CEBPA-dependent HK3 and KLF5 expression in primary AML and during AML differentiation

Abstract

The basic leucine zipper transcription factor CCAAT/enhancer binding protein alpha (CEBPA) codes for a critical regulator during neutrophil differentiation. Aberrant expression or function of this protein contributes to the development of acute myeloid leukemia (AML). In this study, we identified two novel unrelated CEBPA target genes, the glycolytic enzyme hexokinase 3 (HK3) and the krüppel-like factor 5 (KLF5) transcription factor, by comparing gene profiles in two cohorts of CEBPA wild-type and mutant AML patients. In addition, we found CEBPA-dependent activation of HK3 and KLF5 transcription during all-trans retinoic acid (ATRA) mediated neutrophil differentiation of acute promyelocytic leukemia (APL) cells. Moreover, we observed direct regulation of HK3 by CEBPA, whereas our data suggest an indirect regulation of KLF5 by this transcription factor. Altogether, our data provide an explanation for low HK3 and KLF5 expression in particular AML subtype and establish these genes as novel CEBPA targets during neutrophil differentiation.

Figure 1. HK3 and KLF5 expression is significantly downregulated in CEBPA-mutated AML patients.

HK3 (a) and KLF5 (b) mRNA levels were measured by qPCR in total RNA extracted from primary AML (FAB M0-M7) blasts, CD34⁺ samples or granulocytes from healthy donors. Patient characteristics are summarized in Supplementary Table 1. HK3 (c) and KLF5 (d) levels in 154 patients from the Taskesen cohort with normal karyotype, expressing wild type CEBPA (WT), one allele mutated (SM) or two alleles mutated (DM). MWU: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 2. Genetic inhibition of CEBPA impairs HK3 and KLF5 upregulation during neutrophil differentiation of APL cells.

(a) NB4 or HT93 APL cell lines were stably transduced with pLKO.1 lentiviral vectors expressing non-targeting or two independent CEBPA-targeting shRNAs. APL control and CEBPA knockdown cells were differentiated with 1 μM ATRA for 4 days. Knockdown efficiency in NB4 (top panels) or HT93 (bottom panels) APL cells was validated by qPCR. CEBPA mRNA expression was normalized to the housekeeping gene HMBS and is shown as n-fold changes compared to untreated SHC002 control cells. HK3 (b) and KLF5 (c) mRNA expression in NB4 or HT93 APL cells was determined by qPCR and analyzed as in 2a. Impairment in granulocytic differentiation of CEBPA knockdown cells was shown by a reduction of the neutrophil marker granulocyte colony-stimulating factor receptor (GCSFR or CSF3R) (d) Data represent the mean ± s.d. of at least three independent experiments. (e), (f) CEBPA Knockdown efficiency at the protein level in NB4 and HT93 APL cells was confirmed by western blotting. GAPDH is shown as a loading control. MWU: *p < 0.05, **p < 0.01 and ***p < 0.001.

Figure 3. CEBPA binds to and activates the HK3 and the KLF5 promoters.

Schematic representation of a 6 kb human HK3 (a) and a 5 kb human KLF5 (c) genomic regions retrieved from an online database. MatInspector software predicted two putative CEBPA binding sites (squares) in the DNA sequences analyzed. In vivo binding of CEBPA to these CEBPA consensus sites in the HK3 (b) or KLF5 (d) genomic regions was shown by chromatin immunoprecipitation (ChIP) in NB4 APL cells. As a negative control for the different pull downs, absence of GAPDH amplification is shown. *unspecific band, primer dimer. Two HK3 (e–f) and one KLF5 genomic region (g) containing the CEBPA binding sites were PCR amplified from genomic DNA of NB4 cells using proof reading Pfu DNA polymerase and cloned into the pGL4.10-basic vector. H1299 cells were transiently transfected with 40 ng of either HK3 promoter reporter construct A (e), construct A with mutated CEBPA binding site (f, wild-type GAAAGAC, mutated GGTCGAC) or the KLF5 promoter reporter construct (g), together with pcDNA3.1 empty vector or increasing concentrations (40–80–120 ng) (e,g) or 80 ng of CEBPA expression vector (f). The promoter activity is shown as relative light units (RLU) relative to pcDNA3.1 control transfected cells. Results are the means ± s.d. of at least triplicate transfections. MWU: **p < 0.01, ***p < 0.001.

Figure 4. Ectopic expression of CEBPA activates HK3 and KLF5 transcription.

(a–e) HT93 cells were transiently transfected with pcDNA3.1 empty control or a CEBPA expression vector. HK3 (a) and KLF5 (b) mRNA expression was quantified by qPCR. Data were normalized to HMBS and are shown as n-fold regulation as compared to control transfected cells. Induction of CEBPE mRNA, a direct target gene of CEBPA, was measured as a positive control for CEBPA activity (c). Results are the means ± s.d. of at least triplicate transfections. CEBPA transfection efficiency was measured by qPCR (d) and western blotting (e). GAPDH is shown as a loading control. (f–g) Different CEBPA-ER fusion constructs were induced by treating the respective K562 cell lines with 5 μM Tamoxifen for 24 h. HK3 (f) or KLF5 (g) mRNA expression was quantified by qPCR as in 2a. Expression of the CEBPA target CEBPE was measured as positive control for CEBPA activation in wildtype CEBPA p42 expressing K562 cells. MWU: *p < 0.05, **p < 0.01.