Abstract B51: Regulation of HIF1α under hypoxia by APE1/Ref-1 impacts CA9 expression: Dual-targeting in patient-derived 3D pancreatic cancer models
Authors: Logsdon Derek P. , Grimard Michelle , Shahda Safi , Zyromski Nicholas , Schipani Ernestina , Carta Fabrizio , Supuran Claudiu T. , Korc Murray , Ivan Mircea , Kelley Mark R. , Fishel Melissa L. 
Published: December 2016

Abstract
Half of all patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) die within a year despite extensive surgery and/or a highly aggressive chemotherapy regimen. Several mechanisms are proposed to play a role in PDAC therapeutic resistance, including reactive stroma and hypoxia. Cancer-associated fibroblasts (CAFs) contribute to tumor signaling, fibrosis, inflammation, and hypoxia. Hypoxia signaling creates a more aggressive phenotype with increased potential for metastasis and impairs therapeutic efficacy. Carbonic anhydrase IX (CA9) functions as part of the cellular response to hypoxia to regulate intracellular pH to promote cell survival. Apurinic/Apyrimidinic Endonuclease-1-Reduction/oxidation Effector Factor 1 (APE1/Ref-1) is a multi-functional protein that possesses two major activities: an endonuclease function in DNA base excision repair and a redox signaling function that reduces oxidized transcription factors, enabling them to bind to their DNA target sequences. APE1/Ref-1 regulates several transcription factors involved in survival mechanisms, tumor growth, and hypoxia signaling. We explored the mechanisms underlying PDAC cell responses to hypoxia and modulation of APE1/Ref-1 redox signaling control of hypoxia inducible factor 1 alpha (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 and CA9 inhibition will result in enhanced PDAC cell killing under hypoxic conditions.
Methods: We performed qRT-PCR and Western Blots to confirm changes in CA9 expression in PDAC cells and CAFs following APE1/Ref-1 inhibition and hypoxia exposure. We evaluated the effects of dual-targeting APE1/Ref-1 and CA9 on acidification of tumor cells with an intracellular pH indicator. Proliferation assays were used to assess cell killing following inhibition of APE1/Ref-1 and CA9 under hypoxia. Ex vivo 3-Dimensional co-culture models including both tumor and CAFs were used to examine whether we could enhance the efficacy of APE1/Ref-1 and/or CA9 inhibition with a dual-targeting approach to kill tumor spheroids. To delineate which function of APE1/Ref-1 is critical for observed effects, we used the APE1/Ref-1 redox signaling inhibitor, APX3330, an APE1/Ref-1 repair inhibitor, ARI-3, and an APX3330 analog that does not block APE1/Ref-1 redox activity, RN7-58. To inhibit CA9, we used the sulfonamide derivative, SLC-0111 (Clinical Trial NCT02215850).
Results: HIF1α-mediated induction of CA9 is significantly diminished in PDAC cells following APE1/Ref-1 redox inhibition or knock-down in both patient-derived and established cell lines, as well as pancreatic CAFs. Additionally, dual-targeting of APE1/Ref-1 redox signaling and CA9 results in acidification of PDAC cells and reduces PDAC tumor cell growth under hypoxic conditions as well as in a 3D tumor co-culture model.
The results presented here demonstrate potential clinical utility of blocking APE1/Ref-1 and CA9 function for novel PDAC therapeutic treatment. Ongoing experiments will determine the role of APE1/Ref-1 and CA9 in invasion of tumor cells exposed to hypoxic conditions, as well as the efficacy of dual-targeting APE1/Ref-1 and CA9 in in vivo models of PDAC.
Consequently, our findings further demonstrate a combination approach for blocking important signaling pathways in tumor cells. Our results support our previously published studies using APX3330 and STAT3 inhibition as a dual hit strategy in PDAC cells (Cardoso, Jiang et al. 2012) and APX3330 and Avastin (bevacizumab) for HIF1α-VEGF-signaling inhibition as an anti-angiogenesis combination strategy (Luo, Delaplane et al. 2008, Jiang, Gao et al. 2011, Li, Liu et al. 2014). Continued studies centering on the APE1/Ref-1 redox signaling axis and potential clinical partners for pathway inhibition are ongoing in our laboratories. This approach is supported by the planned phase 1 clinical trial for APX3330 scheduled to begin in mid-2016.
Citation Format: Derek P. Logsdon, Michelle Grimard, Safi Shahda, Nicholas Zyromski, Ernestina Schipani, Fabrizio Carta, Claudiu T. Supuran, Murray Korc, Mircea Ivan, Mark R. Kelley, Melissa L. Fishel.{Authors}. Regulation of HIF1α under hypoxia by APE1/Ref-1 impacts CA9 expression: Dual-targeting in patient-derived 3D pancreatic cancer models. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr B51.