Abstract The Epithelial Cell Transforming sequence 2 (ECT2) proto-oncogene is a Guanine Exchange Factor (GEF) for RhoA, Rac1 and Cdc42 and is essential to the regulation of cytokinesis. ECT2 contains the Dbl homology and pleckstrin homology (PH) domains, which are the hallmarks of GEFs. ECT2 is over-expressed in primary non-small cell lung cancer (NSCLC) tumors, and injection of ECT2 transfectants into nude mice efficiently induces tumor formation. High level of ECT2 expression is associated with poor prognosis for patients with NSCLC. Knock down of ECT2 expression by small interfering RNAs effectively suppresses lung cancer cell growth, suggesting a specific role of ECT2 in lung cancer development. Taken together, ECT2 may represent an attractive molecular target for inhibiting lung tumor growth. Our studies are based on the hypothesis that ECT2 plays an important role in lung cancer progression and is a novel target for the development of drugs to treat lung cancer. We have built a model for ECT2 PH domain using protein homology modeling. Docking of new compounds selected from commercial drug-like libraries has led to the identification of novel inhibitors of ECT2 PH domain. We have identified several compounds that bind ECT2 PH domain in the low micromolar range (KD<1μM) as measured using surface plasmon resonance spectroscopy and that inhibit the proliferation of a panel of 23 lung cancer cell lines (EC50<50μM). Importantly, inhibition occurred only in mutant-KRAS cells. Two compounds were shown to inhibit actin polymerization using a phalloidin-rhodamine stain as well as in vitro tumor formation using a colony forming assay and migration using a wound healing assay. These two compounds are being evaluated for their anti-tumor activity against several human lung cancer xenografts in SCID mice. The objectives of our studies are 1) to conduct molecular mechanistic studies of the precise role of ECT2 in the development of lung cancer in vivo animal model of lung cancer; 2) to test the most active compounds in cells in culture to provide a mechanistic rationale for their biological activity and 3) to test the anti-tumor properties of the lead compounds in a in vivo model of lung cancer. The overall goals of our work are to validate ECT2 as a therapeutic target for lung cancer and to develop a novel therapeutic strategy and agents for the improved treatment and the eventual cure of lung cancer. 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 2835. doi:1538-7445.AM2012-2835