Molecular inhibitors of the oncogenic property of AML1/ETO

Coordinator:		Prof. Dr. Manuel Grez
Institution:		Georg-Speyer-Haus Frankfurt
Homepage:		www.georg-speyer-haus.de/grps/

The chromosomal translocation t(8;21) is frequently found in acute myeloid leukemia (AML). This translocation involves the RUNX1 gene, a key regulator of hematopoietic cell differentiation, and the ETO gene, containing a nervy homology region 2 (NHR2) oligomerization domain. Subsets of RUNX1/ETO-dependent AML are associated with unfavorable prognoses and high relapse rates after chemotherapy, e.g. when coinciding with c-Kit mutations. We and others demonstrated that homotetramerization of the -helical NHR2 domain of the RUNX1/ETO fusion protein (also referred to as AML1/ETO) is an essential prerequisite for the onset and maintenance of AML. Thus, interfering with NHR2-mediated tetramerization in oncoproteins is an attractive strategy for molecular intervention and personalized therapy. We reported a 128mer fusion protein containing the complete NHR2 domain (NC128) that disrupts RUNX1/ETO tetramerization and counteracts leukemic cell characteristics. The NHR2 tetramer is a symmetric dimer of dimers, each of which contains two extended -helical monomers that associate in a head-to-tail orientation to form a four-helix bundle. Targeting such a protein-protein interactions (PPI) is considered
difficult because of the size, lack of deep binding pockets, and stability of PPI. We analyzed the energetic contribution of individual amino acids to the NHR2 dimer-tetramer transition (tetramerization) by a structural decomposition of a computed binding free energy and predicted a cluster of five amino acids with strong contributions to the stability of the tetramer (“hot spots”). Mutating the hot spots to alanine abolishes tetramer formation without affecting the dimer formation or helicity of NHR2. Moreover, RUNX1/ETO dimers do not block myeloid differentiation, are unable to enhance the self-renewal capacity of hematopoietic progenitors, and fail to induce leukemia in a murine transplantation model. Scanning the NHR2 dimer-dimer interface for pockets revealed that the deepest and largest pocket is in close proximity of the five hot spots. Such pockets are considered important for the highaffinity binding of PPIM.Thus, our analyses revealed an essential structural motif at the NHR2 interface that is suitable for
intervention in t(8;21) leukemia. Based on these findings small molecular weight compounds were identified by an ELISA assay. One of these compounds X.13 was able to block the proliferation and colony-forming capacity of AML1-ETO dependent cells, induced cell differentition and rescue transcription dysregulation of AML1-ETO traget genes. Moreover, X.13 reduces the tumorforming potential of AML1-ETO transformed cells and block the growth of AML1-ETO immortalized human CD34+ cells.