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. 2011 Oct;96(10):1448-56.
doi: 10.3324/haematol.2011.040535. Epub 2011 Jul 12.

Down-regulation of EVI1 is associated with epigenetic alterations and good prognosis in patients with acute myeloid leukemia

Iria Vázquez  1 , Miren Maicas, José Cervera, Xabier Agirre, Oskar Marin-Béjar, Nerea Marcotegui, Carmen Vicente, Idoya Lahortiga, Maria Gomez-Benito, Claudia Carranza, Ana Valencia, Salut Brunet, Eva Lumbreras, Felipe Prosper, María T Gómez-Casares, Jesús M Hernández-Rivas, María J Calasanz, Miguel A Sanz, Jorge Sierra, María D Odero
Affiliations

Down-regulation of EVI1 is associated with epigenetic alterations and good prognosis in patients with acute myeloid leukemia

Iria Vázquez et al. Haematologica. 2011 Oct.

Abstract

Background: The EVI1 gene (3q26) codes for a zinc finger transcription factor with important roles in both mammalian development and leukemogenesis. Over-expression of EVI1 through either 3q26 rearrangements, MLL fusions, or other unknown mechanisms confers a poor prognosis in acute myeloid leukemia.

Design and methods: We analyzed the prevalence and prognostic impact of EVI1 over-expression in a series of 476 patients with acute myeloid leukemia, and investigated the epigenetic modifications of the EVI1 locus which could be involved in the transcriptional regulation of this gene.

Results: Our data provide further evidence that EVI1 over-expression is a poor prognostic marker in acute myeloid leukemia patients less than 65 years old. Moreover, we found that patients with no basal expression of EVI1 had a better prognosis than patients with expression/over-expression (P=0.036). We also showed that cell lines with over-expression of EVI1 had no DNA methylation in the promoter region of the EVI1 locus, and had marks of active histone modifications: H3 and H4 acetylation, and trimethylation of histone H3 lysine 4. Conversely, cell lines with no expression of EVI1 have DNA hypermethylation and are marked by repressive trimethylation of histone H3 lysine 27 at the EVI1 promoter.

Conclusions: Our results identify EVI1 over-expression as a poor prognostic marker in a large, independent cohort of acute myeloid leukemia patients less than 65 years old, and show that the total absence of EVI1 expression has a prognostic impact on the outcome of such patients. Furthermore, we demonstrated for the first time that an aberrant epigenetic pattern involving DNA methylation, H3 and H4 acetylation, and trimethylation of histone H3 lysine 4 and histone H3 lysine 27 might play a role in the transcriptional regulation of EVI1 in acute myeloid leukemia. This study opens new avenues for a better understanding of the regulation of EVI1 expression at a transcriptional level.

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Figures

Figure 1.
Figure 1.
Survival analysis of a series of patients with acute myeloid leukemia according to EVI1 expression status. (A) In Kaplan-Meier analysis stratified by age, patients <65 years and with EVI1-1C over-expression showed an inferior overall survival in comparison to patients with no EVI1-1C over-expression. (B) In Kaplan-Meier analysis, patients <65 years and no basal expression of EVI1 (-1C/-1D) had a better overall survival in comparison to patients with either expression or over-expression of EVI1, and a trend to better outcome in the global cohort. (C) In Kaplan-Meier analysis, patients <65 years with no basal expression of EVI1 (-1C/-1D) had a better overall survival than patients with EVI1 expression/over-expression. The same results were found in the global cohort.
Figure 2.
Figure 2.
Analysis of the epigenetic status of the EVI1 locus in five myeloid cell lines. (A) Quantification of the relative expression of the EVI1 splice variants, with bone marrow as the control sample. (B) Quantification of the relative expression of EVI1 (EVI1 11–12) after treatment with 5’Aza and TSA. Statistical significance was estimated using the non-parametric Wilcoxon’s matched pairs test; P<0.05 was considered significant (*). (C) Diagram of the methylation status of the EVI1-Island 1 and MDS1EVI1-Island 2 by direct sequencing after bisulfite treatment (white: non-methylated; black: methylated).(D) Diagram of the methylation status of the EVI1-Island 1 and MDS1EVI1-Island 2 after treatment with 5’Aza and TSA (white: non-methylated; black: methylated).
Figure 3.
Figure 3.
Analysis of the epigenetic status of the histones of the EVI1 locus in five myeloid cell lines. (A) Quantitative real-time RT-PCR performed on fragmented chromatin, showing the enrichment of trimethylation of histone H3 lysine 4 (H3K4me3) and histone H3 lysine 27 (H3K27me3) on the EVI1 promoter. (B) Quantitative real-time RT-PCR performed on fragmented chromatin, showing the enrichment of acetylated histones H3 and H4 on the EVI1 promoter. The results were calculated using the ΔΔCt method. They were presented as the fold enrichment of chromatin DNA precipitated by the specific antibody versus chromatin DNA precipitated by no antibody, as control. (C) Quantitative real-time RT-PCR performed on fragmented chromatin, showing the enrichment of acetylated histones H3 and H4 on EVI1 promoter regions after treatment with 5’Aza and TSA. The results were calculated and presented as described above, and comparing with and without the treatment. Statistical significance was estimated using the non-parametric Wilcoxon’s matched pairs test; P<0.05 was considered significant (*), and P<0.01 strongly significant (**).
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