Beyond Cys65: Redox Effector Factor-1 CRISPR-Engineered Cys93 and Cys99 Mutants Reveal Cooperative and Secondary Roles of Redox Signaling and Pancreatic Ductal Adenocarcinoma Progression

Aims: Apurinic/apyrimidinic endonuclease 1/redox effector factor 1 (APE1/Ref-1) is a multifunctional protein implicated in pancreatic ductal adenocarcinoma (PDAC) through its redox regulation of oncogenic transcription factors (TFs) such as hypoxia-inducible factor 1-alpha, nuclear factor kappa-light-chain-enhancer of activated B cells, and others. While Cys65 is recognized as the primary redox-active residue, the functions of other cysteines remain only partially understood. Here, we describe the first CRISPR-engineered homozygous PDAC cell lines with single (C93A, C99A) and double (C93A + C99A) Ref-1 point mutations. This enabled detailed analysis of the individual and combined roles of Cys93 and Cys99 in Ref-1 redox signaling and their impact on various cancer phenotypes. Results: A single mutation at Cys93 decreased TF activation and reduced tumor cell survival in vitro and in vivo . These effects were similar but less pronounced in cells expressing C99A. The C93A + C99A double mutant further impaired redox signaling and decreased tumor growth and metastasis in orthotopic PDAC mouse models. All of these perturbations specifically disrupted Ref-1’s redox activity without impacting its DNA repair function. Due to reduced levels of Ref-1 protein in the C93A + C99A tumor cells, add-back experiments with wild-type Ref-1 and C93A + C99A-Ref-1 examined the impact on cell survival and activation of TFs downstream of Ref-1. Transcriptomic analysis revealed distinct changes in redox and metabolic pathways, although these were less pronounced than those observed with the previously studied C65A mutant. Innovation and Conclusion: These findings demonstrate contributory and cooperative, although secondary, roles for Cys93 and Cys99 in Ref-1 redox activity, establish new genetically engineered PDAC models, and underscore the therapeutic potential of targeting Ref-1 redox function in PDAC. Antioxid. Redox Signal. 00, 000–000.