Chunlu Guo

Implications of Apurinic/Apyrimidinic Endonuclease in Reactive Oxygen Signaling Response after Cisplatin Treatment of Dorsal Root Ganglion Neurons

Peripheral neuropathy is one of the major side effects of the anticancer drug cisplatin. Although previous work suggests that this neuropathy correlates with formation of DNA adducts in sensory neurons, growing evidence suggests that cisplatin also increases the generation of reactive oxygen species (ROS), which could cause DNA damage. Apurinic/apyrimidinic endonuclease/redox factor-1 (Ape1/Ref-1) is a multifunctional protein involved in DNA base excision repair of oxidative DNA damage and in redox regulation of a number of transcription factors. Therefore, we asked whether altering Ape1 functions would influence cisplatin-induced neurotoxicity. Sensory neurons in culture were exposed to cisplatin for 24 hours and several end points of toxicity were measured, including production of ROS, cell death, apoptosis, and release of the immunoreactive calcitonin gene-related peptide (iCGRP). Reducing expression of Ape1 in neuronal cultures using small interfering RNA (siRNA) enhances cisplatin-induced cell killing, apoptosis, ROS generation, and cisplatin-induced reduction in iCGRP release. Overexpressing wild-type Ape1 attenuates all the toxic effects of cisplatin in cells containing normal endogenous levels of Ape1 and in cells with reduced Ape1 levels after Ape1siRNA treatment. Overexpressing the redox deficient/repair competent C65-Ape1 provides partial rescue, whereas the repair-deficient Ape1 (N226A + R177A) does not protect neurons from cisplatin toxicity. We also observe an increase in phosphorylation of p53 after a decrease in Ape1 levels in sensory neuronal cultures. These results strongly support the notion that Ape1 is a potential translational target such that protecting Ape1 levels and particularly its DNA repair function could reduce peripheral neuropathy in patients undergoing cisplatin treatment.

Role of APE1 in differentiated neuroblastoma SH-SY5Y cells in response to oxidative stress; Use of APE1 small molecule inhibitors to delineate APE1 functions

Oxidative DNA damage has been implicated in a number of central nervous system pathologies. The base excision repair (BER) pathway is one of the most important cellular protection mechanisms that respond to oxidative DNA damage. Human apurinic (apyrimidinic) endonuclease/redox effector factor (APE1/Ref-1 or APE1) is an essential enzyme in the BER pathway and is expressed in both mitotic and post-mitotic cells in humans. In neurons, a reduction of APE1 expression increases chemotherapy-induced cytotoxicity, while overexpression of APE1 protects cells against the cytotoxicity. However, given the multiple functions of APE1, knockdown of total APE1 is not completely informative of whether it is the redox or DNA repair activity, or interactions with other proteins. Therefore, the use of selective small molecules that can block each function independent of the other is of great benefit in ascertaining APE1 function in post-mitotic cells. In this study, we chose differentiated SH-SY5Y cells as our post-mitotic cell line model to investigate whether a drug-induced decrease in APE1 DNA repair or redox activity contributes to the growth and survival of post-mitotic cells under oxidative DNA damaging conditions. Here, we demonstrate that overexpression of WT-APE1 or C65-APE1 (repair competent) results in significant increase in cell viability after exposure to H(2)O(2). However, the 177/226-APE1 (repair deficient) did not show a protective effect. This phenomenon was further confirmed by the use of methoxyamine (MX), which blocks the repair activity of APE1 that results in enhanced cell killing and apoptosis in differentiated SH-SY5Y cells and in neuronal cultures after oxidative DNA damaging treatments. Blocking APE1 redox function by a small molecule inhibitor, BQP did not decrease viability of SH-SY5Y cells or neuronal cultures following oxidative DNA damaging treatments. Our results demonstrate that the DNA repair function of APE1 contributes to the survival of nondividing post-mitotic cells following oxidative DNA damage.

The repair function of the multifunctional DNA repair/redox protein APE1 is neuroprotective after ionizing radiation

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.

Nerve Growth Factor Mediates a Switch in Intracellular Signaling for PGE2-Induced Sensitization of Sensory Neurons from Protein Kinase A to Epac

We examined whether nerve growth factor (NGF), an inflammatory mediator that contributes to chronic hypersensitivity, alters the intracellular signaling that mediates the sensitizing actions of PGE2 from activation of protein kinase A (PKA) to exchange proteins directly activated by cAMP (Epacs). When isolated sensory neurons are grown in the absence of added NGF, but not in cultures grown with 30 ng/ml NGF, inhibiting protein kinase A (PKA) activity blocks the ability of PGE2 to augment capsaicin-evoked release of the neuropeptide CGRP and to increase the number of action potentials (APs) evoked by a ramp of current. Growing sensory neurons in culture in the presence of increasing concentrations of NGF increases the expression of Epac2, but not Epac1. An intradermal injection of complete Freunds adjuvant into the rat hindpaw also increases the expression of Epac2, but not Epac1 in the dorsal root ganglia and spinal cord: an effect blocked by intraplantar administration of NGF antibodies. Treating cultures grown in the presence of 30 ng/ml NGF with Epac1siRNA significantly reduced the expression of Epac1, but not Epac2, and did not block the ability of PGE2 to augment capsaicin-evoked release of CGRP from sensory neurons. Exposing neuronal cultures grown in NGF to Epac2siRNAreduced the expression of Epac2, but not Epac1 and prevented the PGE2-induced augmentation of capsaicin and potassium-evoked CGRP release in sensory neurons and the PGE2-induced increase in the number of APs generated by a ramp of current. In neurons grown with no added NGF, Epac siRNAs did not attenuate PGE2-induced sensitization. These results demonstrate that NGF, through increasing Epac2 expression, alters the signaling cascade that mediates PGE2-induced sensitization of sensory neurons, thus providing a novel mechanism for maintaining PGE2-induced hypersensitivity during inflammation.

Identification and Characterization of New Chemical Entities Targeting Apurinic/Apyrimidinic Endonuclease 1 for the Prevention of Chemotherapy-Induced Peripheral Neuropathy.

Chemotherapy-induced peripheral neuropathy (CIPN) is a potentially debilitating side effect of a number of chemotherapeutic agents. There are currently no U.S. Food and Drug Administration–approved interventions or prevention strategies for CIPN. Although the cellular mechanisms mediating CIPN remain to be determined, several lines of evidence support the notion that DNA damage caused by anticancer therapies could contribute to the neuropathy. DNA damage in sensory neurons after chemotherapy correlates with symptoms of CIPN. Augmenting apurinic/apyrimidinic endonuclease (APE)-1 function in the base excision repair pathway reverses this damage and the neurotoxicity caused by anticancer therapies. This neuronal protection is accomplished by either overexpressing APE1 or by using a first-generation targeted APE1 small molecule, E3330 [(2E)-2-[(4,5-dimethoxy-2-methyl-3,6-dioxo-1,4-cyclohexadien-1-yl)methylene]-undecanoic acid; also called APX3330]. Although E3330 has been approved for phase 1 clinical trials (Investigational New Drug application number IND125360), we synthesized novel, second-generation APE1-targeted molecules and determined whether they would be protective against neurotoxicity induced by cisplatin or oxaliplatin while not diminishing the platins’ antitumor effect. We measured various endpoints of neurotoxicity using our ex vivo model of sensory neurons in culture, and we determined that APX2009 [(2E)-2-[(3-methoxy-1,4-dioxo-1,4-dihydronaphthalen-2-yl)methylidene]-N,N-diethylpentanamide] is an effective small molecule that is neuroprotective against cisplatin and oxaliplatin-induced toxicity. APX2009 also demonstrated a strong tumor cell killing effect in tumor cells and the enhanced tumor cell killing was further substantiated in a more robust three-dimensional pancreatic tumor model. Together, these data suggest that the second-generation compound APX2009 is effective in preventing or reversing platinum-induced CIPN while not affecting the anticancer activity of platins.

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 4802: Combination therapy in PDAC involving blockade of the APE1/Ref-1 signaling pathway: An investigation into drug synthetic lethality and anti-neuropathy therapeutic approach

Pancreatic ductal adenocarcinoma (PDAC) is the 4th leading cause of cancer-related mortality in the US. Most patients present with advanced disease and ~93% die within five years, with most surviving less than six months. Combination therapies including Gemcitabine (GemzarTM) and sustained release, nab-paclitaxel (AbraxaneTM) and FOLFIRINOX (5-FU/leucovorin/irinotecan/oxaliplatin) offer modest improvement in survival, albeit at an increase in side effects including chemotherapy-induced peripheral neuropathy. Data is presented on Apurinic/apyrimidinic endonuclease/redox factor-1 (APE1/Ref-1 or APE1) and redox-specific APE1 inhibitor, APX3330 and its effects on tumor cell growth and sensory neuron function. APE1 is a multifunctional protein involved in repairing DNA damage via endonuclease activity and in redox regulation of transcription factors such as HIF-1α, NFkB and STAT3. High expression levels of APE1 indicate decreased survival in PDAC as well as other cancers. Because APE1 is essential for cell viability, generation of APE1 knockout cell lines and determining a comprehensive list of genes regulated by APE1 has been difficult. To circumvent this, we performed single cell RNA-Sequencing on PDAC cells following APE1 knockdown under normoxia and hypoxia to identify differentially expressed genes and further explore APE19s effects on HIF-1α and STAT3 signaling under both conditions. Proteomic analysis on PDAC cells following APE1 knockdown in normoxia and hypoxia revealed changes in signaling downstream of APE1, complementing the transcriptomic data and providing a more complete understanding of pathways affected by APE1. We used the newly identified APE1 targets and pathways along with drug sensitivity data of cancer cell lines from the Cancer Cell Line Encyclopedia (CCLE) to generate potential combination therapies of FDA approved drugs and the APE1 redox inhibitor, APX3330 and next generation analogs. These combinations were tested using an ex vivo 3D tumor-stroma model system using patient derived cells from the tumor as well as cancer-associated fibroblasts. We identified synergy with agents such as Napabucasin and Entinostat. We also tested APX3330 in combination with drugs that are part of PDAC standard of care. In vivo studies combining APX3330 with Gemcitabine showed significantly decreased tumor volume. Combining oxaliplatin (part of FOLFIRINOX) with APX3330 caused a significant reduction in oxaliplatin-induced DNA damage in sensory neurons from a KPC orthotopic graft model, without hindering its anti-cancer activity. With the phase I clinical trial for APX3330 underway (IND 125360), the potential for APE1 targeted therapy enhancing tumor efficacy while providing neuroprotective effects in the sensory neurons provides a win-win scenario. Citation Format: Fenil L. Shah, Nadia Atallah, Michelle Grimard, Chunlu Guo, Chi Zhang, Jill Fehrenbacher, Mark R. Kelley, Melissa Fishel. Combination therapy in PDAC involving blockade of the APE1/Ref-1 signaling pathway: An investigation into drug synthetic lethality and anti-neuropathy therapeutic approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4802.