APE1/Ref-1 protein expression affects CA9 mRNA levels in additional PDAC cell lines

APE1 expression correlates with decreased survival in PDAC and CA9 is upregulated in PDAC

Confirmation of HIF-1 deletion in MEF cells

CA9 protein levels are increased under hypoxia, but APE1 levels are not

Figure Legends for Supplemental Figures

APE1/Ref-1 interactions with STAT3 and NFκB are stimulated by IL-6 and TNFα (respectively) under normoxic conditions

Structures of APE1/Ref-1 and CA9 inhibitors

Cell viability analysis used for normalization of pH data

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 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.

Reduction-oxidation factor 1-apurinic/apyrimidinic endonuclease (Ref-1/APE1) is a critical node in tumor cells, both as a redox regulator of transcription factor activation and as part of the DNA damage response. As a redox signaling protein, Ref-1/APE1 enhances the transcriptional activity of STAT3, HIF-1α, nuclear factor kappa B, and other transcription factors to promote growth, migration, and survival in tumor cells as well as inflammation and angiogenesis in the tumor microenvironment. Ref-1/APE1 is activated in a variety of cancers, including prostate, colon, pancreatic, ovarian, lung and leukemias, leading to increased aggressiveness. Transcription factors downstream of Ref-1/APE1 are key contributors to many cancers, and Ref-1/APE1 redox signaling inhibition slows growth and progression in a number of tumor types. Ref-1/APE1 inhibition is also highly effective when paired with other drugs, including standard-of-care therapies and therapies targeting pathways affected by Ref-1/APE1 redox signaling. Additionally, Ref-1/APE1 plays a role in a variety of other indications, such as retinopathy, inflammation, and neuropathy. In this review, we discuss the functional consequences of activation of the Ref-1/APE1 node in cancer and other diseases, as well as potential therapies targeting Ref-1/APE1 and related pathways in relevant diseases. APX3330, a novel oral anticancer agent and the first drug to target Ref-1/APE1 for cancer is entering clinical trials and will be explored in various cancers and other diseases bringing bench discoveries to the clinic.

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.

AbstractPancreatic 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.

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. Treatment with chemotherapy has not changed the natural course of this disease, and just recently, with combination of chemotherapeutic agents, the median survival reached a year. Several mechanisms are proposed to play a role in the aggressive, treatment-resistant phenotype of PDAC, including adaptation to hypoxia, which leads to increased potential for metastasis and impairs the efficacy of chemotherapy and radiotherapy. Hypoxia-Inducible Factor-1α (HIF1α), a major oxygen sensor in cells, is a transcription factor that is rapidly degraded under normoxic conditions but upregulates a number of genes under hypoxic conditions that contribute to survival, metastasis, and angiogenic signaling in the tumor microenvironment. One of the most notable HIF targets is Carbonic Anhydrase IX (CA9), which promotes tumor cell survival and metastasis by maintaining a steady intracellular pH while acidifying the microenvironment, thereby encouraging epithelial-mesenchymal transition and contributing to extracellular matrix degradation. AP Endonuclease1/ Redox Effector Factor 1 (APE1/Ref-1) is a dual function protein that possesses a DNA repair function as well as the ability to reduce transcription factors and enable them to bind to their DNA target sequences. APE1/Ref-1 regulates several transcription factors involved in preventing apoptosis, survival mechanisms, and hypoxia signaling, including HIF-1α. Therefore, we hypothesized that APE1/Ref-1 inhibition impairs HIF-1α-mediated signaling, and this leads to decreased survival and invasion of tumor cells exposed to hypoxic conditions. Methods: We performed co-immunoprecipitation (co-IP) studies to look at the interaction of APE1/Ref-1 with transcriptional targets, HIF-1α, STAT3, and NFκB along with RT-PCR and Western blotting to confirm expression of hypoxia signaling genes. Luciferase reporter assays were used to quantitate transcriptional activation under hypoxia. Boyden chamber was used to look at migration and invasion as well as proliferation based assays following manipulation of APE1/Ref-1 and hypoxia. Results: HIF-1α and STAT3, but not NFκB associate with APE1/Ref-1 under hypoxia. Moreover, we found that knockdown of APE1/Ref-1 protein diminishes HIF-mediated transcription in hypoxia, as shown by analysis of luciferase reporter assays. Next, we showed that, in hypoxia, APE1/Ref-1 inhibition diminishes HIF-1α-induced downstream targets including CA9 and ANGPTL4 further indicating that APE1/Ref-1 redox activity regulates HIF signaling. Importantly, we found that hypoxia, in the presence or absence of APE1/Ref-1, no longer induced CA9 mRNA levels in HIF-deficient MEFs, proving that hypoxia-dependent regulation of CA9 expression is fully mediated by HIF-1α. A blockade of both CA9 activity via small molecule and CA9 transcription via APE1/Ref-1 leads to decreased PDAC cell proliferation under hypoxia. These data indicate that APE1/Ref-1 inhibition interferes with ηψπoξια-mediated signaling and can further sensitize PDAC cells to CA9/12 inhibition even under the conditions of extreme oxygen deprivation. Ongoing experiments will determine the role APE1/Ref-1 plays in the survival and invasion of tumor cells exposed to hypoxia. Citation Format: Derek P. Logsdon, Huiwen Cheng, Meihua Luo, Safi Shahda, Yangyang Hao, Yan Tong, Zhangsheng Yu, Nicholas Zyromski, Ernestina Schipani, Yunlong Liu, Claudiu T. Supuran, Mircea Ivan, Mark R. Kelley, Melissa L. Fishel. Targeting APE1/Ref-1 results in inhibition of hypoxia signaling genes. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B158.

Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1) (henceforth referred to as Ref-1) is a multifunctional protein that in addition to its base excision DNA repair activity exerts redox control of multiple transcription factors, including nuclear factor κ-light chain enhancer of activated B cells (NF-κB), STAT3, activator protein-1 (AP-1), hypoxia-inducible factor-1 (HIF-1), and tumor protein 53 (p53). In recent years, Ref-1 has emerged as a promising therapeutic target in cancer, particularly in pancreatic ductal carcinoma. Although a significant amount of research has centered on Ref-1, no wide-ranging approach had been performed on the effects of Ref-1 inhibition and transcription factor activity perturbation. Starting with a broader approach, we identified a previously unsuspected effect on the nuclear factor erythroid-related factor 2 (NRF2), a critical regulator of cellular defenses against oxidative stress. Based on genetic and small molecule inhibitor-based methodologies, we demonstrated that repression of Ref-1 potently activates NRF2 and its downstream targets in a dose-dependent fashion, and that the redox, rather than the DNA repair function of Ref-1 is critical for this effect. Intriguingly, our results also indicate that this pathway does not involve reactive oxygen species. The link between Ref-1 and NRF2 appears to be present in all cells tested in vitro, noncancerous and cancerous, including patient-derived tumor samples. In particular, we focused on understanding the implications of the novel interaction between these two pathways in primary pancreatic ductal adenocarcinoma tumor cells and provide the first evidence that this mechanism has implications for overcoming the resistance against experimental drugs targeting Ref-1 activity, with clear translational implications.

Disruptions in DNA repair pathways predispose cells to accumulating DNA damage. A growing body of evidence indicates that tumors accumulate progressively more mutations in DNA repair proteins as cancers progress. DNA repair mechanisms greatly affect the response to cytotoxic treatments, so understanding those mechanisms and finding ways to turn dysregulated repair processes against themselves to induce tumor death is the goal of all DNA repair inhibition efforts. Inhibition may be direct or indirect. This burgeoning field of research is replete with promise and challenge, as more intricacies of each repair pathway are discovered. In an era of increasing concern about healthcare costs, use of DNA repair inhibitors can prove to be highly effective stewardship of R&D resources and patient expenses.

Apurinic/Apyrimidinic endonuclease/Redox factor-1 (APE1/Ref-1) (henceforth referred to as Ref-1) is a multifunctional protein that in addition to its base-excision DNA repair activity exerts redox control of multiple transcription factors, including nuclear factor kappa-lightchain-enhancer of activated B cells (NF-κB), STAT3, activator protein-1 (AP-1), hypoxiainducible factor-1 (HIF-)1 and tumor protein 53 (p53). In recent years, Ref-1 has emerged as a promising therapeutic target in cancer, particularly in pancreatic ductal carcinoma (PDAC). While a significant amount of research has centered on Ref-1, no wideranging approach had been performed on the effects of Ref-1 inhibition and transcription factor activity perturbation. Starting with a broader approach, we identified a previously unsuspected effect on the Nuclear factorerythroid-related factor 2 (NRF2), a critical regulator of cellular defenses against oxidative stress. Based on genetic and small molecule inhibitor-based methodologies, we demonstrated that repression of Ref-1 potently activates NRF2 and its downstream targets in a dose-dependent fashion, and that the redox, rather than the DNA repair function of Ref-1 is critical for this effect. Intriguingly, our results also indicate that this pathway does not involve Reactive Oxygen Species (ROS). The link between Ref-1 and NRF2 appears to be present in all cells tested in vitro, noncancerous and cancerous, including patient-derived tumor samples. In particular, we focused on understanding the implications of the novel interaction between these two pathways in primary PDAC tumor cells and provide the first evidence that this mechanism has implications for overcoming the resistance against experimental drugs targeting Ref-1 activity, with clear translational implications. Redox factor-1 (Ref-1) is a dual function protein which in addition to DNA repair function controls the activity of multiple transcription factors (TFs), including NF-kB (nuclear factor-kB), STAT3, AP-1 (activator protein-1), HIF-1 (hypoxia inducible factor) (14). The redox activity of Ref-1 reduces specific cysteine residues in the DNA binding domain of these TFs, thus stimulating their DNA binding activity (5). As most transcription factors stimulated by Ref-1 are well recognized regulators of tumorigenesis, this protein has emerged as a viable therapeutic target in cancer (3,5-7). Particular attention has been given to pancreatic ductal adenocarcinoma (PDAC) as Ref-1 levels are known to be elevated in a variety of human PDAC-derived cell lines as well as in neoplastic tissue and peri-pancreatic metastases. We and others demonstrated that silencing Ref-1 in pancreatic cancer cells resulted in apoptosis and decreased proliferative capacity (8,9). Furthermore, blockade of Ref-1 redox activity delayed tumor progression in xenograft models of human PDAC, including patient-derived tumor cells (4). However much remains to be elucidated about Ref-1’s functions, particularly with respect to the biochemical consequences of its inhibition. Detailed knowledge at this level is anticipated to increase the effectiveness of Ref-1 inhibitors and help delay/overcome therapeutic resistance to such agents. To this end, we performed a comprehensive survey of the effects of Ref-1 inhibition on the activity of a broad spectrum of transcription factors, using a library of reporters (Attagene, Inc)(10). Our approach led to a novel connection between Ref-1 and Nuclear factor-erythroid-related factor 2 (NRF2), a critical regulator of cellular defenses against oxidative stress (11). 2 at Iniana U niersity Shool of M eicine on July 1, 2015 hp://w w w .jb.org/ D ow nladed from Ref-1 redox function negatively regulates NRF2 MATERIALS AND METHODS Cell lines and patient-derived PDAC cells. MIA-PaCa-2 were purchased from and authenticated by ATCC (Manassas, VA). Pa03C, Panc10.05, Panc 198, and Pa02C were obtained from Dr. Anirban Maitra at The Johns Hopkins University.(12) All cells were maintained at 37°C in 5% CO2 and grown in DMEM (Invitrogen; Carlsbad, CA) with 10% FBS (Hyclone; Logan, UT) and routinely tested for mycoplasma. Inhibitors. E3330 and RN7-58 were synthesized as previously described (13-15), and tin porphyrin (Sn-PP) was purchased from Sigma Aldrich. As a negative control, the E3330 analog, RN7-58 was used. RN7-58, although structurally similar to E3330, does not inhibit Ref-1’s redox activity (16). Transfection of PDAC cells with siRNA. All siRNA transfections (Ref-1 (referred to as siRef-1#1) or scrambled control) were performed as previously described (3,13,17-19). Samples for the Attagene screen and quantitative PCR (QPCR) were collected 72 h after transfection of cancer cells with Ref-1 siRNA. For additional Ref-1 siRNA experiments, we purchased prevalidated siRNAs from LifeTech (#s1446, siRef-1#2, and s1447, siRef-1#3). Attagene cis-FACTORIAL screen. Our initial screen for the effects of Ref-1 on the activity of a diverse panel of transcription factors was performed using Attagene’s cisFACTORIAL technology followed by validation of the NRF2 pathway using the relevant reporter from the library (10). PaCa-2 cells were transfected with siRef-1 or scrambled siRNA as above. Twenty-four hours later cells were washed with fresh medium and transiently transfected with cis-FACTORIAL. Twentyfour hours after transfection cells were supplied with fresh medium (containing 10% FBS) and incubated for an additional 24 h. Profiles of the FACTORIAL endpoint activities were determined as fold-induction values of siRef-1 or scrambled RNA transfected cells divided by values from untransfected cells. The methodology is described in detail in (10,20,21). QPCR Reactions. This method was used to measure the mRNA expression levels of NRF2 and its downstream target genes, HMOX1 (heme oxidase-1), GCLC (glutamate-cysteine ligase, catalytic subunit), and GCLM (glutamate-cysteine ligase, modifier subunit). Total RNA was extracted from cells using the Qiagen RNeasy Mini kit (Valencia, CA) according to the manufacturer's instructions. The extracted RNA was quantified using a Qubit fluorometer (Invitrogen Corp, Carlsbad, CA). First-strand cDNA was prepared from RNA using random hexamers and MultiScribe reverse transcriptase (Applied Biosystems, Foster City, CA). Quantitative PCR was performed using Taqman Gene Expression assays and Universal PCR master mix (Applied Biosystems) in a 7900HT Sequence detection system (Applied Biosystems). The relative quantitative mRNA level was determined using the comparative Ct method using Actin (PaCa-2) or large ribosomal protein, P0 (RPLP0, patient lines) as the reference gene (4). The primers for NRF2, HMOX-1, GCLC, GCLM, Actin, and RPLP0 are commercially available (Applied Biosystems). Experiments were performed in triplicate for each sample. Western blot analysis. Whole cell lysates were prepared by lysing the cells in RIPA buffer (Santa Cruz Biotechnology; Santa Cruz, CA), followed by quantification of protein concentration (Lowry protein assay). Proteins were separated by SDS-PAGE, electroblotted onto nitrocellulose, and immunoblotting was performed using the following antibodies: Ref-1 and NRF2 (abcam 62352), HMOX-1 (Abcam, Cambridge, MA), and Ku70, tubulin or Actin (Sigma Aldrich). Reactive oxygen species (ROS) Measurement. The production of ROS was determined by detecting the fluorescent intensity of the oxidation-sensitive probe dihydrorhodamine 123 (DHR) (Molecular Probes, Invitrogen, Carlsbad, CA). PaCa-2 and Pa03C cells were treated with E3330 for 24 h. As a positive control for ROS production, tertButyl hydroperoxide solution (TBHP, 1 mM, 30 min) was utilized. After washing with PBS, the cells were incubated with 1 μM DHR in fresh PBS for 30 min. Excessive probe was washed off using PBS. Cells were harvested with trypsin, and ROS fluorescence of labeled cells was measured by using a Coulter EPICS XL flow cytometer (Coulter). An average of 10,000 cells from each sample was counted, and each 3 at Iniana U niersity Shool of M eicine on July 1, 2015 hp://w w w .jb.org/ D ow nladed from Ref-1 redox function negatively regulates NRF2 experiment was done in triplicate. Co-immunoprecipitation (Co-IP) Samples were co-immunoprecipitated using the Pierce Co-IP kit (Thermo Scientific) with the following modifications. Cells were washed twice with ice-cold PBS and the proteins were cross-linked using the water soluble, membrane permeable, imidoester crosslinker dimethyl 3,3’-dithiobispropiominidate (DTBP (Thermo Scientific), 5 mM, for 30 min on ice). DTBP was quenched by sequential washing with cold inactivation buffer (100 mM Tris HCl pH 8, 150 mM NaCl) and PBS. Cells were lysed by the addition of IP Lysis buffer supplemented with protease inhibitors (800 μl per 10 cm dish; 4oC, 20 min on rocking platform) and then scraped and transferred to a microcentrifuge tube. Cell debris was removed by centrifugation and the protein concentration of the cleared lysate was determined using the Pierce BCA Protein assay. To reduce non-specific protein binding, 1.4 mg of cell lysate was pre-cleared using the control Agarose resin, prior to adding to columns of either Ref-1 or NRF2 antibody or control rabbit IgG that had been covalently coupled onto an amine-reactive Agarose resin. After extensive washing, the bound proteins were eluted and prepared for SDS-PAGE analysis by the addition of 5x sample buffer containing 100 mM DTT. ChIP assay. We performed ChIP assay according to the manufacturer’s protocol (Millipore). PaCa-2 cells were incubated for 16 h in the presence of 67.5 μM E3330, or 0.17% DMSO in DMEM with 2% FBS. Proteins were crosslinked to DNA by the addition of 37% formaldehyde (to a final concentration of 1%, 10 min, room temperature) and then quenched by adding glycine (125 mM, 5 min, room temperature). Cell lysates were sonicated (Sonic Dismembrator Model 100 (Fisher) setting #4, 12 cycles, 10 sec) until cross-linked DNA was sheared to ac

Severe morbidity and early mortality from iron overload remains a key problem in transfusion‐dependent diseases. The goal of this study is to identify proteins partners to TTC7 which is associated with urinary iron excretion observed in mutant fsn mice in which an abnormal TTC7 protein (7 kDa larger) is produced. The fsn mouse suffers from a severe anemia accompanied by iron deficiency that results from excessive urinary iron excretion. Identification of the normal role of TTC7 protein is critical to understanding the causation of urinary iron excretion. We have identified elements of a leucine‐zipper motif in TTC7 suggesting a role in gene expression. TTC7 is localized to the nucleus of cells suggesting a role in gene regulation. We have generated CMV promoter driven FLAG‐TTC7 transgenic mice for immunoprecipation studies of FLAG‐TTC7 and its partner proteins. These precipitates are used to identify proteins involved in gene expression in association with TTC7 (identified by mass spectrophotometry). The transcriptional mechanism by which the fsn Ttc7 gene defect brings about this loss of urinary iron is expected to unveil novel therapeutic approaches for treatment of iron overload.

The essential base excision repair protein, apurinic/apyrimidinic endonuclease 1 (APE1), plays an important role in redox regulation in cells and is currently targeted for the development of cancer therapeutics. One compound that binds APE1 directly is (E)-3-[2-(5,6-dimethoxy-3-methyl-1,4-benzoquinonyl)]-2-nonylpropenoic acid (E3330). Here, we revisit the mechanism by which this negatively charged compound interacts with APE1 and inhibits its redox activity. At high concentrations (millimolar), E3330 interacts with two regions in the endonuclease active site of APE1, as mapped by hydrogen-deuterium exchange mass spectrometry. However, this interaction lowers the melting temperature of APE1, which is consistent with a loss of structure in APE1, as measured by both differential scanning fluorimetry and circular dichroism. These results are consistent with other findings that E3330 concentrations of >100 μM are required to inhibit APE1's endonuclease activity. To determine the role of E3330's negatively charged carboxylate in redox inhibition, we converted the carboxylate to an amide by synthesizing (E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxocyclohexa-1,4-dien-1-yl)methylene]-N-methoxy-undecanamide (E3330-amide), a novel uncharged derivative. E3330-amide has no effect on the melting temperature of APE1, suggesting that it does not interact with the fully folded protein. However, E3330-amide inhibits APE1's redox activity in in vitro electrophoretic mobility shift redox and cell-based transactivation assays, producing IC(50) values (8.5 and 7 μM) lower than those produced with E3330 (20 and 55 μM, respectively). Thus, E3330's negatively charged carboxylate is not required for redox inhibition. Collectively, our results provide additional support for a mechanism of redox inhibition involving interaction of E3330 or E3330-amide with partially unfolded APE1.

Transgenic mice represent a unique opportunity in biomedical research to discover the genes underlying disease and understand how manipulating the function of single genes and proteins alters physiology in a whole animal system. These advances in biomedical research may accelerate the time between when basic discoveries are made and when the research can be 'translated', that is, when the research will positively impact the lives of patients. The purpose of this article is to present some examples of promising mouse models of human diseases.