PROJECT SUMMARY/ABSTRACT Five-year survival rate of pancreatic ductal adenocarcinoma (PDAC) still remains at a dismal 10%. This poor outcome is attributed to PDAC’s complex tumor microenvironment (TME) containing a desmoplastic stroma, which consists of cancer-associated fibroblasts (CAFs) and a dense extracellular matrix (ECM). This stroma affects proliferation, invasion and drug resistance of pancreatic cancer cells (PCCs). Unexpectedly, the ablation of CAFs or targeting of certain ECM proteins in the stroma led to changes that actually accelerated tumor growth and impaired treatment outcome. To address this challenge, we are pursuing targets that selectively kill PCCs rather than CAFs. Redox effector factor-1 (Ref-1) is upstream of several key signaling pathways as a regulator of transcription factors (TFs). Blockade of Ref-1 redox function with our first-generation inhibitor blocks the activation of key TFs including HIF-1α, NFkB, and AP-1, all of which are implicated in growth and metastasis signaling in the tumor. However more potent second-generation compounds are necessary to effectively treat PDAC. We recently synthesized new analogs and obtained data showing that these are more potent with a significant decrease in tumor cell survival with minimal changes in CAF survival. This selective cancer cell killing is a novel characteristic of our new analogs, as gemcitabine kills both PCCs and CAFs. The underlying mechanism and therapeutic efficacy of these compounds are the focus of this proposal. Our overarching goal is to evaluate second-generation Ref-1 inhibitors for treatment of PDAC by comparing the mechanisms of Ref-1 inhibition in PCCs and CAFs. Our hypothesis is that through inhibition of Ref-1, multiple cancer-associated pathways are simultaneously and effectively targeted leading to suppression of survival signaling in PCCs while sparing the CAFs. The heterogeneity and complexity of PDAC poses a significant challenge in screening and evaluating the efficacy of new compounds on tumors and on stromal components. To overcome these technical challenges and ultimately lead to new treatments, we will perform mechanistic screening of Ref-1 inhibitors using newly developed in vitro tumor models based on patient-derived PCCs and CAFs - 1) 3D co-culture spheroids for screening and tumor-stroma interaction; and 2) a microfluidic tumor-microenvironment-on-chip (T-MOC) model for mechanistic evaluation. Findings using these novel in vitro systems will be validated with orthotopic PDAC models. Specific aims are: Aim 1. Investigate the selectivity of Ref-1 signaling and the effects of its inhibition in PCCs and CAFs using T-MOC models; Aim 2. Characterize the mechanism of Ref-1 inhibition for second-generation compounds; and Aim 3. Validate the efficacy and pharmacodynamic profiles of Ref-1 analogs in orthotopic PDAC models. The aims of this proposal will provide invaluable information regarding both the effectiveness of new Ref-1 analogs in sophis...