Our inhibitors act via an

Our inhibitors act via an allosteric mechanism, i.e. they do not bind at the protein-protein interface. Such a mode of action has been seen with numerous small molecule inhibitors of enzymes, but there have only been limited reports of such allosteric inhibitors of protein-protein interactions (Arkin et al., 2014b). However, this mechanism has clear advantages as the inhibitor does not have to directly compete with the partner protein. Our NMR relaxation data support a model for the activity of these compounds where induction of changes in the dynamics of critical hotspot binding residues on the RUNX binding interface on CBFβ mediate loss of binding. Such a dynamics based mechanism is consistent with numerous NMR studies of allosteric communication which have demonstrated altered dynamics as a mechanism. The interaction between CBFβ and RUNX1 is essential for normal hematopoiesis. The CBFβ inhibitors block the induction of RUNX1-dependent genes during the endothelial-hematopoietic transition (EHT) and inhibit RUNX1 binding to its target genes. Moreover, this result highlights the crucial role of not only the presence of the complete CBF complex, but its actual binding to chromatin for the EHT. At later differentiation stages, RUNX1 inhibition enhances myeloid differentiation of the HPC-7 cell line, which is consistent with the reduction in expression of RUNX1 that occurs upon progressing from the hematopoietic stem cell (HSC) to the myeloid lineage. Based on these results, we explored the biological effects of the CBFβ inhibitors in a disease context. Since all three RUNX proteins (RUNX1, 2, 3) bind CBFβ, the biological effects of these inhibitors likely reflect the inhibition of all the RUNX proteins present in the cell lines used. We have previously shown that this interaction is essential for the transforming properties of the leukemia causing fusion proteins AML1-ETO and TEL-AML1 (Roudaia et al., 2009). Our data therefore suggest a potential therapeutic use of the CBFβ inhibitors in leukemia as they are effective in killing a variety of different leukemia cell lines, consistent with recent results indicating a critical role for both mutated and wild-type RUNX1 in such GW 610 (Ben-Ami et al., 2013; Goyama et al., 2013), while having minimal impact on normal cord blood cells. However, the anti-tumor activity of the compounds is not restricted to hematological malignancies. CBFβ inhibitors increased multiacinar structures in breast cell 3D culture, confirming the crucial role of RUNX1 in this phenotype indicated by previous knock-down experiments, and completely inhibited the growth of a basal-like breast cancer cell line in 3D culture. Currently there is no effective therapy for basal-like breast cancer, so our results support targeting of the RUNX pathway for this very poor prognosis cancer. Our study clearly demonstrates the utility of these compounds for exploring the effects of CBFβ-RUNX inhibition across a broad spectrum of cancers.
Author Contributions
Introduction In epithelial cells planar polarity is maintained by proteins that are encoded by tumor suppressor genes such as SCRIB (Bilder et al., 2000; Bilder and Perrimon, 2000). Scribble, the protein product of SCRIB, is crucial for the proper maintenance of epithelial cell integrity and function (Zhan et al., 2008); it is required for E-cadherin-mediated cell-cell adhesion and, when its expression is down-regulated, epithelial cells acquire mesenchymal appearance and their migration is augmented (Qin et al., 2005). In epithelial cells the orientation of the mitotic spindle is restricted to the plane of the epithelium to ensure that daughter cells will remain within the layer. It was shown recently that Scribble is involved in this process and when its expression is knocked down in Drosophila the orientation of the mitotic spindle becomes random. When combined with inhibition of apoptosis, Scribble knock-down is sufficient to induce epithelial to mesenchymal transition and formation of tumor-like structures (Nakajima et al., 2013). Scribble expression and localization is frequently deregulated in human cancers, including breast cancer, where it is either lost or abnormally overexpressed and localized (Zhan et al., 2008, Feigin et al., 2014). In this study we show that, in human breast cancer, the expression of several exons, which share very little similarity with the sequence from Drosophila, is decreased relative to the exons encoding the rest of the protein. Furthermore, this pattern of exon usage correlates with long-term survival. The underused exons encode part of the C-terminal proline-rich domain of Scribble, which we found to become increasingly phosphorylated in mitosis, leading to association with Numa1, a protein known to be crucial for the proper positioning of the mitotic spindle in polarized cells.