononuclear cells sorted according to Ly5.2⁺ and GFP levels. Original magnification, ×63.

Downregulation of C/EBPγ rescues granulocytic differentiation in primary human AML cells. Since we have identified a subset of patient samples characterized by silencing of CEBPA and upregulation of CEBPG (Figure 1 and Supplemental Figure 1), we next investigated whether downregulation of C/EBPγ in human CEBPA-silenced AML samples would restore granulocytic differentiation. First, we designed and examined the effect of an shRNA sequence specifically targeting human CEBPG (Supplemental Figure 7, A and B). A CEBPA-silenced AML patient sample was then infected with either this C/EBPγ shRNA or an NSC shRNA lentivirus, and cells were transplanted into sublethally irradiated NSG mice. Analysis of the recipient bone marrow cells 16 weeks after transplantation revealed the presence of human CD15-positive cells exclusively in the C/EBPγ shRNA–infected GFP⁺ cells (Figure 6). Further, analysis of the GFP^– fraction in these recipients and the NSC transplanted mice showed absence of CD15 expression, although myeloid identity was observed by CD33 expression (Figure 6A). Morphological analysis of human CD45⁺GFP^– and CD45⁺GFP⁺ sorted cells showed the presence of mature neutrophils only in the C/EBPγ shRNA GFP⁺ infected cells (Figure 6B). Similarly to the flow cytometry data, differential counting of the hCD45⁺ C/EBPγ infected cytospun cells showed 39% versus 1% mature neutrophils in the GFP⁺ and GFP^– fractions, respectively. These experiments indicate that downregulation of C/EBPγ in human CEBPA-silenced AML samples restores granulocytic differentiation in vivo.

Figure 6

Downregulation of C/EBPγ in human AML cells restores neutrophilic differentiation in vivo. (A) Bone marrow analysis of NSG recipient mice 16 weeks after transplantation of human AML cells. Plots represent 2 individual mice transplanted with 1.25 × 10⁶ cells infected with either NSC (nonsilencing control) shRNA lentivirus (n = 2) or a specific human C/EBPγ shRNA lentivirus (n = 2). Plots are gated for human CD45⁺ cells and divided by means of GFP: GFP^– (noninfected) and GFP⁺ (infected cells). Images show human CD33 and CD15 expression and indicate the percentage of cells in each quadrant. (B) Cell morphology on Wright-Giemsa–stained cytospins of sorted human CD45⁺ mononuclear cells according to GFP levels. Original magnification, ×100.

DAC treatment restores the C/EBPα-C/EBPγ balance and promotes expression of myeloid markers in AML samples. Since a subset of the AML patient samples we identified are characterized by silencing of CEBPA due to promoter hypermethylation (refs. 17, 33, Figure 1, and Supplemental Figure 1), we next investigated whether these AML cases could benefit from treatment with demethylating agents. We observed that CEBPA-silenced AML samples treated with 1 μM 5-aza-2′-deoxycitidine (DAC) showed upregulation of CEBPA mRNA expression in comparison with mock control treatment (Figure 7A). In line with our observations above, we determined that this CEBPA upregulation results in a significant reduction of the CEBPG levels in DAC-treated cells (Figure 7A). Further, we observed that these changes in CEBPA and CEBPG expression in C/EBPα-silenced AML upon DAC treatment result in upregulation of myeloid differentiation markers such as CSF3R, Gelatinase A, CEBPE, and neutrophil elastase (Figure 7B). Similarly, flow cytometric analysis showed upregulation of mature granulocytic markers such as CD15 and CD11b upon DAC treatment in the C/EBPα-silenced AML (Figure 7C). In addition, AML patient samples lacking CEBPA promoter hypermethylation did not respond to DAC treatment, and neither CEBPA nor CEBPG levels significantly changed (Supplemental Figure 7C). Bisulfite sequencing demonstrated DAC induced demethylation of the CEBPA promoter (Supplemental Figure 7, D and E). Together, these data indicate that demethylating agents can restore the C/EBPα-C/EBPγ balance and promote myeloid differentiation in certain AML samples.

Figure 7

DAC treatment restores the C/EBPα-C/EBPγ balance and promotes differentiation of primary human AML samples characterized by C/EBPα silencing and C/EBPγ upregulation in vitro. (A) Quantitative RT-PCR in 3 independent AML patient samples treated with either mock control or 1 μM DAC. CEBPA and CEBPG expression are shown relative to 18S expression. The y axis indicates fold change as compared with mock treatment. (B) DAC treatment promotes expression of myeloid differentiation markers in AML patient samples. Quantitative RT-PCR in 2 AML patient samples treated with either mock control or 1 μM DAC. The y axis indicates relative expression of G-CSF–R (CSF3R), gelatinase A (MMP2), CEBPE, and neutrophil elastase (ELANE), calculated as relative to GAPDH expression. (C) C/EBPα silenced patient samples treated with DAC or mock control, and analyzed by flow cytometry. Histograms represent expression of CD15 and CD11b after 8 days of treatment (except those indicated by asterisk, which were analyzed after 6 days). Numbers indicate percentage of positive cells.

Here, in a unique subset of AML defined by the absence of C/EBPα, we found increased CEBPG expression levels. Similar to these human AML cases, in Cebpa conditional KO mice, Cebpa ablation caused an upregulation of Cebpg, demonstrating a direct relation between Cebpg expression and absence of C/EBPα. We showed that overexpression of C/EBPγ in a cell-culture model of granulopoiesis and in murine bone marrow cells induces a neutrophilic differentiation arrest. Accordingly, in C/EBPα-KO mice and in CEBPA silenced human AML, both characterized by a block of neutrophilic development, downregulation of C/EBPγ was sufficient to completely restore granulocytic differentiation (Figure 8). These data point to a role for C/EBPγ in impairment of granulocytic development in a subset of human AML. We therefore hypothesize that these leukemia patients could benefit from therapeutic approaches meant to reduce C/EBPγ levels. We also demonstrate that treatment of these AML patient samples with demethylating agents such as DAC results in hypomethylation and reexpression of C/EBPα, which, as we predicted based in our findings in the murine and cell line models, contributes to the downregulation of C/EBPγ and upregulation of myeloid differentiation markers. This specific subset of AML cases is characterized by an aberrant DNA hypermethylation signature (17, 33), and therefore we assume that the beneficial effect of DAC is not restricted to restoring the C/EBPα-C/EBPγ balance. However, our data support an important role of this balance in myeloid differentiation. Currently, demethylating agents are the standard of care for patients with high-risk myelodysplastic syndromes (MDS) (47, 48), reducing the risk of transformation to AML and improving overall survival. But although these agents are being used as therapeutic agents, very few specific gene targets have been identified, and certainly it is difficult to explain the effect on AML cells based on previously identified methylation targets (49, 50). Therefore, identification of the C/EBPα-C/EBPγ axis as a specific target of DAC contributes to our understanding of the mechanisms of action of these drugs.

Figure 8

Proposed model. The upper panel summarizes the proposed model during normal hematopoiesis, and the lower panel represents the situation in a specific subset of AML patients (CEBPA silenced). In normal hematopoiesis, C/EBPα binds to the proximal promoter of CEBPG and represses its expression. Consequently, myeloid transcription factors drive expression of crucial genes required for neutrophilic differentiation, such as CSF3R (encoding for G-CSF–R) and CEBPE. In contrast, in a subset of AML patient samples characterized by CEBPA silencing due to promoter hypermethylation, we observed that absence of C/EBPα leads to upregulation of C/EBPγ. High C/EBPγ levels prevent transactivation of genes such as CSF3R and CEBPE, resulting in a neutrophilic differentiation block. DAC treatment to restore the C/EBPα-C/EBPγ balance or shRNA approaches to silence C/EBPγ can be applied and restore myeloid differentiation in these particular AMLs.

It is intriguing to ask why CEBPG upregulation is restricted to AML patients with CEBPA methylation/silencing. If CEBPG upregulation is a direct result of defects in C/EBPα, we might expect that other AML subtypes characterized by C/EBPα inactivation would also present elevated CEBPG levels. On one hand, AML cases harboring differentiation-deficient mutations in CEBPA have increased expression of CEBPA and low CEBPG RNA levels. Here, we demonstrate that the N-terminal C/EBPα mutants and the C/EBPα p30 isoform, which is expressed and active in leukemias with mutations in CEBPA (14, 51), are able to suppress Cebpg upregulation and transactivation. On the other hand, FLT3-ITD– and AML1-ETO–positive leukemias present reduced C/EBPα levels and activity (14, 52), but do not show upregulation of CEBPG. Our data indicate that elevated CEBPG levels are directly related to the total absence of CEBPA expression exclusive of CEBPA-silenced leukemias, whereas in the FLT3-ITD and AML1-ETO cases, the CEBPA mRNA levels are reduced, but still present. Accordingly, we demonstrate that in Cebpa^WT/Flox Mx1Cre⁺ LKS cells, which harbor reduced Cebpa levels, Cebpg levels are not increased. Therefore, the critical difference between this particular subtype of AML and the rest of leukemias with C/EBPα inactivation is the absence of C/EBPα versus low or mutated C/EBPα. Nevertheless, we cannot exclude that a yet-unidentified factor in CEBPA-silenced leukemias also contributes to CEBPG upregulation. Our data support that modifications at the chromatin level also regulate murine C/EBPγ expression. It would be of interest to study whether regulation at this level also occurs in the distinct AML types.

We previously reported that activating mutations in NOTCH1 are a key hallmark in CEBPA-silenced leukemias (17). These leukemias are phenotypically characterized by the simultaneous expression of myeloid as well as T-lymphoid markers. Our expression data indicate that Cebpg is highly expressed in lymphoid progenitor cells and that C/EBPα is involved in the active repression of Cebpg during myeloid differentiation. Accordingly, C/EPPγ-KO murine models have shown that C/EBPγ is relevant for proper lymphoid differentiation (30). Together, these observations highlight the possibility that C/EBPγ might be directly involved in the development of these biphenotypic leukemias, either by upregulating lymphoid markers and/or by repressing myeloid differentiation.

Our experiments indicate that granulocytic differentiation requires gradual upregulation of C/EBPα and downregulation of C/EBPγ, as seen in defined progenitor bone marrow populations and in a murine myeloid cell model. These results are in agreement with previous observations showing that C/EBPγ expression is reduced during myeloid differentiation (53). The reciprocal expression of C/EBPα and C/EBPγ suggests that these 2 transcription factors may regulate each other. In fact, our data demonstrate that C/EBPα represses Cebpg expression by affecting E2F1 transcriptional activity, which is in agreement with previous publications (40, 41). Although several reports state that only the C/EBPα p42 isoform represses E2F transcriptional activity (41), it has been reported that C/EBPα p30 can also suppress E2F activity (54). In fact, the luciferase reporter assays using the C/EBPγ proximal promoter indicate that both C/EBPα isoforms can repress C/EBPγ transactivation, consistent with our previously published studies demonstrating that p30 and p42 C/EBPα retain the ability to interact with E2F proteins in myeloid cell extracts (39). These different observations could be explained by a cell-type–dependent effect (54), and indeed the C/EBPα-C/EBPγ ratio seems to correlate with lineage commitment, rather than with cell-cycle activity, suggesting that other mechanisms yet to be identified, and that could work in a cell-specific manner, may also be involved in C/EBPγ repression. In contrast, our preliminary data (unpublished observations) suggest that this regulation is not reciprocal, since we do not observe changes in Cebpa in LKS cells that overexpress C/EBPγ. However, we cannot rule out the possibility that this regulation is cell-type– or differentiation-stage specific. Additionally, other factors could be involved in this C/EBPα-C/EBPγ regulation. In fact, since C/EBPα levels are reduced at the later stages of differentiation and C/EBPγ levels need to be low in order to accomplish neutrophilic differentiation, we hypothesize that other transcription factors may be involved in C/EBPγ downregulation. Further, we previously reported that C/EBPα-deficient progenitor/stem cells have increased Bmi-1 expression (10). Bmi-1 is a component of the polycomb group complex and regulates hematopoietic stem cell self renewal by repressing differentiation-related gene expression (55). Given the similarity between Bmi-1 and C/EBPγ expression during myeloid differentiation and in the C/EBPα conditional KO murine model, it will be intriguing to determine how these components interact during differentiation and stem cell self renewal.

It has previously been shown that ectopic expression of other C/EBP members, such as C/EBPα or C/EBPβ, can induce granulocytic differentiation of cell lines or primary cells in vitro (56–59). In contrast, our experiments demonstrate that C/EBPγ overexpression, either induced by ectopic expression in cell culture or by excision of C/EBPα in KO mice, results in a block of neutrophilic differentiation. Therefore, the effect of C/EBPγ overexpression is opposite to the one observed with other C/EBP members, suggesting different mechanisms of action and activation of distinct target genes. Structurally, C/EBPγ resembles the C/EBPβ isoform liver inhibitory protein (LIP) (7). C/EBPγ and LIP retain the basic region-leucine zipper DNA–binding domain, characteristic of the C/EBP family of transcription factors, but lack the transactivation domains. Similarly to LIP, C/EBPγ acts as a transdominant negative inhibitor of other C/EBP members (27, 29). In addition, C/EBPγ could also functionally mimic the C/EBPα mutant proteins identified in AML patient samples. We and others have shown that loss of C/EBPα is not the key event in leukemogenesis, but rather the synergistic activity of C/EBPα p30 and C-terminal mutants (14, 51). Therefore, C/EBPγ could contribute to the granulocytic differentiation arrest characteristic of AML by performing as a C/EBPα mutant protein. The results reported here demonstrate that downregulation of C/EBPγ by an shRNA approach can induce C/EBPα-independent granulopoiesis in vitro and in vivo. This is in agreement with previous publications in which we and others have shown that C/EBPα-KO cells can generate granulocytes in response to cytokines (19, 38, 60) and that this effect was mediated by C/EBPβ (19). Further, expression of C/EBPβ from the Cebpa locus results in normal granulopoiesis, indicating that C/EBPβ can drive granulopoiesis in the absence of C/EBPα (61). In the present study, we indeed observed that C/EBPγ can regulate C/EBPβ levels in murine LKS (Supplemental Figure 8A), and in line with these results,we observed low C/EBPβ mRNA levels in the AML cases with increased C/EBPγ expression (Supplemental Figure 8B). In addition, C/EBPγ modulates C/EBPβ transactivation activity (Supplemental Figure 8C). Next, we demonstrated that expression of C/EBPβ was able to drive neutrophilic differentiation of C/EBPα KO LKS cells (Supplemental Figure 8, D and E). Thus, we hypothesize that the rescue we observed upon C/EBPγ knockdown in C/EBPα-KO mice as well as in CEBPA-silenced leukemias might be partially mediated by C/EBPβ. Supporting our hypothesis, it was previously reported that C/EBPγ forms heterodimers with C/EBPβ and acts as a dominant negative inhibitor of C/EBPβ (27, 29). Further, since C/EBPγ can bind to G-CSF promoter elements (62), we hypothesize that C/EBPγ could also directly regulate G-CSF production and consequently neutrophilic differentiation. In line with this hypothesis, our experiments demonstrate that C/EBPγ binds to the proximal promoters of Csf3r and Cebpe and that enforced C/EBPγ expression prevents upregulation of these myeloid differentiation factors, which in normal hematopoiesis contribute to the granulocytic differentiation.

In summary, we have here identified C/EBPγ as a protooncogene in AML. Sustained C/EBPγ expression in myeloid precursors prevents myeloid differentiation toward mature granulocytes, and downregulation of C/EBPγ can restore neutrophilic differentiation in a C/EBPα-independent manner. Finally, we demonstrated that DAC treatment restores the C/EBPα-C/EBPγ balance affected in a subset of AML cases, contributing to the upregulation of myeloid differentiation markers in vitro (Figure 8). To date, treatment results with demethylating agents in AML have not been overwhelmingly positive (63, 64). However, these disappointing results could be explained if only certain subsets of AML patients are responsive. A precedent for this is found in treatment of AML patients with retinoids, in which only a specific subset of patients, those harboring the PML/RARα translocation, are responsive (65, 66). Our data supports DAC treatment and C/EBPα-C/EBPγ axis target approaches as a therapy in AML patients with CEBPA hypermethylation and upregulation of CEBPG.

View Supplemental data

We thank Britta Will, David Gonzalez, Hideo Hirai, and all members of the Tenen laboratory for helpful discussions; Pu Zhang, and Susumu S. Kobayashi for careful reading of the manuscript and suggestions; Carol Stocking and Peter F. Johnson for kindly providing us with the RSG-SIN retroviral vector (Stocking), the murine C/EBPγ antibody (Johnson), and the C/EBPβ pCDNA3 plasmid (Johnson); Heike Pahl for providing us with the pGhU6 lentiviral vector; and Li Chai for her generous donation of the NSG mice. We thank Min Deng for providing us with the p30 MSCV plasmid; and John Tigges, Vasilis Toxavidis, and Heidi Mariani from the Harvard Stem Cell Institute/Beth Israel Deaconess Center flow cytometry facility for their help and assistance during cell sorting. This work was supported by NIH R01 grants CA118316 to D.G. Tenen and R. Delwel, HL56745 to D.G. Tenen, a Collegio Ghislieri’s fellowship to G. Amabile, and an EHA research fellowship from the European Hematology Association to M. Alberich-Jordà.

Address correspondence to: Daniel G. Tenen, Center for Life Sciences, 3 Blackfan Circle, Room 437, Boston, Massachusetts 02115, USA. Phone: 617.735.2235; Fax: 617.735.2222; E-mail: dtenen@bidmc.harvard.edu.

Conflict of interest: The authors have declared that no conflict of interest exists.

Reference information: J Clin Invest. 2012;122(12):4490–4504. doi:10.1172/JCI65102.

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