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

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

Targeting Ref-1/APE1 Pathway Inhibition in Pancreatic Cancer Using APX3330 for Clinical Trials

poster abstractPancreatic 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

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 IC50 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

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

Abstract 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

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

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

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

Regulation of HIF1 under Hypoxia by APE1/Ref-1 Impacts CA9 Expression: Dual-Targeting in Patient-Derived 3D Pancreatic Cancer Models

poster abstractAbstract Half of all patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) die within a year despite extensive surgery and/or a highly aggressive chemotherapy regimen. Several mechanisms are proposed to play a role in PDAC therapeutic resistance, including reactive stroma and hypoxia. Hypoxia signaling creates a more aggressive phenotype with increased metastatic potential and impaired therapeutic efficacy. Carbonic anhydrase IX (CA9) functions as part of the cellular hypoxia response to regulate intracellular pH, promoting cell survival. Apurinic/Apyrimidinic Endonuclease-1-Reduction/oxidation Effector Factor 1 (APE1/Ref-1) is a multi-functional protein with two major activities: an endonuclease function in DNA base excision repair and a redox signaling function that reduces 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. We explored the mechanisms underlying PDAC cell responses to hypoxia and modulation of APE1/Ref-1 redox signaling control of hypoxia inducible factor 1 alpha (HIF1), a critical factor in hypoxiainduced CA9 transcription. We hypothesized that obstructing the HIF-CA9 axis at two points via APE1/Ref-1 inhibition and CA9 inhibition will result in enhanced PDAC cell killing under hypoxic conditions. Methods: We performed qRT-PCR and Western Blots to confirm changes in CA9 expression in PDAC cells following APE1/Ref-1 inhibition and hypoxia exposure. Proliferation assays were used to assess cell killing following inhibition of APE1/Ref-1 and CA9 under hypoxia. Ex vivo 3-Dimensional co-culture models including both tumor and CAFs were used to examine whether we could enhance the efficacy of APE1/Ref-1 and/or CA9 inhibition with a dual-targeting approach to kill tumor spheroids. Results: HIF1-mediated induction of CA9 is significantly diminished in PDAC cells following APE1/Ref-1 redox inhibition. Additionally, dual-targeting of APE1/Ref-1 and CA9 reduces PDAC tumor cell growth under hypoxic conditions and in 3D tumor co-cultures

APE1/REF-1 redox signaling regulates HIF1A-mediated CA9 expression in hypoxic pancreatic cancer cells : combination treatment in patient-derived pancreatic tumor model

Indiana University-Purdue University Indianapolis (IUPUI)Pancreatic ductal adenocarcinoma (PDAC) is an extremely deadly disease characterized by aggressive metastasis and therapeutic resistance. Reactive stroma in pancreatic tumors contributes to tumor signaling, 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 multi-functional protein with two major activities: endonuclease activity in DNA base excision repair and a redox signaling activity that reduces oxidized transcription factors, enabling them to bind target sequences in DNA. APE1/Ref-1 is a central node in redox signaling, contributing to the activation of transcription factors involved in tumor survival, growth, and hypoxia signaling. This work evaluates the mechanisms underlying PDAC cell responses to hypoxia and APE1/Ref-1 redox signaling control of hypoxia inducible factor 1 alpha (HIF1a), a critical factor in hypoxia-induced CA9 transcription. We hypothesized that obstructing the HIF-CA9 axis at two points via APE1/Ref-1 inhibition and CA9 inhibition results in enhanced PDAC cell killing under hypoxic conditions. We found that HIF1a-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. Additionally, dual-targeting of APE1/Ref-1 redox signaling activity and CA9 activity results in enhanced 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. Further experiments characterized novel analogs of clinically relevant drugs targeting the key enzymes in this pathway, resulting in improved potency. These results underscore the notion that combination therapy is essential and demonstrate the potential clinical utility of blocking APE1/Ref-1 and CA9 function for novel PDAC therapeutic treatment