AML1/ETO and its function as a regulator of gene transcription via epigenetic mechanisms

Abstract

The chromosomal translocation t(8;21) and the resulting oncofusion gene AML1/ETO have long served as a prototypical genetic lesion to model and understand leukemogenesis. In this review, we describe the wide-ranging role of AML1/ETO in AML leukemogenesis, with a particular focus on the aberrant epigenetic regulation of gene transcription driven by this AML-defining mutation. We begin by analyzing how structural changes secondary to distinct genomic breakpoints and splice changes, as well as posttranscriptional modifications, influence AML1/ETO protein function. Next, we characterize how AML1/ETO recruits chromatin-modifying enzymes to target genes and how the oncofusion protein alters chromatin marks, transcription factor binding, and gene expression. We explore the specific impact of these global changes in the epigenetic network facilitated by the AML1/ETO oncofusion on cellular processes and leukemic growth. Furthermore, we define the genetic landscape of AML1/ETO-positive AML, presenting the current literature concerning the incidence of cooperating mutations in genes such as KIT, FLT3, and NRAS. Finally, we outline how alterations in transcriptional regulation patterns create potential vulnerabilities that may be exploited by epigenetically active agents and other therapeutics.

Fig. 1. Schematic representation of AML1 (RUNX1), ETO (RUNX1T1), and AML1/ETO—structure and function.

A The Runt DNA-binding domain of AML1 and almost the whole co-repressor gene ETO are conserved in the fusion gene including its four functional domains termed Nervy Homology domains 1–4 (NHR 1–4): TATA-binding protein-associated factor homology domain (eTAFH = NHR1), the hydrophobic heptad repeat domain (HHR = NHR2), an α-helical domain (Nervy = NHR3), and the myeloid-Nervy-DEAF1 domain (MYND = NHR4). The AML1/ETO 9a variant contains only the NHR1 and NHR2 functional domains, while the AML1(exon 6)/ETO variant contains 64 additional amino acids downstream of the Runt domain with yet unknown functional consequences. B mRNA splice variants identified in t(8;21) leukemia include the canonical AML1/ETO, the oncogenic AML1/ETO9a, and other rare variants such as AML1(exon 6)/ETO. C The AML1(exon 6)/ETO variant observes a novel breakpoint between AML1 exon 6 and ETO exon 2. Double-stranded synthetic DNA fragments can be utilized to clone novel AML1/ETO splice variants into the retroviral MSCV-IRES-GFP overexpression construct (ref. [21]) by utilizing intrinsic restriction enzyme sites. D RT-PCR using exon-specific and exon-junction-spanning primers for the AML1 exon 6 splice event following retroviral transfection of 293T cells with the novel AE/AE6 and AE9a/A6 constructs, as well as the previously published AE and AE9a constructs and a no transfection control (Empty). AE = AML1/ETO, AE9a = AML1/ETO9a variant, AE/A6: AML1(exon 6)/ETO variant, AE9a/A6 = AML1(exon 6)/ETO9a variant. This figure includes original work (see “Acknowledgements”).

Fig. 2. The AML1/ETO oncofusion protein but not wild type AML1/RUNX1 recruits a repressor complex.

The hematopoietic transcritption factor AML1/RUNX1 binds the consensus sequence TGTGGT on the promoter of its target genes. DNA binding is stabilized by the interaction with CBFß. AML1 recruits the histone acetyltranferases p300 and CBP. The histone acetyltransferases acetylates lysine residues on the histones of its target genes, which induce an open chromatin and activates gene transcription. However, AML1/ETO interacts with NCOR and mSin3, which recruit class I histone deacetyltransferases (HDACs) 1–3. HDACs1–3 deacetylates the lysine residues of histone tails, which change to a closed chromatin conformation resulting in the repression of transcription of target genes. Some evidence supports that DNA methyltransferases (DNMTs) and the polycomb repressor complex 2 (PRC2) including the H3K27 trimethylase EZH2 are directly or indirectly recruited to AML1/ETO target genes.

Fig. 3. Cooperating genetic lesions contribute to AML1/ETO-driven leukemogenesis.

Gene mutations conferring a positive prognostic impact are highlighted in green, while gene mutations with a poor prognostic are indicated in red. Gene mutations with an equivocal prognostic impact are shown in gray. The relative incidence of the respective mutation is depicted in percent. Mutations occurring more frequently in t(8;21) CBF-AML are portrayed to the left, while mutations with a preponderance in inv(16)/t(16;16) CBF-AML are portrayed to the right.