Abstract Pancreatic ductal adenocarcinoma (PDAC) is a therapeutically challenging malignancy notorious for its resilience to therapeutic strategies and ability to thrive under extreme conditions. The cancer's inherent ability to adaptively resist treatments underscores the need for novel and combinatorial therapeutic approaches. Our research focuses on elucidating the role of Apurinic-apyrimidinic endonuclease/Redox effector factor 1 (APE1/Ref-1), a critical multifunctional protein in PDAC pathobiology. APE1 serves two essential functions: as the principal endonuclease in the base excision repair (BER) pathway for DNA repair and as a redox regulator of transcription factors essential for cancer cell proliferation. While the therapeutic targeting of APE1’s redox function has been effective and continues to yield significant advances, the development of treatments aimed at its DNA repair function has proven more challenging with comparatively limited progress. Since standard PDAC treatment regimens involve DNA damaging agents potentially repaired via BER and APE1, we engineered three mutant cell lines with knock-in mutations resulting in a reduction of APE1’s BER activity while maintaining full redox activity. Biochemical evaluations confirmed the effects of the mutation, revealing a 30-fold reduction in DNA repair capacity. Surprisingly, with respect to growth and proliferation, the mutant and wild-type cell lines behaved similarly. Analyses of cytotoxic vulnerabilities revealed similar findings with the mutant lines exhibiting minimal phenotypic differences in sensitivity when challenged with cytotoxic and DNA damaging agents in proliferation-based assays. These findings led us to posit that the mutant cell lines were relying on compensatory DNA repair pathways which were providing sufficient compensation for the decrease in APE1 activity. We further hypothesized that the cell lines may still have hidden, long-term deficiencies in DNA damage processing which are not apparent during the initial stress response. Subsequent colony formation assays revealed that the mutant cell lines do exhibit poor long-term survival even after only short-term exposure to DNA damage. In vivo orthotopic mouse model experiments further corroborated these findings, showing a significant reduction in tumor size and decreased metastasis to the lungs and liver in mutant cell lines compared to wild-type controls. While APE1 is the primary endonuclease in the BER pathway, studies have demonstrated APE1-independent BER in rare, specific contexts. We are probing the hypothesis that these APE1-independent BER pathways function as the compensatory mechanism safeguarding these cells in short-term stress situations. Identifying and characterizing these backup pathways will not only offer a better understanding of APE1’s DNA repair capabilities within PDAC but will also provide a better understanding of how DNA repair mechanisms can be therapeutically targeted for novel therapeutic options or combinations. This opens the path to enhance standard of care treatments such as FOLFIRINOX. Citation Format: Eyram K Kpenu, Randall Wireman, Mahmut Mijiti, Silpa Gampala, Melissa L Fishel, Mark R Kelley. Challenging PDAC adaptability: Uncovering therapeutic vulnerabilities in APE1 DNA repair mechanisms [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C028.