Integrated Post-Translational Modification (PTM) Proteomics Reveals Early Redox and Mitochondrial Remodeling Following Ref-1 Inhibition in Pancreatic Cancer

<jats:p>Background: Apurinic/apyrimidinic endonuclease 1/redox factor-1 (Ref-1/APE1) is a central regulator of redox-dependent transcriptional signaling that promotes pancreatic ductal adenocarcinoma (PDAC) progression, therapeutic resistance, and metabolic adaptation. While pharmacologic inhibition of Ref-1 suppresses tumor growth and alters cellular metabolism, immediate molecular events linking Ref-1 inhibition to downstream cellular adaptation remain poorly understood. We therefore sought to characterize proteome-wide signaling responses induced by second-generation Ref-1 redox inhibitor, APX2014. Methods: We applied an integrated multiplexed proteomics workflow to simultaneously quantify global protein abundance together with cysteine oxidation, phosphorylation, and lysine acetylation in Pa03C PDAC cells following acute treatment (30-120 min) with selective Ref-1 redox inhibitor APX2014. Differential post-translational modification (PTM) analysis, pathway enrichment, structural mapping of regulated sites, and functional mitochondrial substrate utilization assays were performed to define early signaling responses. Results: APX2014 induced rapid and extensive remodeling of PTM landscape while producing minimal changes in global protein abundance. Cysteine oxidation represented the earliest and most sustained response, accompanied by widespread phosphorylation and delayed lysine acetylation. Integrated pathway analyses identified mitochondrial translation, respiratory electron transport, TCA cycle metabolism, and mitochondrial redox homeostasis as the earliest and most consistently regulated processes. Functional mitochondrial assays confirmed impaired utilization of TCA cycle substrates following APX2014 treatment. Coordinated PTM remodeling was observed on Ref-1-associated signaling proteins, including NFkB1 and p53, revealing simultaneous regulation of oxidation, phosphorylation, and acetylation within functionally important domains. Early redox-sensitive protein networks were also associated with subsequent disruption of mitotic organization. Conclusions: Integrated multi-PTM proteomics reveals that pharmacologic Ref-1 redox inhibition rapidly rewires regulatory signaling networks before detectable changes in protein abundance. Our findings identify mitochondrial redox remodeling as an early consequence of Ref-1 inhibition, providing systems-level insight into how Ref-1-targeted therapies disrupt metabolic and stress-adaptive programs in pancreatic cancer. This work establishes a framework for understanding the molecular basis of Ref-1-directed therapeutics and highlights integrated PTM profiling as a powerful strategy for defining early drug response mechanisms. These findings provide a strong translational rationale for advancing next-generation Ref-1 redox inhibitors such as APX2014, developed from the first-in-class inhibitor APX3330 currently in clinical trials, and underscore the broader therapeutic potential of targeting Ref-1 redox signaling in pancreatic cancer.</jats:p>