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...

Persistent chronic inflammation in patients with CGD is associated with hematopoietic proliferative stress, leading to a decrease in the functional activity of HSCs. Our observations have clinical implications for the development of successful autologous cell therapy approaches.

Protection against epigenetic silencing is a desirable feature of future gene therapy vectors, in particular for those applications in which transgene expression will not confer growth advantage to gene-transduced cells. The ubiquitous chromatin opening element (UCOE) consisting of the methylation-free CpG island encompassing the dual divergently transcribed promoters of the human HNRPA2B1-CBX3 housekeeping genes (A2UCOE) has been shown to shield constitutive active heterologous promoters from epigenetic modifications and chromosomal position effects. However, it is unclear if this element can be used to improve expression from tissue-specific enhancer/promoters, while maintaining tissue specificity in hematopoietic cells. Here, we evaluated the potential of the A2UCOE in combination with the myeloid-specific myeloid related protein 8 (MRP8) promoter to target transgene expression specifically to myeloid cells in vitro and in vivo from a self-inactivating lentiviral vector. The inclusion of the A2UCOE did not interfere with specific upregulation of MRP8 promoter activity during myeloid differentiation and mediated sustained and vector copy-dependent expression in myeloid cells. Notably, the A2UCOE did not protect the MRP8 promoter from methylation in the P19 embryonal carcinoma cell line, suggesting that this element maintains the inherent epigenetic state and transcriptional activity of cellular promoters in their native configuration. Thus, the A2UCOE could represent a useful protective genetic element in gene therapy vectors, ensuring physiological transcriptional regulation of tissue-specific promoters independent of the chromosomal integration site.

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.

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.