Dimer-tetramer transition controls RUNX1/ETO leukemogenic activity

Abstract

RUNX1/ETO, the fusion protein resulting from the chromosomal translocation t(8;21), is one of the most frequent translocation products in acute myeloid leukemia. Several in vitro and in vivo studies have shown that the homo-tetramerization domain of ETO, the nervy homology region 2 (NHR2), is essential for RUNX1/ETO oncogenic activity. We analyzed the energetic contribution of individual amino acids within the NHR2 to RUNX1/ETO dimertetramer transition and found a clustered area of 5 distinct amino acids with strong contribution to the stability of tetramers. Substitution of these amino acids abolishes tetramer formation without affecting dimer formation. Similar to RUNX1/ ETO monomers, dimers failed to bind efficiently to DNA and to alter expression of RUNX1-dependent genes. RUNX1/ETO dimers do not block myeloid differentiation, are unable to enhance the selfrenewal capacity of hematopoietic progenitors, and fail to induce leukemia in a murine transplantation model. Our data reveal the existence of an essential structural motif (hot spot) at the NHR2 dimertetramer interface, suitable for a molecular intervention in t(8;21) leukemias. IntroductionChromosomal translocations are frequent events during malignant cell transformation, particularly during leukemogenesis. 1 The translocation t(8;21), one of the most frequent chromosomal anomalies in acute myeloid leukemia (AML), involves the RUNX1 gene (also known as AML1, CBF␣2, or PEBP2␣B) on chromosome 21 and the ETO gene (also known as MTG8 or RUNX1T1) on chromosome 8. The ubiquitously expressed RUNX1 is a transcription factor and belongs to the key regulators of hematopoietic cell differentiation. 2 The fusion protein RUNX1/ETO contains the DNA-binding domain (Runt, RHD) of the RUNX1 transcription factor but lacks the C-terminal transactivation sequence that is replaced by almost the entire ETO protein. 2 forms of RUNX1/ETO coexist in AML-leukemia samples: the originally discovered full-length RUNX1/ETO and a splice variant called RUNX1/ETO9a, which lacks 178 amino acids at the C-terminus. Only RUNX1/ETO9a does not require cooperative events for inducing leukemia development in mice. 3,4 We and others have shown that RUNX1/ETO has a modular structure. Besides the Runt domain, RUNX1/ETO contains 4 functional domains, which are generally referred to as nervy homology region (NHR1 to NHR4). The NHR domains serve as docking interface for a variety of different proteins, including the E-protein HEB, 5,6 the apoptosis-related protein SON, 7 and nuclear corepressor proteins, such as N-CoR, SMRT, mSIN3A, and MTGR1, as well as histone deacetylases (HDACs). [8][9][10][11] In addition, the NHR2 domain mediates tetramer formation through hydrophobic and ionic/polar interactions. Two ␣-helices align in a head-to-tail fashion to form an antiparallel dimer. Two dimers subsequently are positioned on top of each other in a sandwich-like fashion. The total interaction area composing all contact points of the 4 ␣-helices is approximately 10 000 Å. 2 Substitution of 7 leucines withi...

Suppression of RUNX1/ETO oncogenic activity by a small molecule inhibitor of tetramerization

“…4 We analyzed the DNA-binding capacity of fusion-proteins containing the DNA-binding domain of RUNX1/ETO juxtaposed to the oligomerization domain of either RUNX1/ETO (RUNX1/NHR2) or the unrelated oncogenic tyrosine kinase BCR/ABL (RUNX1/BCR). 6 A biotinylated oligonucleotide derived from the RUNX1/ETO binding region within the RUNX3 promoter was used as DNA-binding target.…”

ERG and FLI1 binding sites demarcate targets for aberrant epigenetic regulation by AML1-ETO in acute myeloid leukemia

“…As oligomerization of AML1-ETO has been suggested as essential for leukemogenesis, 33,34 we hypothesized that targeting of AML1-ETO could be dependent on the number of RUNX1 sequences underlying the ERG binding regions. Counting the number of RUNX1 motifs in ERG only binding sites compared with those that bound also AML1-ETO revealed a statistical significant difference (P ϭ 1.8 ϫ 10 Ϫ5 ) in the distribution of the number of binding sites.…”

Revisiting the differentiation paradigm in acute promyelocytic leukemia

“…26,27 Importantly, in the case of the AML1-ETO fusion, auto-aggregation of the protein similarly allows relaxed binding site specificity. 28,29 So, loosening of the DNA recognition specificity through multimerization appears to be a common and critical feature of transcription factor-derived oncogenic fusion proteins in acute myeloid leukemias and may be implicated in the arrest of differentiation.…”