Indiana University-Purdue University Indianapolis (IUPUI) / Pancreatic ductal adenocarcinoma (PDAC) is an extremely deadly disease characterized by aggressive metastasis and therapeutic resistance. Reactive stroma in pancreatic tumors contributes to tumor signaling, 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 multi-functional protein with two major activities: endonuclease activity in DNA base excision repair and a redox signaling activity that reduces oxidized transcription factors, enabling them to bind target sequences in DNA. APE1/Ref-1 is a central node in redox signaling, contributing to the activation of transcription factors involved in tumor survival, growth, and hypoxia signaling. This work evaluates the mechanisms underlying PDAC cell responses to hypoxia and APE1/Ref-1 redox signaling control of hypoxia inducible factor 1 alpha (HIF1a), 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 results in enhanced PDAC cell killing under hypoxic conditions. We found that HIF1a-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. Additionally, dual-targeting of APE1/Ref-1 redox signaling activity and CA9 activity results in enhanced 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. Further experiments characterized novel analogs of clinically relevant drugs targeting the key enzymes in this pathway, resulting in improved potency. These results underscore the notion that combination therapy is essential and demonstrate the potential clinical utility of blocking APE1/Ref-1 and CA9 function for novel PDAC therapeutic treatment.

Apurinic/apyrimidinic endonuclease 1 (APE1) is a key DNA repair factor involved in the DNA base excision repair (BER) pathway that is required for the maintenance of genome stability. In this pathway, APE1 cleaves DNA at an abasic site to generate a DNA single strand break, allowing for repair completion by a DNA polymerase and a DNA ligase. High levels of APE1 have been observed in multiple cancer types however it is not understood if this contributes to cancer onset and development. What is known is that these cancers tend to display increased resistance to DNA damaging treatments and APE1 is therefore considered a key target for inhibition in the treatment of APE1-overexpressing cancers. Considering the relevance of modulating APE1 levels in disease and cancer treatment, very little is known about how cellular APE1 levels are regulated. Our lab has previously shown that the levels of the BER factors Pol β, XRCC1 and DNA Lig IIIα are regulated by ubiquitylation-mediated proteasomal degradation. The aim of this doctoral thesis was therefore to determine if ubiquitylation also regulates APE1 stability in cells. I present evidence that APE1 is ubiquitylated in cells and have identified the UBR3 E3 ligase that is responsible for this activity. Using mouse embryonic fibroblasts generated from Ubr3 knockout mice, I demonstrate that UBR3 regulates APE1 cellular levels. I furthermore show that a loss of cellular UBR3 leads to the formation of DNA double strand breaks and genome instability.