Redox Signaling Activity Research Articles

Drug Inhibition of Redox Factor-1 Restores Hypoxia-Driven Changes in Tuberous Sclerosis Complex 2 Deficient Cells.

Therapies with the mechanistic target of rapamycin complex 1 (mTORC1) inhibitors are not fully curative for tuberous sclerosis complex (TSC) patients. Here, we propose that some mTORC1-independent disease facets of TSC involve signaling through redox factor-1 (Ref-1). Ref-1 possesses a redox signaling activity that stimulates the transcriptional activity of STAT3, NF-kB, and HIF-1α, which are involved in inflammation, proliferation, angiogenesis, and hypoxia, respectively. Here, we demonstrate that redox signaling through Ref-1 contributes to metabolic transformation and tumor growth in TSC cell model systems. In TSC2-deficient cells, the clinically viable Ref-1 inhibitor APX3330 was effective at blocking the hyperactivity of STAT3, NF-kB, and HIF-1α. While Ref-1 inhibitors do not inhibit mTORC1, they potently block cell invasion and vasculature mimicry. Of interest, we show that cell invasion and vasculature mimicry linked to Ref-1 redox signaling are not blocked by mTORC1 inhibitors. Metabolic profiling revealed that Ref-1 inhibitors alter metabolites associated with the glutathione antioxidant pathway as well as metabolites that are heavily dysregulated in TSC2-deficient cells involved in redox homeostasis. Therefore, this work presents Ref-1 and associated redox-regulated transcription factors such as STAT3, NF-kB, and HIF-1α as potential therapeutic targets to treat TSC, where targeting these components would likely have additional benefits compared to using mTORC1 inhibitors alone.

Abstract 1452: Deciphering mechanisms of Ref-1 signaling and its inhibition in aggressive tumor-stroma PDAC models

Abstract Targeted therapy for cancer using small molecules has progressed exponentially, but agents that can affect cancer cells rather than non-tumorigenic cells are crucial to avoid pernicious side effects. AP endonuclease-1/Redox factor-1 (APE1/Ref-1 or Ref-1) is a multifunctional protein with DNA repair activity and redox signaling activity as major functions. The DNA repair function is indispensable for cell survival. Its role as a redox factor that stimulates the DNA binding activity of numerous transcription factors (TFs) such as HIF-1α, NFΚB, STAT3, and AP-1 tends to be dysregulated in cancer cells. Ref-1 is overexpressed in many cancers including, aggressive and invasive pancreatic ductal adenocarcinoma (PDAC), which is the 3rd leading cause of death in U.S. The dense stroma of PDAC primarily constituting cancer-associated fibroblasts (CAFs) makes it highly hypoxic, nutrient poor, and drug resistant. Current treatment regimens offer only modest improvement in survival. Targeting the PDAC stroma or ECM did not result in clinical benefit thus far presumably due to complex interactions between PDAC and its TME. Better treatments options that selectively target the tumor within its protective stroma are hence needed. Our group was able to generate a Ref-1 redox inhibitor, APX3330, that completed Phase I clinical trial (NCT03375086) with a good safety profile, verified target engagement and a recommended phase II dose. However, its potency in preclinical models is in the high µM range indicating the need for more potent second-generation inhibitors. Based on initial SAR studies, we selected 13 second-generation compounds from > 350 that were further characterized for positive properties including increased efficacy for cell killing, mouse and human S9 fraction metabolic stability, plasma half-life, and in silico ADMET properties. Target engagement studies involving blockade of TF activity via luciferase assay and EMSA as well as validation of direct interaction of these inhibitors with Ref-1 using thermal shift assay are ongoing. In two 3D co-culture models, second-generation Ref-1 redox analogs suppressed tumor survival significantly while sparing the CAFs. These findings were confirmed in vivo with xenografts co-implanted with tumor and CAF lines. Gene expression and mitochondria functional data revealed Ref-1's control of TCA cycle in tumor cells, but not in CAFs. To confirm and compare the effects of Ref-1 redox signaling inhibitors in cells, CRISPR editing was used to generate Ref-1 redox deficient cell lines (Ref-1C65A). As confirmation of significantly reduced Ref-1 redox activity, PDAC cells expressing Ref-1C65A did not induce hypoxia marker (CA9) under hypoxia, similar to when Ref-1 was knocked down or blocked via small molecule inhibitor. Ref-1 redox signaling and validation for selective killing of PDAC cells leaving the stomal cells undisturbed paves the way to improved PDAC treatment. Citation Format: Silpa Gampala, Nayela Chowdhury, Olivia Babb, Rachel A. Caston, Randall S. Wireman, Hye-ran Moon, George Sandusky, Emily Hulsey, Bumsoo Han, Millie M. Georgiadis, Sara K. Quinney, Andi R. Masters, James H. Wikel, Mark R. Kelley, Melissa L. Fishel. Deciphering mechanisms of Ref-1 signaling and its inhibition in aggressive tumor-stroma PDAC models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1452.

Effect of combined exposure to environmental aliphatic electrophiles from plants on Keap1/Nrf2 activation and cytotoxicity in HepG2 cells: A model of an electrophile exposome

Electrophiles, ubiquitously found in the environment, modify thiol groups of sensor proteins, leading to activation of redox signaling pathways such as the Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor E2 related factor 2 (Nrf2) pathway. Nrf2 activation by exposure to single electrophiles has been established. However, the effect of exposure to a combination of electrophiles on Nrf2 activation has not been well evaluated. The current study examined whether combined exposure to electrophiles enhances the modification of thiol groups and Keap1/Nrf2 activation in HepG2 cells. Six electrophiles [1,2-naphthoquinone (1,2-NQ), 1,4-NQ, 1,4-benzoquinone, (E)-2-hexenal (hexenal), (E)-2-decenal, and (E)-2-butenal] were tested for S-modification of albumin in vitro and for cytotoxicity to HepG2 cells. Interestingly, a mixture of the electrophiles enhanced S-modification of albumin and cytotoxicity compared with exposure to each electrophile separately. Herein, we focused on 1,2-NQ, 1,4-NQ, and hexenal to clarify the combined effect of electrophiles on Keap1/Nrf2 activation in HepG2 cells. A concentration addition model revealed that 1,2-NQ and/or 1,4-NQ additively enhanced hexenal-mediated S-modification of GSH in vitro, whereas the cytotoxicity of hexenal was synergistically increased by simultaneous exposure of HepG2 cells to the NQs. Furthermore, an NQ cocktail (2.5 μM each) that does not activate Nrf2 enhanced hexenal-mediated Nrf2 activation. These results suggest that combined exposure to electrophiles at low concentrations induces stronger activation of redox signaling compared with exposure to each electrophile alone and worsens their cytotoxicity.

Activation of PTP1B/EGFR signaling and cytotoxicity during combined exposure to ambient electrophiles in A431 cells.

Chemical modification of the thiol group on protein tyrosine phosphatase (PTP) 1B triggers an activation of epidermal growth factor receptor (EGFR) signaling that is mimicked by environmental electrophiles through S-modification of PTP1B. While activation of PTP1B/EGFR by a single exposure to an electrophile has been established, the effects of combined exposure to electrophiles are unknown. Here, we examined the activation of EGFR signaling by combined exposure to ambient electrophiles in human epithelial carcinoma A431 cells. Simultaneous exposure to 1,2- and 1,4-naphthoquinone (NQ) augmented the S-modification of endogenous and recombinant human PTP1B (hPTP1B). Combined exposure of hPTP1B or A431 cells to 1,2- and 1,4-NQ escalated the inactivation of PTP compared with individual exposure. Phosphorylation of EGFR and its downstream kinase extracellular signal-regulated kinase (ERK) 1/2 by 1,2-NQ exposure was facilitated by simultaneous exposure to 1,2-NQ with 10 µM 1,4-NQ. An EGFR inhibitor diminished the phosphorylation of ERK1/2, indicating that ERK was phosphorylated following EGFR activation by the NQ cocktail. The combined exposure to NQs also accelerated cell death in A431 cells compared with each NQ alone. While no EGFR/ERK activation was seen following 1,4-benzoquinone (BQ) treatment, exposure to 1,4-NQ in the presence of 1,4-BQ increased 1,4-NQ-mediated activation of EGFR. This suggests that the enhancement of 1,4-NQ-dependent EGFR activation by 1,4-BQ is caused by a different mechanism than 1,2-NQ with 1,4-NQ. These results suggest that combined exposure to ambient electrophiles, even at low concentrations, can induce stronger activation of redox signaling than individual exposure. Our findings indicate that combining different electrophiles may produce unexpected effects.

Redox Signaling and Reactive Sulfur Species to Regulate Electrophilic Stress

Humans are exposed to various xenobiotic electrophiles on a daily basis. Electrophiles form covalent adducts with nucleophilic residues of proteins. Redox signaling, which consists of effector molecules (e.g., kinases and transcription factors) and redox sensor proteins with low pKa cysteine residues, is involved in cell survival, cell proliferation, quality control of cellular proteins and oxidative stress response. Herein, we showed that at a low dose, xenobiotic electrophiles selectively modified redox sensor proteins through covalent modification of their reactive thiols, resulting in activation of a variety of redox signaling pathways. However, increasing the dose of xenobiotic electrophiles caused non-selective and extensive modification of cellular proteins involved in toxicity. Of interest, reactive sulfur species (RSS), such as hydrogen sulfide (H2S), cysteine persulfide (CysSSH), glutathione persulfide (GSSH) and even synthetic polysulfide (e.g., Na2S4), readily captured xenobiotic electrophiles, forming their sulfur adducts, which was associated with inactivation of the electrophiles. Our findings suggest that an adaptive response through redox signaling activation and RSS-mediated electrophile capturing is involved in the regulation of electrophilic stress.

Cross-Talk between NADPH Oxidase and Mitochondria: Role in ROS Signaling and Angiogenesis.

Angiogenesis, a new vessel formation from the pre-existing ones, is essential for embryonic development, wound repair and treatment of ischemic heart and limb diseases. However, dysregulated angiogenesis contributes to various pathologies such as diabetic retinopathy, atherosclerosis and cancer. Reactive oxygen species (ROS) derived from NADPH oxidase (NOX) as well as mitochondria play an important role in promoting the angiogenic switch from quiescent endothelial cells (ECs). However, how highly diffusible ROS produced from different sources and location can communicate with each other to regulate angiogenesis remains unclear. To detect a localized ROS signal in distinct subcellular compartments in real time in situ, compartment-specific genetically encoded redox-sensitive fluorescence biosensors have been developed. Recently, the intercellular communication, “cross-talk”, between ROS derived from NOX and mitochondria, termed “ROS-induced ROS release”, has been proposed as a mechanism for ROS amplification at distinct subcellular compartments, which are essential for activation of redox signaling. This “ROS-induced ROS release” may represent a feed-forward mechanism of localized ROS production to maintain sustained signaling, which can be targeted under pathological conditions with oxidative stress or enhanced to promote therapeutic angiogenesis. In this review, we summarize the recent knowledge regarding the role of the cross-talk between NOX and mitochondria organizing the sustained ROS signaling involved in VEGF signaling, neovascularization and tissue repair.

Abstract PR01: A phase I study targeting the APE1/Ref-1 DNA repair-redox signaling protein with the APX3330 inhibitor

Abstract APX3330 is an orally administered anti-cancer small molecule that targets the APE1/Ref-1 protein (apurinic endonuclease 1 / redox effector factor 1). APE1/Ref-1 is both a DNA repair and reduction-oxidation (redox) signaling protein that participates in the DNA base excision repair (BER) pathway. Additionally, APE1/Ref-1 regulates the redox status of a number of important transcription factors (TFs) in tumor cells. These include NFkB, STAT3, and HIF-1a. APE1/ref-1 converts these TFs from an off (oxidized) to an on (reduced) state allowing them to bind to the promoter sequence of genes regulating tumor growth, metastasis, metabolism and survival of tumor cells. APX3330 selectively binds to APE1/Ref-1, specifically inhibiting its redox signaling activity. APE1/Ref-1 also plays a key role in a variety of inflammatory disorders beyond cancers including diabetic macular edema, inflammatory bowel disorders and others. Data will be presented for study NCT03375086 evaluating APX3330 in patients with incurable malignancies. Eligibility required adequate organ function, PS 0-2 and tumors not amenable to curative therapy. Primary and secondary objectives included determining the recommended phase 2 dose (RP2D), the safety and PK/PD profiles of APX3330 and reporting any RECIST anti-tumor activity. Patients received APX3330 b.i.d., in 21-day cycles. APX3330 was well tolerated at dose levels from 240-600 mg/d. The most frequent treatment-related adverse events (all grades) included G1 nausea (16%) and fatigue (16%). Six subjects had disease stabilization for > 4 cycles, and of these, four subjects with the following diagnosis, RECIST response and days on study included: colorectal cancer, PR (partial response), 357 days; endometrial, SD (stable disease), 421days; melanoma, SD, 337 days; prostate, SD, 252 days. All study objectives were completed and determined that APX3330 is safe for chronic dosing from 240 up to and including 600 mg/day. It provides clinical benefit to patients with a variety of tumor types (e.g., endometrial, colorectal, prostate and melanoma cancer). Pharmacodynamic (PD) studies from patient biopsy evaluation indicates APX3330-mediated effect upon cancer cells, including decrease in transcription factors activity for those regulated by the APE1/Ref-1 protein. Differentially expressed proteins (DEPs) were analyzed comparing pre-treatment and on-treatment tumor biopsies. Specifically studied were NFkB, HIFa and STAT3 genes downstream of these three TFs. Circulating tumor cell (CTC) analysis was performed on sixteen patients who had baseline and on-treatment samples that were evaluated longitudinally. Of these, 7/16 (44%) of patients showed a reduction in CTCs after initiation of treatment with APX3330. APE1/Ref-1 serum levels were determined to be elevated in aggressive tumors and were reduced following treatment with APX3330 in SD patients (past four treatment cycles). Pharmacokinetic (PK) data indicate confirmation of pre-clinical data. Conclusions: APX3330 is an orally administered inhibitor of APE1/Ref-1 specifically targeting the redox signaling function of this protein. This phase I study identified 600 mg PO daily as the RP2D for further development. RECIST evaluation identified signs of clinical activity in this un-selected population of patients with advanced cancer. PD analyses (proteomics, CTC, serum) indicate APX3330 mediated targeting of the APE1/Ref-1 protein. Citation Format: Mark R. Kelley, Safi Shahda, Nehal J. Lakhani, Bert O'Neil, Lincy Chu, Amanda K. Anderson, Jun Wan, Amber L. Mosley, Hao Liu, Richard A. Messmann. A phase I study targeting the APE1/Ref-1 DNA repair-redox signaling protein with the APX3330 inhibitor [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PR01. doi:10.1158/1535-7163.TARG-19-PR01

Blocking HIF signaling via novel inhibitors of CA9 and APE1/Ref-1 dramatically affects pancreatic cancer cell survival

Pancreatic ductal adenocarcinoma (PDAC) has reactive stroma that promotes tumor signaling, fibrosis, inflammation, and hypoxia, which activates HIF-1α to increase tumor cell metastasis and therapeutic resistance. Carbonic anhydrase IX (CA9) stabilizes intracellular pH following induction by HIF-1α. Redox effector factor-1 (APE1/Ref-1) is a multifunctional protein with redox signaling activity that converts certain oxidized transcription factors to a reduced state, enabling them to upregulate tumor-promoting genes. Our studies evaluate PDAC hypoxia responses and APE1/Ref-1 redox signaling contributions to HIF-1α-mediated CA9 transcription. Our previous studies implicated this pathway in PDAC cell survival under hypoxia. We expand those studies, comparing drug responses using patient-derived PDAC cells displaying differential hypoxic responses in 3D spheroid tumor-stroma models to characterize second generation APE1/Ref-1 redox signaling and CA9 inhibitors. Our data demonstrates that HIF-1α-mediated CA9 induction differs between patient-derived PDAC cells and that APE1/Ref-1 redox inhibition attenuates this induction by decreasing hypoxia-induced HIF-1 DNA binding. Dual-targeting of APE1/Ref-1 and CA9 in 3D spheroids demonstrated that this combination effectively kills PDAC tumor cells displaying drastically different levels of CA9. New APE1/Ref-1 and CA9 inhibitors were significantly more potent alone and in combination, highlighting the potential of combination therapy targeting the APE1-Ref-1 signaling axis with significant clinical potential.

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

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.

Superoxide‐Dependent Redox Signaling in the Supraoptic Nucleus Is Associated with the Neuroendocrine Response to Water and Electrolyte Imbalance

In models of altered osmolality and water balance, including two‐day salt loading and water deprivation, previous studies have suggested that intracellular redox signaling in the supraoptic nucleus (SON) contributes to the neuroendocrine response. However, the precise redox‐dependent signaling pathways and reactive oxygen species (ROS) involved in this response remain unclear. Herein, we tested the hypothesis that water deprivation increases specific ROS, particularly superoxide, and stimulates the redox‐sensitive transcription factor, nuclear factor erythroid 2‐related factor 2 (Nrf2) in the SON and that these responses are dependent on the duration of water restriction. To test this hypothesis, we used rodent models deprived of water for 24 and 48 hours (24h WD and 48h WD, respectively). We observed an increase in Nrf2 mRNA in the SON of rodents subjected to 24h WD (Control: 0.84 ± 0.08 vs 24h WD: 1.16 ± 0.09 arbitrary units) suggesting the activation of redox signaling during dehydration. In addition, at 48h WD, levels of hemeoxygenase‐1 (HO‐1), a Nrf2 target gene, were elevated in the SON (Control: 0.98 ± 0.1 vs 48h WD: 1.46 ± 0.1 arbitrary units). HO‐1 activity was also increased at both time points of water restriction (F2,34= 7.718, p < 0.05). To determine if levels of specific ROS are altered during water deprivation, we used the electron paramagnetic resonance (EPR) spectroscopy. After 24h or 48h of water deprivation in C57BL/6 mice, fresh non‐fixed brains were collected and the SON was harvested via micropunch. Superoxide levels were measured in the SON micropunches by EPR. We also measured plasma osmolality and volume. We observed a dehydration period‐dependent increase in superoxide levels (reported at EPR spectra amplitude in arbitrary units) in the SON (Control: 7150 ± 3856; 24h WD: 14240 ± 9931; 48h WD: 15795 ± 7400 arbitrary units/mg of tissue) along with plasma osmolality (Control: 317 ± 2.0; 24h WD: 326.8 ± 1.3; 48h WD: 330.7 ± 1.3 mOsm/kg H2O); protein (Control: 3.56 ± 0.16; 24h WD: 4.36 ± 0.16; 48h WD: 3.94 ± 0.14 g/dL plasma); and hematocrit (Control: 35.8 ± 5.1; 24h WD: 42.4 ± 0.9; 48h WD: 45.7 ± 0.9 %). Together, these data suggest that the neuroendocrine system is associated with superoxide‐dependent redox signaling and activation of Nrf2 in the SON to cope with dehydration.Support or Funding InformationFAPESPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Exposure to Electrophiles Impairs Reactive Persulfide-Dependent Redox Signaling in Neuronal Cells.

Electrophiles such as methylmercury (MeHg) affect cellular functions by covalent modification with endogenous thiols. Reactive persulfide species were recently reported to mediate antioxidant responses and redox signaling because of their strong nucleophilicity. In this study, we used MeHg as an environmental electrophile and found that exposure of cells to the exogenous electrophile elevated intracellular concentrations of the endogenous electrophilic molecule 8-nitroguanosine 3',5'-cyclic monophosphate (8-nitro-cGMP), accompanied by depletion of reactive persulfide species and 8-SH-cGMP which is a metabolite of 8-nitro-cGMP. Exposure to MeHg also induced S-guanylation and activation of H-Ras followed by injury to cerebellar granule neurons. The electrophile-induced activation of redox signaling and the consequent cell damage were attenuated by pretreatment with a reactive persulfide species donor. In conclusion, exogenous electrophiles such as MeHg with strong electrophilicity impair the redox signaling regulatory mechanism, particularly of intracellular reactive persulfide species and therefore lead to cellular pathogenesis. Our results suggest that reactive persulfide species may be potential therapeutic targets for attenuating cell injury by electrophiles.

Abstract B88: Clinical trials targeting APE1/Ref-1 in pancreatic cancer with APX3330

Abstract APE1/Ref-1 function is upstream of numerous transcription factors and impinges on several different pathways through its transcriptional regulatory function. These include HIF1, STAT3, NFkB, AP-1 and NRF2. These signaling pathways are known oncogenic drivers in many malignancies but particularly in pancreatic cancer (Zou et al. 2009; Fishel et al. 2011; Cardoso et al. 2012). APX3330 (formerly E3330) selectively and directly binds to APE1/Ref-1 thus inhibiting its APE1/Ref-1 redox signaling activity. Importantly, while APX3330 blocks APE1/Ref-1’s redox function, it has no effect on APE1/Ref-1 endonuclease DNA repair activity. APX3330 has been shown in multiple in vivo models of pancreatic cancer to be effective in reducing tumor volume and metastases as both a single agent and in combination with gemcitabine. Additional preclinical data will be presented. APX3330 was recently shown to augment 5-flurouracil (5-FU) in a colon cancer xenograft model as well as increased cytotoxicity in colon cancer stem cells (Lou et al. 2014). The safety and oral dosing administration schedule of APX3330 has been previously established through a prior development program that evaluated the preclinical toxicology and phase 1 and phase 2 safety and clinical profile in more than 400 non-cancer patients with active hepatitis C and abnormal liver function tests. These studies demonstrated an apparent lack of significant acute toxicity on neurologic, cardiovascular, or pulmonary function at doses up to 240 mg/day. Gastrointestinal symptoms and symptoms related to skin rash or irritation were identified as adverse events of particular interest. A Phase 1 dose-escalation study of APX3330 in patients with advanced solid tumors and a dose-expansion cohort of patients with advanced ductal adenocarcinoma of the pancreas (PDAC) is planned. In Phase 1a, we will determine the maximum-tolerated-dose and recommended Phase 2 dose for APX3330 as a single agent in patients with advanced solid tumors (~6 to 30 patients). Phase 1b, will be to characterize preliminary antitumor activity of APX3330 in a PDAC-specific dose expansion cohort and exploratory biomarker endpoints (~10 PDAC patients). The exploratory/correlative objectives for the study include analysis of peripheral blood mononuclear cells (PBMCs) for evidence of APX3330-mediated pharmacodynamic effect. Analysis will include assays for biomarkers previously identified from preclinical studies of APX3330 including TNFα; CAIX, and IL-6. In addition, evaluation of patients’ plasma CRP levels will be performed, as this is a marker of inflammation and may identify a patient population that would respond more favorably to APX3330. Archival tumor tissue samples will be analyzed for CDA polymorphisms, CXCL12 and CXCR4. Citation Format: Mark R. Kelley, Safi Shahda, Bert H. O’Neil, Homer Pearce, Jackie Walling.{Authors}. Clinical trials targeting APE1/Ref-1 in pancreatic cancer with APX3330. [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 B88.

Regulation of HIF1α under Hypoxia by APE1/Ref-1 Impacts CA9 Expression: Dual Targeting in Patient-Derived 3D Pancreatic Cancer Models.

Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer-related mortality in the United States. Aggressive treatment regimens have not changed the disease course, and the median survival has just recently reached a year. Several mechanisms are proposed to play a role in PDAC therapeutic resistance, including hypoxia, which creates a more aggressive phenotype with increased metastatic potential and impaired therapeutic efficacy. AP Endonuclease-1/Redox Effector Factor 1 (APE1/Ref-1) is a multifunctional protein possessing a DNA repair function in base excision repair and the ability to reduce 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. Here, we explore the mechanisms underlying PDAC cell responses to hypoxia and modulation of APE1/Ref-1 redox signaling activity, which regulates the transcriptional activation of hypoxia-inducible factor 1 alpha (HIF1α). Carbonic anhydrase IX (CA9) is regulated by HIF1α and functions as a part of the cellular response to hypoxia to regulate intracellular pH, thereby promoting cell survival. We hypothesized that modulating APE1/Ref-1 function will block activation of downstream transcription factors, STAT3 and HIF1α, interfering with the hypoxia-induced gene expression. We demonstrate APE1/Ref-1 inhibition in patient-derived and established PDAC cells results in decreased HIF1α-mediated induction of CA9. Furthermore, an ex vivo three-dimensional tumor coculture model demonstrates dramatic enhancement of APE1/Ref-1-induced cell killing upon dual targeting of APE1/Ref-1 and CA9. Both APE1/Ref-1 and CA9 are under clinical development; therefore, these studies have the potential to direct novel PDAC therapeutic treatment. Mol Cancer Ther; 15(11); 2722-32. ©2016 AACR.

Abstract 4740: Targeting Ref-1/APE1 pathway inhibition in pancreatic cancer using APX3330 for clinical trials

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related mortality in the US. Most patients present with advanced disease and ∼95% die within five years, with most surviving less than six months. Targeted therapies including Gemcitabine (GemzarTM), FOLFIRINOX (5-FU/leucovorin/irinotecan/oxaliplatin), and sustained release, nab-paclitaxel (AbraxaneTM) offer modest improvement in survival, albeit at an increase in side effects and unwanted toxicities. Data is presented on redox factor-1 (Ref-1) and specific Ref-1 inhibitor APX3330. Ref-1 regulates multiple transcription factors involved in pancreatic cancer survival signaling due to its redox-coactivator activity on HIF-1α, NFkB, NRF2 and STAT3. High expression levels of Ref-1 indicate decreased survival in PDAC as well as other cancers. APX3330 has been shown in multiple in vitro and in vivo pancreatic cancer models to be effective in reducing tumor growth and metastases as a single agent. The mechanism of action has been extensively investigated and characterized for its specific activity on Ref-1, as well as its preclinical PK/PD and ADME. The safety and dose administration of APX3330 have been established by Eisai pharmaceutical company through a previous development program including toxicology, phase I, and phase II clinical evaluation in non-cancer patients in Japan. We have partnered with ApeX Therapeutics to develop APX3330 for cancer treatment (phase I trial anticipated start date early 2016). While developing APX3330 for single agent use, we studied interactions of Ref-1, APX3330, convergent pathways; i.e. HIF-1 α and STAT3, and downstream targets like CAIX. Initially, we performed in vivo studies demonstrating single and combination effects of APX3330 with Gemcitabine (Gem) showing significantly decreased tumor volume in the APX3330 and Gem combination treatments compared to the single-agents alone. We also tested single and combination studies of APX3330 in an ex vivo 3-D tumor-stroma model system using patient derived tumor cells along with patient derived cancer-associated fibroblasts (CAFs). We used the CAIX inhibitor SLC-0111 and JAK2 inhibitor, Ruxolitinib; both agents in clinical trials. In our ex vivo 3D co-culture system, APX3330 decreases the tumor area and intensity in a dose-dependent manner. The combination of APX3330 with Gem demonstrated an additive enhancement effect in the tumor. Blocking both Ref-1 redox-signaling activity with APX3330 and CAIX activity via SLC-0111 demonstrated enhanced tumor killing in our models. APX3330 along with Ruxolitinib also demonstrated enhanced tumor killing. These data demonstrate APX3330 single agent efficacy in our 3D patient PDAC model and enhanced tumor killing when pathways regulated by Ref-1, HIF-1 α and STAT3 are blocked. Additional drug combinations focused on pathways that are dependent on Ref-1 signaling will also be presented. Citation Format: Melissa L. Fishel, Derek P. Logsdon, Michelle L. Grimard, Claudiu T. Supuran, Nicholas Zyromski, Mircea Ivan, Mark R. Kelley, Fenil Shah. Targeting Ref-1/APE1 pathway inhibition in pancreatic cancer using APX3330 for clinical trials. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4740.

Microtubule stability is affected by oxidative stress and contributes to lung injury and barrier dysfunction

Pathologic activation of redox signaling by inflammatory agents causes endothelial cell (EC) activation and barrier dysfunction. Microtubules (MT) play an important role in the regulation of vascular endothelial permeability, but their impact in modulation of inflammation and EC barrier is not well understood. This study examined effects of LPS‐induced oxidative stress on MT dynamics and involvement of MT in the LPS‐induced mechanisms of Rho GTPase activation, EC permeability and lung injury. LPS treatment of pulmonary vascular EC induced elevation of reactive oxygen species (ROS) and oxidative stress leading to cytoskeletal remodeling, EC hyper‐permeability, increased neutrophil adhesion and activation of inflammatory signaling. LPS also triggered ROS‐dependent disassembly of the microtubule network leading to activation of MT‐dependent signaling. Stabilization of MT or inhibition of MT‐associated Rho‐specific GEF activity suppressed LPS‐induced EC barrier dysfunction in vitro and attenuated vascular leak and lung inflammation in vivo. These studies demonstrate for the first time the mechanism of ROS‐induced Rho activation via destabilization of MT and GEF‐H1‐dependent activation of Rho signaling leading to pulmonary EC barrier dysfunction and exacerbation of LPS‐induced inflammation.Funding: NHLBI HL089257, HL107920

Acute ethanol intake induces superoxide anion generation and mitogen-activated protein kinase phosphorylation in rat aorta: A role for angiotensin type 1 receptor

Ethanol intake is associated with increase in blood pressure, through unknown mechanisms. We hypothesized that acute ethanol intake enhances vascular oxidative stress and induces vascular dysfunction through renin–angiotensin system (RAS) activation. Ethanol (1g/kg; p.o. gavage) effects were assessed within 30min in male Wistar rats. The transient decrease in blood pressure induced by ethanol was not affected by the previous administration of losartan (10mg/kg; p.o. gavage), a selective AT1 receptor antagonist. Acute ethanol intake increased plasma renin activity (PRA), angiotensin converting enzyme (ACE) activity, plasma angiotensin I (ANG I) and angiotensin II (ANG II) levels. Ethanol induced systemic and vascular oxidative stress, evidenced by increased plasma thiobarbituric acid-reacting substances (TBARS) levels, NAD(P)H oxidase‐mediated vascular generation of superoxide anion and p47phox translocation (cytosol to membrane). These effects were prevented by losartan. Isolated aortas from ethanol-treated rats displayed increased p38MAPK and SAPK/JNK phosphorylation. Losartan inhibited ethanol-induced increase in the phosphorylation of these kinases. Ethanol intake decreased acetylcholine-induced relaxation and increased phenylephrine-induced contraction in endothelium-intact aortas. Ethanol significantly decreased plasma and aortic nitrate levels. These changes in vascular reactivity and in the end product of endogenous nitric oxide metabolism were not affected by losartan. Our study provides novel evidence that acute ethanol intake stimulates RAS activity and induces vascular oxidative stress and redox-signaling activation through AT1-dependent mechanisms. These findings highlight the importance of RAS in acute ethanol-induced oxidative damage.

Oxidative Stress Contributes to Lung Injury and Barrier Dysfunction via Microtubule Destabilization

Oxidative stress is an important part of host innate immune response to foreign pathogens, such as bacterial LPS, but excessive activation of redox signaling may lead to pathologic endothelial cell (EC) activation and barrier dysfunction. Microtubules (MTs) play an important role in agonist-induced regulation of vascular endothelial permeability, but their impact in modulation of inflammation and EC barrier has not been yet investigated. This study examined the effects of LPS-induced oxidative stress on MT dynamics and the involvement of MTs in the LPS-induced mechanisms of Rho activation, EC permeability, and lung injury. LPS treatment of pulmonary vascular EC induced elevation of reactive oxygen species (ROS) and caused oxidative stress associated with EC hyperpermeability, cytoskeletal remodeling, and formation of paracellular gaps, as well as activation of Rho, p38 stress kinase, and NF-κB signaling, the hallmarks of endothelial barrier dysfunction. LPS also triggered ROS-dependent disassembly of the MT network, leading to activation of MT-dependent signaling. Stabilization of MTs with epothilone B, or inhibition of MT-associated guanine nucleotide exchange factor (GEF)-H1 activity by silencing RNA-mediated knockdown, suppressed LPS-induced EC barrier dysfunction in vitro, and attenuated vascular leak and lung inflammation in vivo. LPS disruptive effects were linked to activation of Rho signaling caused by LPS-induced MT disassembly and release of Rho-specific GEF-H1 from MTs. These studies demonstrate, for the first time, the mechanism of ROS-induced Rho activation via destabilization of MTs and GEF-H1-dependent activation of Rho signaling, leading to pulmonary EC barrier dysfunction and exacerbation of LPS-induced inflammation.

Antitumor Indolequinones Induced Apoptosis in Human Pancreatic Cancer Cells via Inhibition of Thioredoxin Reductase and Activation of Redox Signaling

Indolequinones (IQs) were developed as potential antitumor agents against human pancreatic cancer. IQs exhibited potent antitumor activity against the human pancreatic cancer cell line MIA PaCa-2 with growth inhibitory IC(50) values in the low nanomolar range. IQs were found to induce time- and concentration-dependent apoptosis and to be potent inhibitors of thioredoxin reductase 1 (TR1) in MIA PaCa-2 cells at concentrations equivalent to those inducing growth-inhibitory effects. The mechanism of inhibition of TR1 by the IQs was studied in detail in cell-free systems using purified enzyme. The C-terminal selenocysteine of TR1 was characterized as the primary adduction site of the IQ-derived reactive iminium using liquid chromatography-tandem mass spectrometry analysis. Inhibition of TR1 by IQs in MIA PaCa-2 cells resulted in a shift of thioredoxin-1 redox state to the oxidized form and activation of the p38/c-Jun NH(2)-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) signaling pathway. Oxidized thioredoxin is known to activate apoptosis signal-regulating kinase 1, an upstream activator of p38/JNK in the MAPK signaling cascade and this was confirmed in our study providing a potential mechanism for IQ-induced apoptosis. These data describe the redox and signaling events involved in the mechanism of growth inhibition induced by novel inhibitors of TR1 in human pancreatic cancer cells.

Functional Analysis of Novel Analogues of E3330 That Block the Redox Signaling Activity of the Multifunctional AP Endonuclease/Redox Signaling Enzyme APE1/Ref-1

APE1 is a multifunctional protein possessing DNA repair and redox activation of transcription factors. Blocking these functions leads to apoptosis, antiangiogenesis, cell-growth inhibition, and other effects, depending on which function is blocked. Because a selective inhibitor of the APE redox function has potential as a novel anticancer therapeutic, new analogues of E3330 were synthesized. Mass spectrometry was used to characterize the interactions of the analogues (RN8-51, 10-52, and 7-60) with APE1. RN10-52 and RN7-60 were found to react rapidly with APE1, forming covalent adducts, whereas RN8-51 reacted reversibly. Median inhibitory concentration (IC(50) values of all three compounds were significantly lower than that of E3330. EMSA, transactivation assays, and endothelial tube growth-inhibition analysis demonstrated the specificity of E3330 and its analogues in blocking the APE1 redox function and demonstrated that the analogues had up to a sixfold greater effect than did E3330. Studies using cancer cell lines demonstrated that E3330 and one analogue, RN8-51, decreased the cell line growth with little apoptosis, whereas the third, RN7-60, caused a dramatic effect. RN8-51 shows particular promise for further anticancer therapeutic development. This progress in synthesizing and isolating biologically active novel E3330 analogues that effectively inhibit the APE1 redox function validates the utility of further translational anticancer therapeutic development.

Redox Buffer Capacity of the Cell: Theoretical and Experimental Approach

Reactive oxygen species (ROS) are involved in a variety of biological phenomena, such as mutation, carcinogenesis, inflammation, aging, development, and signal transduction. Intracellular generation of ROS might lead to the activation of redox signaling or oxidative stress. Nonetheless, it is difficult to estimate whether ROS-induced intracellular events are beneficial or deleterious to the cell. The quantitative basis of changes in the intracellular redox state of cells is not well-defined, thus leading to the dilemma that redox changes induced by oxidants in distinct cell types cannot be predicted. To overcome this limitation this study undertakes to analyze on a theoretical as well as on an experimental basis the intracellular redox state changes occurring inside cells upon addition of oxidants or reductants. 2,7-Dichlorodihydrofluorescein (H(2)DCF) was used to characterize the redox buffer capacity in erythrocytes. It was shown that the redox buffer capacity of erythrocytes in the relation to peroxynitrite (ONOO(-)) is 2.1times lower than the redox buffer capacity of erythrocytes in the relation to hydrogen peroxide (H(2)O(2)). The feasibility of redox buffer capacity assessment as an innovative tool for investigation and description of redox signaling events in cells is discussed.