Chimeric transcription factors are a hallmark of human leukemia, but the molecular mechanisms by which they block differentiation and promote aberrant self-renewal remain unclear. Here, we demonstrate that the ETO2-GLIS2 fusion oncoprotein, which is found in aggressive acute megakaryoblastic leukemia, confers megakaryocytic identity via the GLIS2 moiety while both ETO2 and GLIS2 domains are required to drive increased self-renewal properties. ETO2-GLIS2 directly binds DNA to control transcription of associated genes by upregulation of expression and interaction with the ETS-related ERG protein at enhancer elements. Importantly, specific interference with ETO2-GLIS2 oligomerization reverses the transcriptional activation at enhancers and promotes megakaryocytic differentiation, providing a relevant interface to target in this poor-prognosis pediatric leukemia.
SummaryOncogenic transcription factors such as the leukemic fusion protein RUNX1/ETO, which drives t(8;21) acute myeloid leukemia (AML), constitute cancer-specific but highly challenging therapeutic targets. We used epigenomic profiling data for an RNAi screen to interrogate the transcriptional network maintaining t(8;21) AML. This strategy identified Cyclin D2 (CCND2) as a crucial transmitter of RUNX1/ETO-driven leukemic propagation. RUNX1/ETO cooperates with AP-1 to drive CCND2 expression. Knockdown or pharmacological inhibition of CCND2 by an approved drug significantly impairs leukemic expansion of patient-derived AML cells and engraftment in immunodeficient murine hosts. Our data demonstrate that RUNX1/ETO maintains leukemia by promoting cell cycle progression and identifies G1 CCND-CDK complexes as promising therapeutic targets for treatment of RUNX1/ETO-driven AML.
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...
The t(8;21) translocation is one of the most frequent cytogenetic abnormalities in acute myeloid leukaemia (AML) and results in the RUNX1/RUNX1T1 rearrangement. Despite the causative role of the RUNX1/RUNX1T1 fusion gene in leukaemia initiation, additional genetic lesions are required for disease development. Here we identify recurring ZBTB7A mutations in 23% (13/56) of AML t(8;21) patients, including missense and truncating mutations resulting in alteration or loss of the C-terminal zinc-finger domain of ZBTB7A. The transcription factor ZBTB7A is important for haematopoietic lineage fate decisions and for regulation of glycolysis. On a functional level, we show that ZBTB7A mutations disrupt the transcriptional repressor potential and the anti-proliferative effect of ZBTB7A. The specific association of ZBTB7A mutations with t(8;21) rearranged AML points towards leukaemogenic cooperativity between mutant ZBTB7A and the RUNX1/RUNX1T1 fusion.
About 12% of all de novo acute myeloid leukemias are characterized by the translocation t(8;21), which generates the oncogenic fusion protein RUNX1/ETO. RUNX1/ETO has a modular structure and contains several docking sites for heterologous proteins, including transcriptional corepressors like N-CoR, SMART, and mSIN3A. RUNX1/ETO is found in high molecular weight complexes, which are crucial for the block in myeloid differentiation observed in RUNX1/ETO-transformed cells. Essential for high molecular weight complex formation is the nervy homology region 2 (NHR2) within ETO, which serves as interacting surface for oligomerization as well as association with members of the ETO protein family. Here, we show that the expression of a fusion peptide consisting of 128 amino acids (NC128), including the entire NHR2 domain of ETO, disrupts the stability of the RUNX1/ETO high molecular weight complexes, restores transcription of RUNX1/ETO target genes, and reverts the differentiation block induced by RUNX1/ETO in myeloid cells. In the presence of NC128, RUNX1/ETO-transformed cells lose their progenitor cell characteristics, are arrested in cell cycle progression, and undergo cell death. Our results indicate that selective interference with the oligomerization domain of ETO could provide a promising strategy to inhibit the oncogenic properties of the leukemia-associated fusion protein RUNX1/ ETO. [Cancer Res 2007;67(5):2280-9]
We identified the first small-molecule protein-protein interaction inhibitors of RUNX1/ETO tetramerization applying structure-based virtual screening guided by predicted hot spots and pockets in the interface. A 3D similarity screening revealed specific hot spot mimetics, one of which prevents the proliferation of RUNX1/ETO-dependent SKNO-1 cells at low micromolar concentration. Using solely a protein-protein complex structure to start with, this strategy can be the first step in any comparable structure-based endeavor to identify protein-protein interaction inhibitors.
The RUNX1/ETO (RE) fusion protein, which originates from the t(8;21) chromosomal rearrangement, is one of the most frequent translocation products found in de novo acute myeloid leukemia (AML). In RE leukemias, activated forms of the c-KIT tyrosine kinase receptor are frequently found, thereby suggesting oncogenic cooperativity between these oncoproteins in the development and maintenance of t(8;21) malignancies. In this report, we show that activated c-KIT cooperates with a C-terminal truncated variant of RE, REtr, to expand human CD34+ hematopoietic progenitors ex vivo. CD34+ cells expressing both oncogenes resemble the AML-M2 myeloblastic cell phenotype, in contrast to REtr-expressing cells which largely undergo granulocytic differentiation. Oncogenic c-KIT amplifies REtr-depended clonogenic growth and protects cells from exhaustion. Activated c-KIT reverts REtr-induced DNA damage and apoptosis. In the presence of activated c-KIT, REtr-downregulated DNA-repair genes are re-expressed leading to an enhancement of DNA-repair efficiency via homologous recombination. Together, our results provide new mechanistic insight into REtr and c-KIT oncogenic cooperativity and suggest that augmented DNA repair accounts for the increased chemoresistance observed in t(8;21)-positive AML patients with activated c-KIT mutations. This cell-protective mechanism might represent a new therapeutic target, as REtr cells with activated c-KIT are highly sensitive to pharmacological inhibitors of DNA repair.
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