Abstract 2941: APE1/Ref-1 redox signaling regulates HIF1a-mediated CA9 expression in hypoxic pancreatic cancer cells: Combination treatment in patient-derived pancreatic tumor models
Authors: Logsdon Derek , Shah Fenil , Carta Fabrizio , Supuran Claudiu , Fishel Melissa , Kelley Mark R. 
Published: August 2018

Abstract
Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease characterized by aggressive metastasis and therapeutic resistance. Reactive stroma in PDAC tumors leads to fibrosis, inflammation, and hypoxia. Hypoxia signaling creates a more aggressive phenotype with increased potential for metastasis and decreased therapeutic efficacy. Carbonic anhydrase IX (CA9) functions as part of the cellular response to hypoxia by regulating intracellular pH to promote cell survival. Apurinic/Apyrimidinic Endonuclease-1-Reduction/oxidation Effector Factor 1 (APE1/Ref-1) is a multifunctional protein with endonuclease activity in DNA base excision repair and redox signaling activity. This redox activity is responsible for reducing oxidized cysteines on specific transcription factors, including hypoxia inducible factor 1 alpha (HIF1α), enabling them to bind target sequences in DNA. We evaluated the mechanisms underlying PDAC cell responses to hypoxia and APE1/Ref-1 redox signaling control of HIF1α, a critical factor in hypoxia-induced CA9 transcription. We hypothesized that obstructing the HIF-CA9 axis at two points via APE1/Ref-1 inhibition (which results in a decrease in CA9 expression) and direct CA9 inhibition results in enhanced PDAC cell killing under hypoxic conditions. In our studies, HIF1α-mediated induction of CA9 is significantly attenuated following APE1/Ref-1 knock down or redox signaling inhibition in patient-derived PDAC cells and pancreatic cancer-associated fibroblast cells using the APE1/Ref-1 redox signaling inhibitor APX3330 (currently in clinical trials). Additionally, dual-targeting of APE1/Ref-1 redox signaling activity and CA9 activity results in additive-to-synergistic enhancement of acidification and cytotoxicity of PDAC cells under hypoxic conditions as well as decreased tumor growth in an ex vivo 3-dimensional tumor co-culture model. These studies are clinically relevant as we used the CA9 inhibitor SLC-0111 (phase I clinical trial completed), as well as APX3330 (Apexian Pharmaceuticals: IND 125360), for which a phase I clinical trial has opened. Further experiments characterized novel analogs of APX3330: APX2009 and APX2014, which demonstrated up to 50-fold improved potency as measured by pH reduction, cytotoxicity, and inhibition of hypoxia-induced CA9 expression. An SLC-0111 analog, FC12-531A, demonstrated up to 75-fold improved potency as measured by cytotoxicity. In combination, these analogs resulted in synergistic inhibition of 3D tumor spheroid growth at nanomolar-to-low-micromolar concentrations. These results underscore the concept that proper combination therapy has significant clinical utility of blocking APE1/Ref-1 and CA9 function for novel PDAC therapeutic treatment.
Citation Format: Derek Logsdon, Fenil Shah, Fabrizio Carta, Claudiu Supuran, Melissa Fishel, Mark R. Kelley. APE1/Ref-1 redox signaling regulates HIF1a-mediated CA9 expression in hypoxic pancreatic cancer cells: Combination treatment in patient-derived pancreatic tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2941.