Expression of CEBPA is reduced in RUNX1-mutated acute myeloid leukemia

Abstract

CEBPA (CCAAT/enhancer-binding protein alpha) is a member of the C/EBP family of bZIP transcription factors encoding two different translational protein isoforms. The CEBPA transcription factor is involved in cell cycle arrest, repression of self-renewal and myeloid differentiation during normal hematopoiesis. In acute myeloid leukemia (AML), mutations in CEBPA result in a cellular differentiation block. They occur in around 8% of normal karyotype AML (CN-AML). Approximately half of the patients harbor two CEPBA mutations associated to a favorable prognosis. CEBPA function can also be affected by promoter methylation or alterations in other oncogenes, for example through the t(8;21)(q22;q22)/RUNX1–RUNX1T1, which suppresses CEBPA mRNA expression. The t(8;21)(q22;q22) translocation replaces the C terminus, including the transactivation domain (TAD) of RUNX1, with RUNX1T1. RUNX1–RUNX1T1 in t(8;21) blocks CEBPAdependent activation of its own (CEBPA) promoter and thereby inhibits autoregulation. Other leukemic fusion proteins involving core-binding factor (CBF) family members, for example t(3;21)(q26;q22)/RUNX1–EVI1 or inv(16)(p13q22)/CBFB–MYH11, did not suppress CEBPA mRNA, indicating a RUNX1–RUNX1T1-specific effect on CEBPA transcriptional control. Intragenic RUNX1 mutations confer an adverse prognosis in AML. Previously, we identified RUNX1 mutations in 32.7% of CNAML or with non-complex chromosomal imbalances. RUNX1 mutations are absent in CBF–AML and acute promyelocytic leukemia. They are inversely correlated with CEBPA mutations. To clarify whether intragenic RUNX1 mutations such as RUNX1– RUNXT1 fusions also result in CEBPA mRNA downregulation, we investigated 359 AML patients consisting of two independent cohorts: cohort 1 with 209 AML cases (109 males/100 females; median age 65.4 years; 19.7–88.1 years) from different cytogenetic subgroups (normal karyotype (n1⁄4 93), t(8;21)(q22;q22)/RUNX1– RUNX1T1 (n1⁄4 16), t(15;17)(q22;q12)/PML-RARA (n1⁄4 15), sole þ 8 (n1⁄4 12), sole þ 13 (n1⁄4 10), complex karyotypes (n1⁄4 10) and other rare noncomplex genetic abnormalities (n1⁄4 53)). Cohort 2 comprised 150 normal karyotype patients selected according to RUNX1 mutation status (92 males/58 females; median age, 69.7 years; 18.3–88.1 years; 81/150 (54.0%) RUNX1 mutated) with survival data in 124 cases. Bone marrow and/or peripheral blood samples were sent to the MLL Munich Leukemia Laboratory in 2005–2011. All patients gave their written informed consent to genetic analysis and scientific studies. Chromosome-banding analysis was performed in all cases, when needed, combined with fluorescent in situ hybridization. RUNX1 mutations were analyzed by Sanger sequencing or an amplicon-based high-throughput deep-sequencing assay (454 Life Sciences, Branford, CT, USA). CEBPA (mRNA) expression was quantified in cohort 1 by gene expression microarray profiling (Affymetrix HG-U133 Plus 2.0 microarrays; Santa Clara, CA, USA). The gene expression raw data were processed according to the manufacturer’s recommendations. Detection calls, that is present, marginal, or absent expression, were determined by default parameters. For measurement of CEBPA expression in cohort 2, a quantitative real-time reverse transcriptase PCR (RT-PCR) assay was established (Taqman, Life Technologies, Carlsbad, CA, USA; CEBPA TaqMan Gene Expression Assay: HS00269972_S1). mRNA expression of CEBPA was normalized against expression of ABL1; ratios were given as %CEBPA/ABL1. First, we investigated 209 AML cases from different cytogenetic subgroups using gene expression microarray profiling (Table 1a). The RUNX1 mutation status was analyzed in 178 cases (RUNX1– RUNX1T1 or PML-RARA-mutated cases had been excluded), in 41/ 178 (23%) of patients RUNX1 was mutated. The median CEBPA expression intensity value in all patients was 670 (range 48–5244). RUNX1-mutated cases showed a lower CEBPA expression than RUNX1 wild-type cases (n1⁄4 41 vs 137, mean±s.d. 429±395 vs 998±717; Po0.001). Cases harboring a t(8;21)/RUNX1–RUNX1T1 presented a lower CEBPA expression than patients without (n1⁄4 16 vs 193, mean±s.d. 292±216 vs 950±808; Po0.001), whereas t(15;17)/PML-RARA-mutated cases showed enhanced CEBPA expression (n1⁄4 15 vs 194, mean±s.d. 1940±1290 vs 819±690; P1⁄4 0.005) (Figure 1a). As reported previously, cases with a sole þ 13 showed lower expression than cases without (n1⁄4 10 vs 199, mean±s.d. 326±406 vs 929±803; P1⁄4 0.020); however, all þ 13 cases were RUNX1-mutated. For validation, an independent cohort of 150 normal karyotype AML was investigated for RUNX1 mutations (Table 1b), CEBPA expression was quantified using real-time RT-PCR. RUNX1

DOI: 10.1038/bcj.2012.34

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Cite this paper

@inproceedings{Grossmann2012ExpressionOC, title={Expression of CEBPA is reduced in RUNX1-mutated acute myeloid leukemia}, author={Vera Grossmann and Ulrike Bacher and Alexander Kohlmann and K Butschalowski and Andreas Roller and Sabine Jeromin and Frank Dicker and Winfried Vinzenz Kern and Susanne Schnittger and Torsten Haferlach and Claudia Haferlach}, booktitle={Blood cancer journal}, year={2012} }