Abstract B102: Targeting the SIX1/EYA transcriptional complex as a potential anti-cancer therapy.
Authors: Patrick Aaron , Blevins Melanie , Krueger Aaron , Cabrera Josh , Backos Don , Reigan Phil , Zhao Rui , Ford Heide 
Published: August 2014

The SIX1 homeoprotein and members of the EYA family of co-activators form a bipartite transcription factor important in development and disease. SIX1 and EYA are highly expressed during development, but their expression is lost in most adult tissues. However, SIX1 and EYA are re-expressed in a variety of cancers, where they promote transformation and metastasis. Interestingly, the EYA proteins have a C-terminal EYA Domain (ED) that contains protein tyrosine phosphatase activity, representing the first transcription factor family to contain intrinsic phosphatase activity. Intriguingly, the EYA tyrosine phosphatase activity is critical for breast cancer metastasis. Transcription factors and phosphatases have traditionally been difficult to target. However, great strides have been made recently in developing small molecule protein-protein interaction inhibitors. In addition, the active site of EYA presents a unique opportunity to target the complex, as it uses Asp as the active site residue instead of the more common Cys found in most cellular phosphatases. We thus used a multipronged approach to target the SIX1/EYA pathway, through inhibiting SIX1/EYA complex formation and through inhibiting the protein phosphatase activity of EYA. To gain a better understanding of the SIX1-EYA interface, we determined the 2.0 Å resolution crystal structure of the SIX1/EYA2 complex, revealing that the interaction between SIX1 and EYA2 resembles that of the p53/HDM2 complex, with a single helix of SIX1 binding in a hydrophobic groove in EYA2. A single amino acid substitution in this SIX1 helix is sufficient to inhibit complex formation and to inhibit SIX1-induced tumor metastasis. As the interface looks amenable to small molecule targeting we used both virtual screening (VS) of a 400K compound library, and a high throughput screen (HTS) performed with a 370K compound library, to identify novel inhibitors of the SIX1-EYA interaction. VS identified 2 compounds with IC50s of ∼30μM. HTS identified 11 compounds with IC50s &lt;10μM that are able to enter the cell and inhibit SIX1/EYA2 mediated transcription. HTS carried out against the phosphatase activity of EYA2 led to the identification of a lead compound (E01) with an IC50 &lt;5μM. Subsequent characterization has identified E01 as an allosteric inhibitor and functional analyses reveal E01 is able to inhibit breast cancer cell migration. Since SIX1 and EYA family members are regulators of embryonic development and are scarcely expressed in adult tissues, inhibition of the SIX1/EYA pathway will likely be a potent approach to inhibit tumor progression with limited side effects.

Citation Information: Mol Cancer Ther 2013;12(11 Suppl):B102.

Citation Format: Aaron Patrick, Melanie Blevins, Aaron Krueger, Josh Cabrera, Don Backos, Phil Reigan, Rui Zhao, Heide Ford. Targeting the SIX1/EYA transcriptional complex as a potential anti-cancer therapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr B102.