Biophysical and pharmacokinetic characterization of a small-molecule inhibitor of RUNX1/ETO tetramerization with anti-leukemic effects.

Acute myeloid leukemia (AML) is a malignant disease of immature myeloid cells and the most prevalent acute leukemia among adults. The oncogenic homo-tetrameric fusion protein RUNX1/ETO results from the chromosomal translocation t(8;21) and is found in AML patients. The nervy homology region 2 (NHR2) domain of ETO mediates tetramerization; this oligomerization is essential for oncogenic activity. Previously, we identified the first-in-class small-molecule inhibitor of NHR2 tetramer formation, 7.44, which was shown to specifically interfere with NHR2, restore gene expression down-regulated by RUNX1/ETO, inhibit the proliferation of RUNX1/ETO-depending SKNO-1 cells, and reduce the RUNX1/ETO-related tumor growth in a mouse model. However, no biophysical and structural characterization of 7.44 binding to the NHR2 domain has been reported. Likewise, the compound has not been characterized as to physicochemical, pharmacokinetic, and toxicological properties. Here, we characterize the interaction between the NHR2 domain of RUNX1/ETO and 7.44 by biophysical assays and show that 7.44 interferes with NHR2 tetramer stability and leads to an increase in the dimer population of NHR2. The affinity of 7.44 with respect to binding to NHR2 is Klig = 3.75 ± 1.22 µM. By NMR spectroscopy combined with molecular dynamics simulations, we show that 7.44 binds with both heteroaromatic moieties to NHR2 and interacts with or leads to conformational changes in the N-termini of the NHR2 tetramer. Finally, we demonstrate that 7.44 has favorable physicochemical, pharmacokinetic, and toxicological properties. Together with biochemical, cellular, and in vivo assessments, the results reveal 7.44 as a lead for further optimization towards targeted therapy of t(8;21) AML.

Gopalswamy M ，Kroeger T ，Bickel D ，Frieg B ，Akter S ，Schott-Verdugo S ，Viegas A ，Pauly T ，Mayer M ，Przibilla J ，Reiners J ，Nagel-Steger L ，Smits SHJ ，Groth G ，Etzkorn M ，Gohlke H ... -  《Scientific Reports》

Compatibility of RUNX1/ETO fusion protein modules driving CD34+ human progenitor cell expansion.

Chen-Wichmann L ，Shvartsman M ，Preiss C ，Hockings C ，Windisch R ，Redondo Monte E ，Leubolt G ，Spiekermann K ，Lausen J ，Brendel C ，Grez M ，Greif PA ，Wichmann C ... -  《-》

Targeting the oligomerization domain of ETO interferes with RUNX1/ETO oncogenic activity in t(8;21)-positive leukemic cells.

Wichmann C ，Chen L ，Heinrich M ，Baus D ，Pfitzner E ，Zörnig M ，Ottmann OG ，Grez M ... -  《CANCER RESEARCH》

RUNX1-ETO Depletion in t(8;21) AML Leads to C/EBPα- and AP-1-Mediated Alterations in Enhancer-Promoter Interaction.

Acute myeloid leukemia (AML) is associated with mutations in transcriptional and epigenetic regulator genes impairing myeloid differentiation. The t(8;21)(q22;q22) translocation generates the RUNX1-ETO fusion protein, which interferes with the hematopoietic master regulator RUNX1. We previously showed that the maintenance of t(8;21) AML is dependent on RUNX1-ETO expression. Its depletion causes extensive changes in transcription factor binding, as well as gene expression, and initiates myeloid differentiation. However, how these processes are connected within a gene regulatory network is unclear. To address this question, we performed Promoter-Capture Hi-C assays, with or without RUNX1-ETO depletion and assigned interacting cis-regulatory elements to their respective genes. To construct a RUNX1-ETO-dependent gene regulatory network maintaining AML, we integrated cis-regulatory element interactions with gene expression and transcription factor binding data. This analysis shows that RUNX1-ETO participates in cis-regulatory element interactions. However, differential interactions following RUNX1-ETO depletion are driven by alterations in the binding of RUNX1-ETO-regulated transcription factors.

Ptasinska A ，Pickin A ，Assi SA ，Chin PS ，Ames L ，Avellino R ，Gröschel S ，Delwel R ，Cockerill PN ，Osborne CS ，Bonifer C ... -  《Cell Reports》

Depletion of RUNX1/ETO in t(8;21) AML cells leads to genome-wide changes in chromatin structure and transcription factor binding.

Ptasinska A ，Assi SA ，Mannari D ，James SR ，Williamson D ，Dunne J ，Hoogenkamp M ，Wu M ，Care M ，McNeill H ，Cauchy P ，Cullen M ，Tooze RM ，Tenen DG ，Young BD ，Cockerill PN ，Westhead DR ，Heidenreich O ，Bonifer C ... -  《-》