Summary Pancreatic cancer (PC) has the highest mortality of any cancer, with a 5-year survival rate of less than 10%. Despite progress in the development of targeted therapies, the survival rate is still unacceptably low. It is, therefore, important to identify novel therapeutic approaches that could lead to a more effective treatment for this malignant disease. This proposal explores the novel concept that a core 2 mucin synthesis gene, GCNT3, regulates PC growth and survival, ascribing a new and critical role to GCNT3. We have shown that GCNT3 is overexpressed in PC patients and contributes to PC pathogenesis, indicating that GCNT3 is a promising therapeutic target. However, how GCNT3 regulates PC is not clear. Using in silico small molecular docking simulation approaches, we discovered that talniflumate is a novel inhibitor that selectively binds to GCNT3. Examining the molecular mechanisms of GCNT3-mediated PC growth, we found that siRNA-mediated knockdown of GCNT3 or treatment with talniflumate, inhibited xenograft tumors. Talniflumate also reduced GCNT3 expression, leading to reduced production of mucins in vivo and in vitro, and improved cellular gemcitabine uptake. Taken together, our findings strongly suggest that targeting mucin biosynthesis through GCNT3 may improve responsiveness to drug treatment. We, therefore, hypothesize that aberrant expression of GCNT3 will lead to excessive mucin synthesis during PC development and cause chemoresistance. Hence, the inhibition of aberrant mucin synthesis is sufficient to disrupt the barrier properties of the mucin mesh to efficiently increase the access of chemotherapeutics to target sites. Three independent, yet interrelated, specific aims are proposed to address this hypothesis. Aim 1: Generate GCNT3-deficient KPC mice and determine whether targeting GCNT3 ablates the mucin barrier and inhibits PC progression/metastasis by increasing gemcitabine and nab-paclitaxel (NPT) efficacy. Aim 2: Determine whether combinational therapy of talniflumate plus NPT are effective in treating PC by a) evaluating the efficacy and dose-response effects of the combination administered in vivo in a clinically relevant KPC model of spontaneous PC and b) determining mucin disruption and gemcitabine or NPT uptake in combination-treated tumors. c) We will establish the GCNT3 and NPT levels in tumor tissue and their correlation with mucin subtypes (Mucs) and PC treatment efficacy via whole genome transcriptome sequencing and PET imaging. Aim 3: We will investigate the role of GCNT3 in gemcitabine resistance, mucin overexpression and PC growth. a) For clinical relevance, we will assess the inhibition of human patient- derived xenografts (PDX) and PC slice cultures by combinational therapy. b) Evaluate the efficacy of combination against GCNT3 overexpressing human PC PDX tumors in vivo. Veterans Health Relevance: Pancreatic cancer major health problem and many veterans and their families suffered from this neoplastic d...