Browsing by Subject "Ref-1"

Now showing 1 - 8 of 8
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    Activation of the integrated stress response (ISR) pathways in response to Ref-1 inhibition in human pancreatic cancer and its tumor microenvironment
    (Frontiers Media, 2023-04-27) Mijit, Mahmut; Boner, Megan; Cordova, Ricardo A.; Gampala, Silpa; Kpenu, Eyram; Klunk, Angela J.; Zhang, Chi; Kelley, Mark R.; Staschke, Kirk A.; Fishel, Melissa L.; Pediatrics, School of Medicine
    Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment.
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    APE1/Ref-1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single-cell RNA sequencing
    (Wiley, 2017-12) Shah, Fenil; Goossens, Emery; Atallah, Nadia M.; Grimard, Michelle; Kelley, Mark R.; Fishel, Melissa L.; Department of Pediatrics, School of Medicine
    Apurinic/apyrimidinic endonuclease 1/redox factor-1 (APE1/Ref-1 or APE1) is a multifunctional protein that regulates numerous transcription factors associated with cancer-related pathways. Because APE1 is essential for cell viability, generation of APE1-knockout cell lines and determining a comprehensive list of genes regulated by APE1 has not been possible. To circumvent this challenge, we utilized single-cell RNA sequencing to identify differentially expressed genes (DEGs) in relation to APE1 protein levels within the cell. Using a straightforward yet novel statistical design, we identified 2837 genes whose expression is significantly changed following APE1 knockdown. Using this gene expression profile, we identified multiple new pathways not previously linked to APE1, including the EIF2 signaling and mechanistic target of Rapamycin pathways and a number of mitochondrial-related pathways. We demonstrate that APE1 has an effect on modifying gene expression up to a threshold of APE1 expression, demonstrating that it is not necessary to completely knockout APE1 in cells to accurately study APE1 function. We validated the findings using a selection of the DEGs along with siRNA knockdown and qRT-PCR. Testing additional patient-derived pancreatic cancer cells reveals particular genes (ITGA1, TNFAIP2, COMMD7, RAB3D) that respond to APE1 knockdown similarly across all the cell lines. Furthermore, we verified that the redox function of APE1 was responsible for driving gene expression of mitochondrial genes such as PRDX5 and genes that are important for proliferation such as SIPA1 and RAB3D by treating with APE1 redox-specific inhibitor, APX3330. Our study identifies several novel genes and pathways affected by APE1, as well as tumor subtype specificity. These findings will allow for hypothesis-driven approaches to generate combination therapies using, for example, APE1 inhibitor APX3330 with other approved FDA drugs in an innovative manner for pancreatic and other cancer treatments.
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    Drug Inhibition of Redox Factor-1 Restores Hypoxia-Driven Changes in Tuberous Sclerosis Complex 2 Deficient Cells
    (MDPI, 2022-12-15) Champion, Jesse D.; Dodd, Kayleigh M.; Lam, Hilaire C.; Alzahrani, Mohammad A. M.; Seifan, Sara; Rad, Ellie; Scourfield, David Oliver; Fishel, Melissa L.; Calver, Brian L.; Ager, Ann; Henske, Elizabeth P.; Davies, David Mark; Kelley, Mark R.; Tee, Andrew R.; Pediatrics, School of Medicine
    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.
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    An 'environment to nucleus' signaling system operates in B lymphocytes: redox status modulates BSAP/Pax-5 activation through Ref-1 nuclear translocation
    (2000-03) Tell, Gianluca; Zecca, Alessandro; Pellizzari, Lucia; Spessotto, Paola; Colombatti, Alfonso; Kelley, Mark R.; Damante, Giuseppe; Pucillo, Carlo
    The Ref-1 (also called APE or HAP1) protein is a bifunctional enzyme impacting on a wide variety of important cellular functions. It acts as a major member of the DNA base excision repair pathway. Moreover, Ref-1 stimulates the DNA-binding activity of several transcription factors (TFs) through the reduction of highly reactive cysteine residues. Therefore, it represents a mechanism that regulates eukaryotic gene expression in a fast way. However, it has been demonstrated that external stimuli directly act on Ref-1 by increasing its expression levels, a time-consuming mechanism representing a paradox in terms of rapidity of TF regulation. In this paper we demonstrate that this is only an apparent paradox. Exposure of B lymphocytes to H2O2 induced a rapid and sustained increase in Ref-1 protein levels in the nucleus as evaluated by both western blot analysis and by pulse–chase experiments. A time course, two color in situ immunocytochemistry indicated that the up-regulation of Ref-1 in the nucleus at <30 min was primarily the consequence of translocation of its cytoplasmic form. This early nuclear accumulation is effective in modulating the DNA-binding activity of the B cell-specific activator protein BSAP/Pax-5. In fact, EMSA experiments demonstrate that a transient interaction with Ref-1 up-regulates the DNA-binding activity of BSAP/Pax-5. Moreover, in a co-transfection experiment, Ref-1 increased the BSAP/Pax-5 activating effect on an oligomerized BSAP/Pax-5 binding site of the CD19 promoter by 5- to 8-fold. Thus, Ref-1 mediates its effect by up-regulating the DNA-binding activity of BSAP/Pax-5, accounting for a new and fast outside/inside pathway of signaling in B cells.
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    Ref-1 redox activity alters cancer cell metabolism in pancreatic cancer: exploiting this novel finding as a potential target
    (BMC, 2021-08-10) Gampala, Silpa; Shah, Fenil; Lu, Xiaoyu; Moon, Hye-ran; Babb, Olivia; Umesh Ganesh, Nikkitha; Sandusky, George; Hulsey, Emily; Armstrong, Lee; Mosely, Amber L.; Han, Bumsoo; Ivan, Mircea; Yeh, Jing-Ruey Joanna; Kelley, Mark R.; Zhang, Chi; Fishel, Melissa L.; Pediatrics, School of Medicine
    Background: Pancreatic cancer is a complex disease with a desmoplastic stroma, extreme hypoxia, and inherent resistance to therapy. Understanding the signaling and adaptive response of such an aggressive cancer is key to making advances in therapeutic efficacy. Redox factor-1 (Ref-1), a redox signaling protein, regulates the conversion of several transcription factors (TFs), including HIF-1α, STAT3 and NFκB from an oxidized to reduced state leading to enhancement of their DNA binding. In our previously published work, knockdown of Ref-1 under normoxia resulted in altered gene expression patterns on pathways including EIF2, protein kinase A, and mTOR. In this study, single cell RNA sequencing (scRNA-seq) and proteomics were used to explore the effects of Ref-1 on metabolic pathways under hypoxia. Methods: scRNA-seq comparing pancreatic cancer cells expressing less than 20% of the Ref-1 protein was analyzed using left truncated mixture Gaussian model and validated using proteomics and qRT-PCR. The identified Ref-1's role in mitochondrial function was confirmed using mitochondrial function assays, qRT-PCR, western blotting and NADP assay. Further, the effect of Ref-1 redox function inhibition against pancreatic cancer metabolism was assayed using 3D co-culture in vitro and xenograft studies in vivo. Results: Distinct transcriptional variation in central metabolism, cell cycle, apoptosis, immune response, and genes downstream of a series of signaling pathways and transcriptional regulatory factors were identified in Ref-1 knockdown vs Scrambled control from the scRNA-seq data. Mitochondrial DEG subsets downregulated with Ref-1 knockdown were significantly reduced following Ref-1 redox inhibition and more dramatically in combination with Devimistat in vitro. Mitochondrial function assays demonstrated that Ref-1 knockdown and Ref-1 redox signaling inhibition decreased utilization of TCA cycle substrates and slowed the growth of pancreatic cancer co-culture spheroids. In Ref-1 knockdown cells, a higher flux rate of NADP + consuming reactions was observed suggesting the less availability of NADP + and a higher level of oxidative stress in these cells. In vivo xenograft studies demonstrated that tumor reduction was potent with Ref-1 redox inhibitor similar to Devimistat. Conclusion: Ref-1 redox signaling inhibition conclusively alters cancer cell metabolism by causing TCA cycle dysfunction while also reducing the pancreatic tumor growth in vitro as well as in vivo.
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    Ref-1/APE1 as Transcriptional Regulator and Novel Therapeutic Target in Pediatric T-cell Leukemia
    (AACR, 2017-01-01) Ding, Jixin; Fishel, Melissa L.; Reed, April M.; McAdams, Erin; Czader, Magdalena; Cardoso, Angelo A.; Kelley, Mark R.; Department of Pediatrics, School of Medicine
    The increasing characterization of childhood acute lymphoblastic leukemia (ALL) has led to the identification of multiple molecular targets, but have yet to translate into more effective targeted therapies, particularly for high-risk, relapsed T-cell ALL. Searching for master regulators controlling multiple signaling pathways in T-ALL, we investigated the multi-functional protein redox factor-1 (Ref-1/APE1), which acts as a signaling "node" by exerting redox regulatory control of transcription factors important in leukemia. Leukemia patients' transcriptome databases showed increased expression in T-ALL of Ref-1 and other genes of the Ref-1/SET interactome. Validation studies demonstrated that Ref-1 is expressed in high-risk leukemia T-cells, including in patient biopsies. Ref-1 redox function is active in leukemia T-cells, regulating the Ref-1 target NF-kB, and inhibited by the redox-selective Ref-1 inhibitor E3330. Ref-1 expression is not regulated by Notch signaling, but is upregulated by glucocorticoid treatment. E3330 disrupted Ref-1 redox activity in functional studies and resulted in marked inhibition of leukemia cell viability, including T-ALL lines representing different genotypes and risk groups. Potent leukemia cell inhibition was seen in primary cells from ALL patients, relapsed and glucocorticoid-resistant T-ALL cells, and cells from a murine model of Notch-induced leukemia. Ref-1 redox inhibition triggered leukemia cell apoptosis and down-regulation of survival genes regulated by Ref-1 targets. For the first time, this work identifies Ref-1 as a novel molecular effector in T-ALL and demonstrates that Ref-1 redox inhibition results in potent inhibition of leukemia T-cells, including relapsed T-ALL. These data also support E3330 as a specific Ref-1 small molecule inhibitor for leukemia.
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    The repair function of the multifunctional DNA repair/redox protein APE1 is neuroprotective after ionizing radiation
    (2011-09) Vasko, Michael R.; Guo, Chunlu; Thompson, Eric L.; Kelley, Mark R.
    Although exposure to ionizing radiation (IR) can produce significant neurotoxicity, the mechanisms mediating this toxicity remain to be determined. Previous studies using neurons isolated from the central nervous system show that IR produces reactive oxygen species and oxidative DNA damage in those cells. Because the base excision DNA repair pathway repairs single-base modifications caused by ROS, we asked whether manipulating this pathway by altering APE1 expression would affect radiation-induced neurotoxicity. In cultures of adult hippocampal and sensory neurons, IR produces DNA damage as measured by phosphorylation of histone H2A.X and results in dose-dependent cell death. In isolated sensory neurons, we demonstrate for the first time that radiation decreases the capsaicin-evoked release of the neuropeptide CGRP. Reducing APE1 expression in cultured cells augments IR-induced neurotoxicity, whereas overexpressing APE1 is neuroprotective. Using lentiviral constructs with a neuronal specific promoter that selectively expresses APE1s different functions in neurons, we show that selective expression of the DNA repair competent (redox inactive) APE1 constructs in sensory neurons resurrects cell survival and neuronal function, whereas use of DNA-repair deficient (redox active) constructs is not protective. Use of an APE1 redox-specific inhibitor, APX3330, also facilitates neuronal protection against IR-induced toxicity. These results demonstrate for the first time that the repair function of APE1 is required to protect both hippocampal and DRG neuronal cultures—specifically neuronal cells—from IR-induced damage, while the redox activity of APE1 does not appear to be involved.
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    Targeting Ref-1/APE1 Pathway Inhibition in Pancreatic Cancer Using APX3330 for Clinical Trials
    (Office of the Vice Chancellor for Research, 2016-04-08) Fishel, Melissa L.; Logsdon, Derek P.; Supuran, Claudiu T.; Zyromski, Nicholas; Ivan, Mircea; Kelley, Mark R.; Shah, Fenil
    Pancreatic ductal adenocarcinoma is the 4th leading cause of cancer-related mortality in the US. Most patients present with advanced disease and ~95% die within five years, most surviving under six months. Targeted therapies offer modest improvement in survival, albeit at an increase in side effects and unwanted toxicities. Ref-1 regulates transcription factors involved in pancreatic cancer cell survival signaling due to its redox-coactivator activity, such as HIF-1α, NFκB, NRF2 and STAT3. High expression levels of Ref-1 indicate decreased survival in PDAC and other cancers. APX3330, a specific Ref-1 inhibitor, has been shown in multiple in vitro and in vivo pancreatic cancer models to be effective in reducing tumor growth and metastases. The safety and dose administration of APX3330 have been previously established, including toxicology, phase I, and phase II clinical evaluation in non-cancer patients in Japan (Eisai). We have partnered with ApeX Therapeutics to develop APX3330 for cancer treatment (phase I trial anticipated early 2016). We studied interactions of Ref-1, APX3330, convergent pathways; i.e. HIF-1α and STAT3, and downstream targets like CAIX. We performed in vivo studies demonstrating single and combination effects of APX3330 with Gemcitabine (Gem) showing significantly decreased tumor volume in the combination treatments. 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. We used the CAIX inhibitor SLC-0111 and JAK2 inhibitor, Ruxolitinib; both in clinical trials. In our 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, and APX3330 with SLC-0111/Ruxolitinib enhanced tumor killing. These data demonstrate APX3330 single agent efficacy in our 3D patient model and enhanced tumor killing when pathways regulated by Ref-1, HIF-1 and STAT3 are blocked.