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Apurinic/apyrimidinic (AP) endonuclease-reduction/oxidation factor 1 (APE1/Ref-1, also called APE1) is a multifunctional enzyme with crucial roles in DNA repair and reduction/oxidation (redox) signaling. APE1 was originally described as an endonuclease in the Base Excision Repair (BER) pathway. Further study revealed it to be a redox signaling hub regulating critical transcription factors (TFs). Although a significant amount of focus has been on the role of APE1 in cancer, recent findings support APE1 as a target in other indications, including ocular diseases [diabetic retinopathy (DR), diabetic macular edema (DME), and age-related macular degeneration (AMD)], inflammatory bowel disease (IBD) and others, where APE1 regulation of crucial TFs impacts important pathways in these diseases. The central responsibilities of APE1 in DNA repair and redox signaling make it an attractive therapeutic target for cancer and other diseases.

In the realm of DNA repair, base excision repair (BER) protein, APE1/Ref-1 (Apurinic/Apyrimidinic Endonuclease 1/Redox Effector - 1, also called APE1) has been studied for decades. However, over the past decade, APE1 has been established as a key player in reduction-oxidation (redox) signaling. In the review by Caston et al. (The multifunctional APE1 DNA repair-redox signaling protein as a drug target in human disease), multiple roles of APE1 in cancer and other diseases are summarized. In this Review, we aim to expand on the contributions of APE1 to various diseases and its effect on disease progression. In the scope of cancer, more recent roles for APE1 have been identified in cancer cell metabolism, as well as chemotherapy-induced peripheral neuropathy (CIPN) and inflammation. Outside of cancer, APE1 signaling may be a critical factor in inflammatory bowel disease (IBD) and is also an emergent area of investigation in retinal ocular diseases. The ability of APE1 to regulate multiple transcription factors (TFs) and therefore multiple pathways that have implications outside of cancer, makes it a particularly unique and enticing target. We discuss APE1 redox inhibitors as a means of studying and potentially combating these diseases. Lastly, we examine the role of APE1 in RNA metabolism. Overall, this article builds on our previous review to elaborate on the roles and conceivable regulation of important pathways by APE1 in multiple diseases.

Targeted therapy for cancer using small molecules has progressed exponentially, but agents that can affect cancer cells rather than non-tumorigenic cells are crucial to avoid pernicious side effects. AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein with DNA repair activity and redox signaling activity as major functions. The DNA repair function is indispensable for cell survival. Its role as a redox factor that stimulates the DNA binding activity of numerous transcription factors (TFs) such as HIF-1α, NFΚB, STAT3, and AP-1 tends to be dysregulated in cancer cells. Ref-1 is overexpressed in many cancers including, aggressive and invasive pancreatic ductal adenocarcinoma (PDAC), which is the 3rd leading cause of death in U.S. The dense stroma of PDAC primarily constituting cancer-associated fibroblasts (CAFs) makes it highly hypoxic, nutrient poor, and drug resistant. Current treatment regimens offer only modest improvement in survival. Targeting the PDAC stroma or ECM did not result in clinical benefit thus far presumably due to complex interactions between PDAC and its TME. Better treatments options that selectively target the tumor within its protective stroma are hence needed. Our group was able to generate a Ref-1 redox inhibitor, APX3330, that completed Phase I clinical trial (NCT03375086) with a good safety profile, verified target engagement and a recommended phase II dose. However, its potency in preclinical models is in the high µM range indicating the need for more potent second-generation inhibitors. Based on initial SAR studies, we selected 13 second-generation compounds from > 350 that were further characterized for positive properties including increased efficacy for cell killing, mouse and human S9 fraction metabolic stability, plasma half-life, and in silico ADMET properties. Target engagement studies involving blockade of TF activity via luciferase assay and EMSA as well as validation of direct interaction of these inhibitors with Ref-1 using thermal shift assay are ongoing. In two 3D co-culture models, second-generation Ref-1 redox analogs suppressed tumor survival significantly while sparing the CAFs. These findings were confirmed in vivo with xenografts co-implanted with tumor and CAF lines. Gene expression and mitochondria functional data revealed Ref-1's control of TCA cycle in tumor cells, but not in CAFs. To confirm and compare the effects of Ref-1 redox signaling inhibitors in cells, CRISPR editing was used to generate Ref-1 redox deficient cell lines (Ref-1C65A). As confirmation of significantly reduced Ref-1 redox activity, PDAC cells expressing Ref-1C65A did not induce hypoxia marker (CA9) under hypoxia, similar to when Ref-1 was knocked down or blocked via small molecule inhibitor. Ref-1 redox signaling and validation for selective killing of PDAC cells leaving the stomal cells undisturbed paves the way to improved PDAC treatment. Citation Format: Silpa Gampala, Nayela Chowdhury, Olivia Babb, Rachel A. Caston, Randall S. Wireman, Hye-ran Moon, George Sandusky, Emily Hulsey, Bumsoo Han, Millie M. Georgiadis, Sara K. Quinney, Andi R. Masters, James H. Wikel, Mark R. Kelley, Melissa L. Fishel. Deciphering mechanisms of Ref-1 signaling and its inhibition in aggressive tumor-stroma PDAC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1452.