ABSTRACT Pancreatic ductal adenocarcinoma (PDAC) is particularly resistant to therapy and typically presents as metastatic disease. Characterized by hypoxia, dense stroma, and metabolic rewiring, original approaches and combination strategies are desperately needed. We propose to investigate inhibition of a redox signaling protein and drug combinations that selectively kill the tumor by impinging on critical pathways the tumor is using to survive. Redox factor-1 (Ref-1) regulates the activity of various transcription factors that drive pancreatic cancer cell proliferation and drug resistance as well as genes involved in cellular metabolism. Under hypoxia, inhibition of Ref-1 significantly perturbed metabolic pathways (TCA cycle and OXPHOS) and HIF-regulated genes, and thus slowed the growth of pancreatic cancer co-culture spheroids and xenografts. The first-generation Ref-1 inhibitor (APX3330) completed phase I trial and demonstrated 32% response, predicted PK, and target engagement with no significant toxicities. There was disease stabilization in six patients with four on treatment for an extended time (>250 days). Based on encouraging phase I data and a detailed structural-activity relationship (SAR) program, we have also identified next generation Ref-1 inhibitors that are at lead optimization stage, a strategy to screen for patients that have sensitivity to Ref-1 inhibition, and molecular targets that are likely to synergize with Ref-1 inhibition. However, adaptive mechanisms of resistance eventually emerge with targeted therapy, therefore we will also focus on the development of novel combinations. Our hypothesis is that targeting the redox function of Ref-1 alone and in mechanistically designed combination therapies will induce metabolic lethality and inhibit pancreatic cancer growth and metastasis. In Aim 1, identification of metabolic characteristics of cancer cells/tissues that associate with the outcome of Ref-1 inhibition and prediction of new metabolic targets to improve the efficacy of Ref-1 inhibition. Our recently developed computational predictor of cell-wise metabolic flux will be used to study the metabolic changes due to Ref-1 inhibition in PDAC cells at the single cell level. In Aim 2, NMR to establish direct interactions of Ref-1 and the new analogues, efficacy, toxicity, and metabolic stability studies will allow us to advance the top lead candidate(s) for in vivo studies for Candidate Selection (NIH Milestone 4) and IND (Investigational New Drug) submission leading to eventual Phase I trial. Lastly in Aim 3, evaluation of Ref-1 in preclinical combination therapy will be used to overcome adaptive resistance. To further predict metabolic nodes that could be perturbed to synergize with Ref-1 inhibition, creating a metabolic lethality, computational predictor of cell-wise metabolic flux described in Aim1 will be used. The efficacy of Ref-1 alone and in new combinations will be investigated using organoids in vitro and the mous...