Derek P. Logsdon

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 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.

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

Background: The redox activity of Ref-1 activates the binding of several transcription factors important in cancer. Results: Repression of Ref-1 potently activates NRF2 resulting in up-regulation of target gene expression. Conclusion: Activation of NRF2 is a potential mechanism of resistance to therapies based on Ref-1 inhibition. Significance: Dual blockade of Ref-1 and NRF2 or the specific downstream target, HMOX-1, represents a strategy for overcoming resistance. 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.

Regulation of HIF1a 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.

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.

Abstract B51: 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.

Abstract 4740: Targeting Ref-1/APE1 pathway inhibition in pancreatic cancer using APX3330 for clinical trials

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.