Abstract About 70% of breast cancer patients with progressive disease are resistant to conventional cancer treatments such as adjuvant chemotherapy and/or radiation therapy. Recently, the STAT1 signaling pathway has been identified as a predictive marker of poor outcome in breast cancer patients and as a factor responsible for radiation and chemotherapy resistance. STAT1 was initially considered as a tumor suppressor, since it causes growth arrest and apoptosis after stimulation with interferons and in response to DNA damage. However, constitutive activation/overexpression of STAT1 results in pro-survival cellular signaling via enhancement of expression of pro-survival proteins Mcl-1 and Hsp27 that might be responsible for observed radiation resistance of breast cancer cells. It may therefore be beneficial to sensitize patients with resistant disease to radiation or chemotherapy by inhibiting STAT1 signaling, yet there has been little effort to develop direct inhibitors of STAT1. We have recently developed a selective inhibitor ST1-Hel2A-2 that directly binds to the N-terminal domain of STAT1, but not other STAT family members, and inhibits STAT1 transcriptional activity (ACS Chem Biol, 2007. 2(12): 799). The inhibitor kills breast cancer cells at nanomolar concentrations but does not effect survival of normal mammary epithelial cells. We have now found that inhibition of STAT1 N-domain can further sensitize breast cancer cells to ionizing radiation. The mechanism for observed sensitization at least partially depends on down-regulation of STAT1 target genes, including pro-survival Hsp27. In addition, ST1-Hel2A-2 is capable of disrupting the STAT1 N-domain protein-protein interactions. Yeast two-hybrid screening has revealed that the STAT1 N-domain associates with double-stranded RNA-activated protein kinase (PKR) and DNA glycosylase NEIL1. Both PKR and NEIL1 facilitate DNA repair after ionizing radiation and protect mammalian cells from radiation-mediated cell death. ST1-Hel2A-2 disrupts STAT1 interactions with both proteins resulting in a reduced DNA repair capacity. Our data suggest that a novel inhibitor of STAT1 decreases radiation resistance of breast cancer cells through downregulation of pro-survival genes and inhibition of DNA repair. Inhibition of STAT1 signaling appears to be a promising strategy in overcoming radiation resistance and can be used as an effective treatment of metastatic breast cancer when combined with conventional therapies. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 489.