The leukemogenicity of AML1-ETO is dependent on site-specific lysine acetylation

Abstract

The chromosomal translocations found in acute myelogenous leukemia (AML) generate oncogenic fusion transcription factors with aberrant transcriptional regulatory properties. Although therapeutic targeting of most leukemia fusion proteins remains elusive, the posttranslational modifications that control their function could be targetable. We found that AML1-ETO, the fusion protein generated by the t(8;21) translocation, is acetylated by the transcriptional coactivator p300 in leukemia cells isolated from t(8;21) AML patients, and that this acetylation is essential for its self-renewal-promoting effects in human cord blood CD34(+) cells and its leukemogenicity in mouse models. Inhibition of p300 abrogates the acetylation of AML1-ETO and impairs its ability to promote leukemic transformation. Thus, lysine acetyltransferases represent a potential therapeutic target in AML.

Fig. 1

The enhanced self-renewal capacity and transcriptional activation induced by A-E in human HSPCs requires the NHR1 domain. (A) The NHR1 domain (amino acids 245 to 436) is required for A-E to increase CAFC formation by human CD34⁺ cells transduced with the indicated retroviral vector. The number of cobblestone areas at week 5 is shown (± SD; n = 3). (B) Deletion of NHR1 affects the ability of A-E to promote self-renewal in liquid culture. CD34 expression was examined weekly in MIGR1-, A-E–, and A-E ΔNHR1–transduced human CD34⁺ cells (± SD; n = 3). (C) M-CSFR, GM-CSF, and IL-3 promoter activity was examined in cells transfected with MIGR1, A-E, or A-E ΔNHR1 (± SD; n = 3). All values were standardized to the level of Renilla luciferase activity.

Fig. 2

The NHR1 domain of A-E interacts with p300, which potentiates its transcriptional activating properties. (A) The NHR1 domain is required for the interaction of A-E with p300. An antibody to Flag was used for immunoprecipitation (IP); antibodies to p300 or Flag were used for Western blotting (WB). (B) AE9a also interacts with p300. An antibody to hemagglutinin (HA) was used for immunoprecipitation; antibodies to p300 or HA were used for Western blotting. (C) Knockdown of p300 decreased the expression of A-E–activated target genes. Kasumi-1 cells were transduced with shRNAs against p300 or a control shRNA. Quantitative polymerase chain reaction (qPCR) was performed to quantify the level of target gene expression (± SD; n = 3). (D) A-E and p300 colocalize on some A-E up-regulated genes, as shown by ChIP-seq assays. Representative examples of A-E and p300 co-occupancy are shown as custom tracks in the UCSC genome browser. The locations of the Runx1 consensus binding sites are shown by green lines on the x axis. DNA binding was also analyzed by qPCR amplification of the regions indicated by the red and green arrows on the x axis (± SD; n = 3).

Fig. 3

The acetylation of A-E Lys⁴³ by p300 is required for A-E–induced leukemogenesis. (A) Acetylation of Flag-tagged A-E by p300. Antibodies to Flag or to acetyl lysine were used for immunoprecipitation or Western blotting. (B) The ability of A-E to bind p300 is required for its lysine acetylation. Antibodies to Flag or to acetyl A-E Lys²⁴ or Lys⁴³ were used for immunoprecipitation or Western blotting. (C) Lethally irradiated recipient mice were injected with HSPCs transduced with AE9a or AE9a ΔNHR1. The number of GFP⁺ c-kit⁺ cells in the peripheral blood is shown 15 weeks after transplant. (D) Survival of mice receiving AE9a-transduced or AE9a mutant–transduced HSPCs (P < 0.0001, n = 15 per group). (E) Acetylation of A-E Lys⁴³ was detected in blast cells from t(8;21)⁺ leukemia patients. Antibodies to ETO, acetyl A-E Lys⁴³, or AML1 were used for immunoprecipitation or Western blotting (29).

Fig. 4

Inhibition of p300 or lysine acetylation blocks leukemogenesis by inhibiting cell growth of A-E–activated target genes. (A) Lys-CoA-Tat (50 μM) inhibited the growth of primary t(8;21)⁺ leukemia cells and Kasumi-1 cells (± SD; n = 3) but not HL-60 cells or normal human CD34⁺ HSPCs. (B) Lys-CoA-Tat (50 μM) blocks A-E Lys⁴³ and histone H3 acetylation in Kasumi-1 cells, as detected by antibodies to acetyl A-E Lys⁴³ or to histone H3. (C) The binding of TAF_II250 and TAF7 to A-E peptides that contain acetylated or unacetylated Lys²⁴ or Lys⁴³ was studied using a peptide pull-down assay. Antibodies to TAF_II250 or TAF7 were used for Western blotting. (D) A hypothetical model showing how p300, and acetylation of A-E by p300, might cooperate to induce leukemia.