PROJECT SUMMARY/ABSTRACT Antisense oligonucleotides (ASO) are the ultimate platform technology that could cripple lethal, drug-resistant tumors by targeting “undruggable” oncogenes. ASO are 16-20 bp nuclease-resistant oligonucleotides that base pair with a target RNA and can elicit its degradation or alter its splicing. Despite its great potential in cancer therapy, poor uptake into tumor cells currently limits the clinical use of ASO in cancer patients. The long-term goal of the Edinger lab is to use our knowledge of endolysosomal trafficking to develop novel, effective, and minimally toxic therapies for cancer and other diseases. The overall objective of this proposal is to develop novel approaches that increase ASO activity in tumors and normal tissues. The central hypothesis is that simultaneously inhibiting endocytic recycling and lysosomal fusion will increase ASO activity in tumor and normal tissues by trapping ASO in the pre-lysosomal compartment from which ASO escape is most efficient. Under the first aim, it will be determined whether oral administration of a small molecule that enhances ASO activity in vitro also increases ASO activity in tumors. Under the second aim, a candidate approach will be used in conjunction with cutting-edge genetic tools to identify which of the known molecular targets of a second molecule are responsible for ASO potentiation and endolysosomal trafficking disruption. The rationale for this project is that small molecules that increase ASO activity in tumors could yield major benefits to patients with aggressive, drug-resistant tumors by improving the efficacy of oncology ASO in late stages of clinical development and stimulating development of new ASO. The proposed research is expected to have a profound positive impact by establishing the feasibility of using small molecules to make oncology ASO clinically viable and stimulating development of new ASO for cancer therapy. The overall training objective in this application is to develop and cultivate the management, networking, translational, and writing skills that are necessary to be successful in a postdoctoral fellowship and as an academic PI at a major research institution. The Training Plan addresses these training goals by taking advantage of critical resources provided by UCI and its vibrant, collaborative cancer research community.