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.