Project Summary Triple negative breast cancer (TNBC) has the worst prognosis and survival in all breast cancer subtypes with currently available standard therapies, representing an unmet therapeutic challenge. Significant heterogeneity (with six genetically defined subgroups) and a lack of FDA-approved targeted therapeutics and are the major reasons contributing to poor prognosis and high mortality rates and have prevented identification of common molecular targets and development of targeted therapeutics. To meet this challenge, we extensively analyzed breast cancer patient databases and discovered that expression of Eukaryotic Elongation Factor 2-Kinase (EF2K) gene encoding an unusual alpha kinase has a dramatic effect on survival rates of TNBC patients. Remarkably, TNBC patients with low EF2K expression had no mortality up to 10 years. More importantly, we found that EF2K is highly overexpressed in the majority of TNBC patients and showed that it is an important driver for TNBC tumorigenesis, invasion and progression; and validated it as a potential molecular target using genetic silencing technology in multiple preclinical animal models. Based on our preliminary studies, we hypothesize that EF2K represents an Achilles’ heel for targeting TNBC and if effectively targeted, it will have a tremendous impact on patient survival. Considering that EF2K knock-out mice are healthy and have no signs of toxicity, EF2K-targeted therapies are expected to be safe. However, currently there is no specific or potent inhibitors for targeting EF2K. Thus, in collaboration with a team of investigators, including medicinal chemists and computational chemists, we recently synthesized a serious of potential small molecule inhibitors of EF2K based on computational modeling and identified a lead compound that significantly inhibits EF2K activity. Based on the lead compound, we further designed and synthesized more than a hundred of second generation of novel compounds and identified a more potent inhibitor that is effective in inhibiting EF2K at with nanomolar concentrations and demonstrated remarkable in vivo efficacy in TNBC tumors when formulated in long-acting single-lipid based nanoparticles, with no observed toxic effects. In this proposed project, we will characterize and optimize the EF2K-inhibitor based therapy using clinically relevant TNBC models with the goal of developing highly specific strategies for TNBC. Our long-term goal is to develop safe, highly effective therapies and translate them to the clinic for TNBC patients, who lack targeted treatment options.