Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1) redox function negatively regulates NRF2.

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.

Targeting DNA repair pathways for cancer treatment: what's new?

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.

Inhibition of apurinic/apyrimidinic endonuclease I's redox activity revisited.

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.

Research from the bedside to the lab bench & back.

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.

Mutation in erythroid specific transcription factor KLF1 causes Hereditary Spherocytosis in the Nan hemolytic anemia mouse model.

KLF1 regulates definitive erythropoiesis of red blood cells by facilitating transcription through high affinity binding to CACCC elements within its erythroid specific target genes including those encoding erythrocyte membrane skeleton (EMS) proteins. Deficiencies of EMS proteins in humans lead to the hemolytic anemia Hereditary Spherocytosis (HS) which includes a subpopulation with no known genetic defect. Here we report that a mutation, E339D, in the second zinc finger domain of KLF1 is responsible for HS in the mouse model Nan. The causative nature of this mutation was verified with an allelic test cross between Nan/+ and heterozygous Klf1(+/-) knockout mice. Homology modeling predicted Nan KLF1 binds CACCC elements more tightly, suggesting that Nan KLF1 is a competitive inhibitor of wild-type KLF1. This is the first association of a KLF1 mutation with a disease state in adult mammals and also presents the possibility of being another causative gene for HS in humans.

Hematologic characterization and chromosomal localization of the novel dominantly inherited mouse hemolytic anemia, neonatal anemia (Nan).

One of the most commonly inherited anemias in man is Hereditary Spherocytosis (HS) with an incidence of 1 in 2000 for persons of Northern European descent. Mouse models of HS include spontaneous inherited hemolytic anemias and those generated by gene targeting. The Neonatal anemia (Nan) mouse is a novel model of HS generated by N-ethyl-N-nitrosurea mutagenesis and suffers from a severe neonatal anemia. Adult Nan mice have a lifelong hemolytic anemia with decreased red blood cell numbers, hematocrit, and hemoglobin, but elevated zinc protoporphyrin levels. Blood smears taken from Nan mice show a hypochromic anemia characterized by poikilocytosis, anisocytosis and polychromasia. The Nan phenotype can be transferred by bone marrow transplantation indicating that the defect is intrinsic to bone marrow. The hemolytic anemia in adult Nan mice can be identified by osmotic fragility testing. Examination of the erythrocyte membrane skeleton proteins (EMS) reveals a global deficiency of these proteins with protein 4.1a being completely absent. The Nan locus maps to mouse Chromosome 8 and does not co-localize with any known EMS genes. The identification of the Nan gene will likely uncover a novel protein that contributes to the stability of the EMS and may identify a new mutation for HS.

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.

Exploiting the Ref-1-APE1 node in cancer signaling and other diseases: from bench to clinic.

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.

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.