Abstract The Rho GTPase Rac is a pivotal regulator of cancer cell migration and invasion; processes required for metastatic progression. We previously characterized the small molecule EHop-016 as a novel Rac inhibitor in metastatic breast cancer cells and recently reported that EHop-016 was effective at reducing tumor growth, metastasis, and angiogenesis in nude mice at 25 mg/kg bodyweight (BW) (Castillo-Pichardo, et al. 2014). We also determined the pharmacokinetics and bioavailability of EHop-016, and reported that EHop-016 is rapidly cleared from mouse plasma with a half-life of ∼5 hrs and ∼30% bioavailability (Humphries-Bickley, et al., 2015). To improve the bioavailability and efficacy of EHop-016, we synthesized and screened a number of derivatives, from which EHop-016A was identified as a potent Rac inhibitor at nM concentrations. Moreover, as determined from immunofluorescence and brightfield microscopy of several breast cancer cell lines, EHop-016A has a dramatic effect on cell morphology by inducing a loss of cell polarity, and inhibiting cell surface actin-based extensions and focal adhesions, to ultimately result in the detachment of cells from the extracellular matrix (ECM). In addition, EHop-016A reduces breast cancer cell migration in a transwell assay. EHop-016A also decreases mammosphere formation, indicating an inhibitory effect on breast cancer stem cell-like properties. The effect of EHop-016A on metastatic cancer cell viability was determined via MTT assays. EHop-016A decreases cell viability with a GI50 of 150 nM and 110 nM in MDA-MB-435 and MDA-MB-231 human metastatic cancer cell lines respectively. Western blotting demonstrated that EHop-016A decreases anti-apoptotic proteins BCL-2 and BCL-xL without affecting their gene expression, as quantified by qPCR. Consequently, EHop-016A increases pro-apoptotic caspase 3/7 activity. These results indicate that the EHop-016A induced cell rounding and detachment from the substratum results in anoikis (apoptosis due to dissolution of integrin-mediated cell to ECM attachments). Therefore, this new small molecule compound has potential as an inhibitor of metastatic breast cancer progression, and warrants further investigation as an anticancer agent. Citation Format: Tessa Humphries-Bickley, Linette Castillo-Pichardo, Luis Borrero-Garcia, Ingrid Forestier-Roman, Luis Cubano, Eliud Hernandez-O'Farrill, Cornelis Vlaar, Suranganie Dharmawardhane. A novel inhibitor of malignant signaling in metastatic breast cancer. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr A141.