APE 1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cell RNA sequencing

Apurinic/apyrimidinic endonuclease 1/redox factor‐1 ( APE 1/Ref‐1 or APE 1) is a multifunctional protein that regulates numerous transcription factors associated with cancer‐related pathways. Because APE 1 is essential for cell viability, generation of APE 1‐knockout cell lines and determining a comprehensive list of genes regulated by APE 1 has not been possible. To circumvent this challenge, we utilized single‐cell RNA sequencing to identify differentially expressed genes (DEGs) in relation to APE 1 protein levels within the cell. Using a straightforward yet novel statistical design, we identified 2837 genes whose expression is significantly changed following APE 1 knockdown. Using this gene expression profile, we identified multiple new pathways not previously linked to APE 1, including the EIF 2 signaling and mechanistic target of Rapamycin pathways and a number of mitochondrial‐related pathways. We demonstrate that APE 1 has an effect on modifying gene expression up to a threshold of APE 1 expression, demonstrating that it is not necessary to completely knockout APE 1 in cells to accurately study APE 1 function. We validated the findings using a selection of the DEGs along with si RNA knockdown and qRT ‐ PCR . Testing additional patient‐derived pancreatic cancer cells reveals particular genes ( ITGA 1 , TNFAIP 2 , COMMD 7 , RAB 3D ) that respond to APE 1 knockdown similarly across all the cell lines. Furthermore, we verified that the redox function of APE 1 was responsible for driving gene expression of mitochondrial genes such as PRDX 5 and genes that are important for proliferation such as SIPA 1 and RAB 3D by treating with APE 1 redox‐specific inhibitor, APX 3330. Our study identifies several novel genes and pathways affected by APE 1, as well as tumor subtype specificity. These findings will allow for hypothesis‐driven approaches to generate combination therapies using, for example, APE 1 inhibitor APX 3330 with other approved FDA drugs in an innovative manner for pancreatic and other cancer treatments.