Abstract Eyes Absent Homolog 4 (EYA4) is a dual-specificity phosphatase re-expressed in triple-negative breast cancer (TNBC) through methylation of repressive transcription factor marks. Its overexpression correlates with advanced disease and promotes genomic instability by deregulating Rad51-mediated homologous recombination (HR) and non-homologous end joining (NHEJ), throughout the cell cycle. Here, we characterize a novel property of EYA4: its ability to bind single-stranded DNA (ssDNA), and present a small-molecule inhibitor that abolishes its tyrosine phosphatase activity, possibly through the weakening of the DNA binding. Using a combination of chimeric EYA4 fragments and mutants, mobility shift assays, and biophysical methods including nuclear magnetic resonance (NMR) spectroscopy and microscale thermophoresis (MST), we mapped the DNA-binding interface and identified key residues mediating this interaction. ssDNA binding was found to stimulate the tyrosine phosphatase activity of EYA4, and our small-molecule inhibitor disrupts the phosphatase activity with an IC50 of 1 µM, indirectly suppressing enzymatic function. Functional assays, including homologous recombination reporter systems, immunofluorescence for DNA damage markers, MTT viability assays, and live-cell imaging using the Incucyte system (Essen), showed that EYA4 inhibition impairs HR, leads to DNA double-strand break accumulation, reduces proliferation, and increases apoptosis in MDA-MB-231 TNBC and TKCC PDAC models at concentrations of 1-5 µM. Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) imaging further revealed cell cycle defects, including mitotic failure and chromosomal segregation errors, consistent with disrupted repair during replication. Testing conducted on large panels of patient derived cell lines with the EYA4 inhibitor alone and in combination with standard of care demonstrated broader therapeutic relevance. These findings support targeting EYA4’s DNA-binding function as a valid strategy to suppress tumor growth and enhance the efficacy of DNA-damaging therapies in HR-deficient (HRD+) cancers. Citation Format: Daniela A. Garcia, Chandler Heupel, Hannah Palacios, Eloise Dray. The protein phosphatase EYA4 is a druggable target for the treatment of breast cancer abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 1288.
Garcia et al. (Fri,) studied this question.