Targeted Inactivation of APE1 Endonuclease by E96A Mutation Sensitizes Pancreatic Ductal Adenocarcinoma to DNA-Damagin Therapy

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by genomic instability, therapeutic resistance, and limited effective therapies. Chronic inflammatory and oxidative stress expose PDAC cells to persistent DNA damage, making DNA repair pathways essential for tumor survival. Apurinic/apyrimidinic endonuclease 1 (APE1/Ref-1) is a multifunctional protein with critical roles in base excision repair (BER) and redox regulation of transcription factors. This dissertation tests the hypothesis that selective disruption of APE1 endonuclease activity destabilizes PDAC tumor homeostasis and sensitizes tumors to genotoxic therapy. To test this hypothesis, PDAC cell models with selective reduction of APE1 endonuclease activity were generated by introducing a separation-of-function E96A mutation. Three independent homozygous APE1 E96A knock-in cell lines were engineered in a patient-derived PDAC cell line via CRISPR/Cas9. These cells retained normal APE1 protein expression but exhibit an approximately 150-fold reduction in incision activity, confirming severe impairment of APE1-mediated BER while preserving normal redox function. While short-term viability assays detected minimal differences, long-term clonogenic survival assays revealed marked hypersensitivity to alkylating agents and oxidative stress. Mechanistically, the cells failed to tolerate genotoxic stress and accumulated DNA damage, resulting in increased cell death following genotoxic challenge. Transcriptomic profiling revealed baseline downregulation of genes involved in extracellular matrix (ECM) remodeling and cell adhesion consistent with a less aggressive phenotype. In vivo, orthotopic mouse models demonstrated that APE1 E96A cells formed significantly smaller tumors and exhibited reduced metastasis, even in the absence of treatment. Circulating pro-oncogenic microRNAs were correspondingly reduced. Therapeutically, E96A tumors displayed heightened sensitivity to DNA-damaging agents. Temozolomide (TMZ) produced supra-additive tumor suppression when combined with APE1 endonuclease deficiency, with intermediate-dose TMZ in mutant tumors achieving regression comparable to high-dose TMZ in controls. Enhanced sensitivity was also observed with platinum-based agents. Notably, E96A cells failed to compensate for the reduction in APE1-mediated BER, showing no induction of alternative DNA repair or stress response pathways. In summary, this work establishes APE1-mediated BER as a critical survival mechanism in PDAC and identifies APE1 endonuclease function as a therapeutically exploitable vulnerability.

Author dc:creator

• Kpenu, Eyram Kwaku

Advisor dc:contributor.advisor

• Kelley, Mark R.

• APE1 (Apurinic/apyrimidinic endonuclease 1)
• Base excision repair (BER)
• DNA damage response
• Mitochondrial DNA damage (mtDNA)
• Pancreatic Cancer
• Translational research

Language dc:language.iso

en_US

Handle dc:identifier.uri

https://hdl.handle.net/1805/56404

OAI identifier oai:identifier

oai:scholarworks.indianapolis.iu.edu:1805/56404