Abstract BACKGROUND: The MUC16 gene encodes the CA125 ovarian cancer antigen. This glycoprotein is expressed in the majority of Epithelial Ovarian Cancers (EOC) but has limited expression elsewhere in the adult human. Expression of the carboxy-portion of MUC16 results in increased invasion, soft agar growth and in vivo growth. OBJECTIVES:. We seek to identify MUC16 specific small molecules as chemotherapy candidate drugs for development in ovarian cancer. METHODS: Our previously reported study of dual fluorescence isogenic high content screening for MUC16/CA125 selective agents (Mol.Can.Ther, 2011) was extended to a library of 74,942 compounds from NCI, to identify small molecules which have a differential effects against MUC16 positive cells compared to isogenic MUC16 negative ovarian cancer cell lines Carboxy-terminus portions of the human MUC16 gene were introduced into A2780 EOC cell lines labeled with mCherry and compared to GFP labeled isogenic controls in a 384 well co-culture assay for cellular viability by computerized fluorescence intensities and compared for the relative toxicity against MUC16+/MUC16- isogenic cell lines. Compounds with differential activity were selected, resupplied and examined by Vi Cell counting and Alamar Blue cytotoxic assays for confirmation in SK-OV-3 cell line pairs. RESULTS: A2780 isogenic pair was initially used and thirty compounds which selectively targeted MUC16 positive cells were identified. These 30 compounds were tested for growth inhibition with a secondary SKOV3 isogenic screen for confirmation. Two compounds were among the screen identified in the secondary SKOV3 isogenic pair testing. These compounds were studied using Alamar Blue Assay with a panel of ovarian cell lines including CAOV3; OV433; OV432; SKOV8 (WT and MUC16 knockdown and an isogenic T80 MUC16+/− pair (in vitro transformed normal ovarian surface epithelial cell line) and IGROV1-R10. We are in the process of examining the Structural Activity Relationship around these 2 compounds and will present their response. CONCLUSIONS: These MUC16 +/− dual fluorescence screens can be used as an example of single gene target, high throughput screening strategies to identify potentially specific therapeutics for key oncogenic targets. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1828. doi:1538-7445.AM2012-1828