April Reed

Role of the Multifunctional DNA Repair and Redox Signaling Protein Ape1/Ref-1 in Cancer and Endothelial Cells: Small-Molecule Inhibition of the Redox Function of Ape1

The DNA base excision-repair pathway is responsible for the repair of DNA damage caused by oxidation/alkylation and protects cells against the effects of endogenous and exogenous agents. Removal of the damaged base creates a baseless (AP) site. AP endonuclease1 (Ape1) acts on this site to continue the BER-pathway repair. Failure to repair baseless sites leads to DNA strand breaks and cytotoxicity. In addition to the repair role of Ape1, it also functions as a major redox-signaling factor to reduce and activate transcription factors such as AP1, p53, HIF-1alpha, and others that control the expression of genes important for cell survival and cancer promotion and progression. Thus, the Ape1 protein interacts with proteins involved in DNA repair, growth-signaling pathways, and pathways involved in tumor promotion and progression. Although knockdown studies with siRNA have been informative in studying the role of Ape1 in both normal and cancer cells, knocking down Ape1 does not reveal the individual role of the redox or repair functions of Ape1. The identification of small-molecule inhibitors of specific Ape1 functions is critical for mechanistic studies and translational applications. Here we discuss small-molecule inhibition of Ape1 redox and its effect on both cancer and endothelial 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.

APE1/Ref-1 Regulates STAT3 Transcriptional Activity and APE1/Ref-1-STAT3 Dual-Targeting Effectively Inhibits Pancreatic Cancer Cell Survival

Pancreatic cancer is a largely incurable disease, and increasing evidence supports strategies targeting multiple molecular mediators of critical functions of pancreatic ductal adenocarcinoma cells. Intracellular redox state modulates the activity of various signal transduction pathways and biological processes, including cell survival, drug resistance and responsiveness to microenvironmental factors. Recently, it has been shown that the transcription factor STAT3 is under redox control, but the mechanisms involved in its regulation are unknown. Here, we demonstrate for the first time that STAT3 DNA binding and transcriptional activity is directly regulated by the redox function of the APE1/Ref-1 endonuclease, using overexpression and redox-specific mutational strategies, and gene knockdown. Also, pharmacological blockade of APE1/Ref-1 by the redox-selective inhibitor E3330 abrogates STAT3 DNA binding. Since APE1/Ref-1 also exerts redox control on other cancer-associated transcription factors, we assessed the impact of dual-targeting of STAT3 signaling and APE1/Ref-1 redox on pancreatic cancer cell functions. We observed that disruption of APE1/Ref-1 redox activity synergizes with STAT3 blockade to potently inhibit the proliferation and viability of human PDAC cells. Mechanistically, we show that STAT3–APE1/Ref-1 dual targeting promotes marked tumor cell apoptosis, with engagement of caspase-3 signaling, which are significantly increased in comparison to the effects triggered by single target blockade. Also, we show that STAT3–APE1/Ref-1 dual blockade results in significant inhibition of tumor cell migration. Overall, this work demonstrates that the transcriptional activity of STAT3 is directly regulated by the redox function of APE1/Ref-1, and that concurrent blockade of STAT3 and APE1/Ref-1 redox synergize effectively inhibit critical PDAC cell functions.

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 APE1s endonuclease activity. To determine the role of E3330s 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 APE1s 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, E3330s 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.

Small-molecule inhibitors of proteins involved in base excision repair potentiate the anti-tumorigenic effect of existing chemotherapeutics and irradiation

There has been a recent upsurge in the development of small-molecule inhibitors specific to DNA repair proteins or proteins peripherally involved in base excision repair and the DNA damage response. These specific, nominally toxic inhibitors are able to potentiate the effect of existing cancer cell treatments in a wide array of cancers. One of the largest obstacles to overcome in the treatment of cancer is incomplete killing with initial cancer treatments, leading to resistant cancer. The progression of our understanding of cancer and normal cell responses to DNA damage has allowed us to develop biomarkers that we can use to help us predict responses of cancers, more specifically target cancer cells and overcome resistance. Initial successes using these small-molecule DNA repair inhibitors in target-validation experiments and in the early stages of clinical trials indicate an important role for these inhibitors, and allow for the possibility of a future in which cancers are potentially treated in a highly specific, individual manner.

Accelerated repair and reduced mutagenicity of oxidative DNA damage in human bladder cells expressing the E. coli FPG protein

Repair of some oxidized purines such as 8‐oxo‐7,8‐dihydroguanine (8‐oxoG) is inefficient in human cells in comparison to repair of other major endogenous lesions (e.g. uracil, abasic sites or oxidized pyrimidines). This is due to the poor catalytic properties of hOGG1, the major DNA glycosylase involved in 8‐oxoG removal. The formamidopyrimidine DNA glycosylase (FPG) protein from E. coli is endowed with a potent 8‐oxoG glycolytic activity coupled with a β,δ‐AP lyase. In this study, we have expressed FPG fused to the enhanced green fluorescent protein (EGFP) in human bladder cells to accelerate the repair of oxidative DNA damage. Cells expressing the fusion protein EGFP–FPG repaired 8‐oxoG and AP sites at accelerated rates, in particular via the single‐nucleotide insertion base excision repair (BER) pathway and were resistant to mutagenicity of the oxidizing carcinogen potassium bromate. FPG may stably protect human cells from some harmful effects of oxidative DNA damage.

Base excision repair apurinic/apyrimidinic endonucleases in apicomplexan parasite Toxoplasma gondii.

DNA repair is essential for cell viability and proliferation. In addition to reactive oxygen produced as a byproduct of their own metabolism, intracellular parasites also have to manage oxidative stress generated as a defense mechanism by the host. The spontaneous loss of DNA bases due to hydrolysis and oxidative DNA damage in intracellular parasites is great, but little is known about the type of DNA repair machineries that exist in these early-branching eukaryotes. However, it is clear, processes similar to DNA base excision repair (BER) must exist to rectify spontaneous and host-mediated damage in Toxoplasma gondii. Here we report that T. gondii, an opportunistic protozoan pathogen, possesses two apurinic/apyrimidinic (AP) endonucleases that function in DNA BER. We characterize the enzymatic activities of Toxoplasma exonuclease III (ExoIII, or Ape1) and endonuclease IV (EndoIV, or Apn1), designated TgAPE and TgAPN, respectively. Over-expression of TgAPN in Toxoplasma conferred protection from DNA damage, and viable knockouts of TgAPN were not obtainable. We generated an inducible TgAPN knockdown mutant using a ligand-controlled destabilization domain to establish that TgAPN is critical for Toxoplasma to recover from DNA damage. The importance of TgAPN and the fact that humans lack any observable APN family activity highlights TgAPN as a promising candidate for drug development to treat toxoplasmosis.

Small molecule activation of apurinic/apyrimidinic endonuclease 1 reduces DNA damage induced by cisplatin in cultured sensory neurons

Although chemotherapy-induced peripheral neuropathy (CIPN) affects approximately 5-60% of cancer patients, there are currently no treatments available in part due to the fact that the underlying causes of CIPN are not well understood. One contributing factor in CIPN may be persistence of DNA lesions resulting from treatment with platinum-based agents such as cisplatin. In support of this hypothesis, overexpression of the base excision repair (BER) enzyme, apurinic/apyrimidinic endonuclease 1 (APE1), reduces DNA damage and protects cultured sensory neurons treated with cisplatin. Here, we address stimulation of APE1s endonuclease through a small molecule, nicorandil, as a means of mimicking the beneficial effects observed for overexpression of APE1. Nicorandil, was identified through high-throughput screening of small molecule libraries and found to stimulate APE1 endonuclease activity by increasing catalytic efficiency approximately 2-fold. This stimulation is primarily due to an increase in kcat. To prevent metabolism of nicorandil, an approved drug in Europe for the treatment of angina, cultured sensory neurons were pretreated with nicorandil and daidzin, an aldehyde dehydrogenase 2 inhibitor, resulting in decreased DNA damage but not altered transmitter release by cisplatin. This finding suggests that activation of APE1 by nicorandil in cisplatin-treated cultured sensory neurons does not imbalance the BER pathway in contrast to overexpression of the kinetically faster R177A APE1. Taken together, our results suggest that APE1 activators can be used to reduce DNA damage induced by cisplatin in cultured sensory neurons, although further studies will be required to fully assess their protective effects.

Abstract B32: First-in-class Ref-1 redox inhibitors for the multipathway targeting of survival signals for relapsed childhood acute lymphoblastic leukemia

Acute lymphoblastic leukemia (ALL) is the most frequent pediatric cancer and, despite significant treatment advances, relapsed ALL remains the second leading cause of childhood death. In T-cell ALL, more than 10% of the patients show poor response to frontline therapy and about a third of the patients in remission develop recurrence of their leukemia. Importantly, effective, curative therapies are lacking for children with relapse or refractory disease. Our strategy for T-ALL was to develop agents that target master molecular regulators controlling the activity of multiple complementary, non-recurrent signaling pathways, integrating oncogenic signals and microenvironment cues. Ref-1/APE1 (Ref-1) is a multi-function protein that exerts redox control of multiple transcription factors (TFs), regulating their DNA binding and downstream transcriptional programs. These include TFs playing important roles in T-cell ALL, namely NF-κB, AP-1 and STAT3, the latter of which we have shown recently to be required for T-cell leukemogenesis. We showed that Ref-1 is expressed by leukemia T-cells in the malignant bone marrow (BM), and that its expression is increased in drug-resistant T-ALL cells. Analyses of multiple leukemia transcriptome databases showed significant increased expression of Ref-1 in T-cell ALL specimens, as well as of other genes in the Ref-1/SET molecular axis. Molecular and functional studies showed that disruption of Ref-1 redox function markedly inhibits leukemia T-cell survival and proliferation, triggering molecular changes promoting cell apoptosis. We identified three new small molecule chemical entities - APX2007, APX2009 and APX2032, that significantly inhibit the reduction of Ref-1 and the DNA binding of Ref-1-regulated TFs as assessed by EMSA. These compounds (cpds) are significantly more potent than a previously identified Ref-1 redox antagonist, E3330. Functional studies showed that all three APX cpds markedly inhibit leukemia cell survival. Potent inhibition of tumor cell viability was seen in primary cells from ALL patients, relapsed T-ALL, and cells from a murine model of Notch-induced leukemia. Blockade of Ref-1 redox triggers significant leukemia cell apoptosis, and correlates with down-regulation of survival genes regulated by the Ref-1 ‘targets’ STAT3 and NF-kB. Blockade of Ref-1 redox by the APX cpds markedly inhibits the viability of drug- resistant T-ALL cells, with an antitumor efficacy comparable to chemotherapy-sensitive leukemia cells. This is significant since glucocorticoid-resistance is predictive of ALL relapse, and glucocorticoid-resistant leukemia T-cells show reduced sensitivity to inhibitors of other leukemia-associated signaling pathways (as PI3K/Akt, mTOR). Preliminary safety studies in mice using a clinical formulation demonstrated the systemic administration of the APX cpds do not result in acute adverse reactions or significant hematological toxicities. Importantly, studies in a xenograft model of glucocorticoid-, doxorubicin-resistant human T-ALL showed that treatment with the APX cpds result in significant decrease in leukemia blasts in the peripheral blood and in the BM. In summary, we developed novel, first-in-class inhibitors of Ref-1, which show acceptable in vivo PK and toxicity, and that potently inhibit T-ALL, including patient9s specimens and drug-resistant leukemia T-cells. These new chemical entities target a unique molecular regulator, as Ref-1 redox function controls multiple TFs involved in leukemogenesis and disease progression. Studies are underway to further define the therapeutic efficacy and long-term remission potential of ApeX compounds in animal models of T-ALL, including a model of leukemia recurrence post-frontline chemotherapy. These studies should support the selection of a candidate for development and progression to clinical trials in pediatric patients with refractory, relapsed ALL. Citation Format: Angelo A. Cardoso, James H. Wikel, Jixin Ding, April M. Reed, Meihua Luo, Mark R. Kelley. First-in-class Ref-1 redox inhibitors for the multipathway targeting of survival signals for relapsed childhood acute lymphoblastic leukemia. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr B32.

Abstract C173: APE1/Ref-1-STAT3 dual-targeting synergize to effectively inhibit pancreatic cancer cell survival.

Pancreatic cancer remains a largely incurable disease, with patients facing the worst 5-year survival rate of any cancer. The challenge is to identify the molecular effectors that critically regulate the survival of pancreatic ductal adenocarcinoma (PDAC) cells, to devise effective molecular-targeted strategies that can prevent or minimize the selection of resistant tumor variants, and overcome the protective role of the tumor-associated fibrosis and stroma. Increasing evidence supports the need for strategies targeting multiple molecular effectors in PDAC. Thus, a strategy is to identify critical molecules that regulate multiple signaling mediators (as transcription factors), and on intracellular mechanisms with direct effects on multiple pathways (as antioxidant and redox mediators) critical for PDAC functions. APE1/Ref-1 is a dual function protein, which in addition to DNA repair activity also exerts redox control of transcription factors, including NF- B, p53, AP-1, CREB, HIF-1 and others. Treatment with E3330, a unique small molecule redox signaling inhibitor that recognizes an alternate, redox active conformation of APE1/Ref-1, markedly inhibits the DNA binding and transcriptional activity of NF-κB, AP-1, and HIF-1. Our previous work established APE1/Ref-1 as a potential molecular target in pancreatic cancer. We demonstrated that human adenocarcinoma and peri-pancreatic metastases exhibit increased APE1/Ref-1 expression, and blocking APE1/Ref-1 redox activity delays tumor progression in xenograft models of PDAC, including patient-derived tumor cells. STAT3 is a transcription factor that regulates critical cell functions and has been shown to play important roles in several cancers. STAT3 signaling has been implicated in pancreatic cancer biology, namely by mediating or regulating cell survival, proliferation, tumor angiogenesis and metastasis. Although STAT3 signaling can be engaged and modulated by different processes, the mechanisms regulating STAT3 transcriptional activity in PDAC cells are largely unknown, namely the impact of oxidative stress and its redox status. A recent report demonstrated that STAT3 activity is under redox control and identified the critical oxidation-sensitive cysteines in the STAT3 DNA binding domain. However, the modifier of STAT3 which converts it from an oxidized to a reduced form was not identified. It has been shown that APE1/Ref-1 physically interacts with STAT3 on the VEGF promoter and enhances IL-6-induced DNA binding activity of STAT3 in HepG2 cells. However, it is unknown whether APE-1/Ref-1 is involved in the redox control of STAT3 activity, and whether the cellular redox status affects STAT3 signaling in PDAC cells. Here, we demonstrate that APE1/Ref-1 redox activity regulates the STAT3 DNA binding and transcriptional activity using gene silencing, overexpression of WT or redox-defective APE1/Ref-1, and redox-selective pharmacological inhibition. Blockade of APE1/Ref-1 redox synergizes with STAT3 selective antagonists to markedly inhibit the proliferation and survival of human PDAC cells, inducing cell apoptosis. These studies identify the mechanism by which APE1/Ref-1 regulates STAT3 signaling, and establishes the rationale for the development of APE1/STAT3 dual-targeting strategies for the treatment of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C173.