Abstract 2366: Inhibition of Ref-1/APE1 redox activity with APX3330 enhances Ref-1/APE1 protein unfolded conformation in human PDAC cells
Authors: Mijit Mahmut , Babb Olivia , Gampala Silpa , Wireman Randall , Georgiadis Millie M. , Fishel Melissa L. , Kelley Mark R. 
Published: June 2022

Abstract
Ref-1/APE1 (Redox factor-1, Ref-1; Apurinic/apyrimidinic endonuclease 1, APE1) is a redox signaling enzyme that biochemically converts several transcription factors (TFs), such as NF-kB, STAT3, HIF-1α from an oxidized to a reduced state, allowing TFs to bind DNA and activate various biological responses such as cell growth and inflammation. Ref-1 and its transcriptional targets are highly active in Pancreatic Ductal Adenocarcinoma (PDAC) which often presents with tumor metastasis and therapy resistance. The redox activity of Ref-1 is determined by the redox status of the relevant cysteine residues (Cys), particularly Cys residue 65 (C65), which is critical among seven Cys residues for Ref-1’s redox activity. Using CRISPR editing in PDAC cells, we changed the C65 to Ala (C65A) in Ref-1 to evaluate the effects of loss of Ref-1 redox activity on cell signaling pathways and how Ref-1 activity regulates critical transcription factors. Using Ref-1 redox inhibitor, APX3330, we previously investigated interactions between Ref-1 and APX3330 in a cell-free system in which Ref-1 underwent conformational changes in the presence of APX3330. This resulted in complete exposure of all seven Cys residues causing oxidation of the Ref-1 protein and redox signaling inactivity. However, how these events translate in the cell has not been completely established. Using a gel-based mobility shift assay, we examined the redox-modified Cys residues of Ref-1 in response to APX3330 in PDAC cell lines over time. We observed a gradual increase in labeling of Cys in Ref-1 following APX3330 treatment between 10min to 6h which returned to control levels at 48h in both wild-type control (Cas9) and C65A-expressing cells. This reduction in the redox status of Ref-1 was likely due to unfolding alteration like the cell-free system. To confirm this, both cell lines were treated with N-ethylmaleimide (NEM) prior to cell lysing, which blocks free thiols in the protein. We detected dramatic decreases in reduced forms of Ref-1 after APX3330 treatment (between 10min-6h) in both cell lines, demonstrating that C65 is not essential for the unfolded conformational changes of Ref-1. However, C65A-Ref-1 protein demonstrated a distinctly different redox pattern upon APX3330 treatment in comparison to WT Ref-1 in the Cas9 control line. C65A-expressing cells also had reduced mitochondrial function compared to Cas9 control PDAC cells. In vivo studies demonstrated significant reduction in tumor size, weight, and growth in C65A PDAC cells compared to the Cas9 control lines, further demonstrating the critical role of C65 in the Ref-1 redox signaling and downstream pathways including metabolism and proliferation. In conclusion, we demonstrate direct interactions between Ref-1 and APX3330 in PDAC cells and subsequent biological results confirm a critical involvement of Ref-1-C65 in the redox signaling and tumor phenotype.
Citation Format: Mahmut Mijit, Olivia Babb, Silpa Gampala, Randall Wireman, Millie M. Georgiadis, Melissa L. Fishel, Mark R. Kelley. Inhibition of Ref-1/APE1 redox activity with APX3330 enhances Ref-1/APE1 protein unfolded conformation in human PDAC cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2366.