Abstract Breast cancer often arises from inappropriate activation of transcription factors involved in normal mammary development, such as the signal transducers and activators of transcription (STAT) family of transcription factors. STAT3, which plays an important role in mammary remodeling, is activated (as assessed by tyrosine phosphorylation) in approximately 70% of primary breast cancers, with many of these tumors being highgrade. Furthermore, inhibition of STAT3 leads to a loss of tumorigenicity of breast cancer cells. Therefore, targeting STAT3 in breast cancer would be a potentially beneficial form of therapy. To identify small molecule STAT3 inhibitors, we developed a high throughput cell-based assay to identify compounds that inhibit STAT3- dependent transcriptional activity. We screened approximately 200,000 compounds, and specificity for STAT3 was ensured by eliminating molecules that inhibited either STAT1- or NF-kappaB-dependent transcriptional activity. From this screen, we identified 4-[4-(phenylmethyl) piperidin-1-yl] thieno[2,3-d]pyrimidine (PMPTP) (Probe ID ML116) as a potent inhibitor of STAT3 transcriptional activity, which has no effect on STAT1 or NF-kappaB-dependent transcriptional activity. Treatment with PMPTP of MDA-MB-468 breast cancer cells, which contain constitutively activated STAT3, leads to inhibition of expression of STAT3 target genes including bcl-x and mcl1. Furthermore, PMPTP is a potent inhibitor of viability of MDA-MB-468 breast cancer cells. By contrast, SK-BR-3 cells, which do not contain constitutively active STAT3, were only minimally affected. This suggests that PMPTP reduces the viability of cells that depend on constitutively active STAT3. PMPTP has no effect on STAT3 tyrosine or serine phosphorylation or STAT3 nuclear translocation, suggesting that PMPTP may be inhibiting STAT3 at the level of DNA binding or co-factor recruitment. Importantly, PMPTP lowers the apoptotic potential of breast cancer cells thereby making them more susceptible to other forms of therapy. Building on this observation, we identified a number of beneficial combinations of PMPTP with cytotoxic drugs currently used in breast cancer therapy as well as novel anti-cancer agents. Additionally, we analyzed a number of structural analogues of PMPTP, and we identified two compounds that had approximately 4-fold increased potency in inhibiting the viability of breast cancer cells containing STAT3 activation. Taken together, these findings identify PMPTP as a potential new form of therapy for breast cancers containing activated STAT3, which can be enhanced using conventional and non-conventional chemotherapy. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-15-12.