Michelle Grimard

Applying Small Molecule Signal Transducer and Activator of Transcription-3 (STAT3) Protein Inhibitors as Pancreatic Cancer Therapeutics

Constitutively activated STAT3 protein has been found to be a key regulator of pancreatic cancer and a target for molecular therapeutic intervention. In this study, PG-S3-001, a small molecule derived from the SH-4-54 class of STAT3 inhibitors, was found to inhibit patient-derived pancreatic cancer cell proliferation in vitro and in vivo in the low micromolar range. PG-S3-001 binds the STAT3 protein potently, Kd = 324 nmol/L by surface plasmon resonance, and showed no effect in a kinome screen (>100 cancer-relevant kinases). In vitro studies demonstrated potent cell killing as well as inhibition of STAT3 activation in pancreatic cancer cells. To better model the tumor and its microenvironment, we utilized three-dimensional (3D) cultures of patient-derived pancreatic cancer cells in the absence and presence of cancer-associated fibroblasts (CAF). In this coculture model, inhibition of tumor growth is maintained following STAT3 inhibition in the presence of CAFs. Confocal microscopy was used to verify tumor cell death following treatment of 3D cocultures with PG-S3-001. The 3D model was predictive of in vivo efficacy as significant tumor growth inhibition was observed upon administration of PG-S3-001. These studies showed that the inhibition of STAT3 was able to impact the survival of tumor cells in a relevant 3D model, as well as in a xenograft model using patient-derived cells. Mol Cancer Ther; 15(5); 794–805. ©2016 AACR.

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.

APE1/Ref‐1 knockdown in pancreatic ductal adenocarcinoma – characterizing gene expression changes and identifying novel pathways using single‐cell RNA sequencing

Apurinic/apyrimidinic endonuclease 1/redox factor‐1 (APE1/Ref‐1 or APE1) is a multifunctional protein that regulates numerous transcription factors associated with cancer‐related pathways. Because APE1 is essential for cell viability, generation of APE1‐knockout cell lines and determining a comprehensive list of genes regulated by APE1 has not been possible. To circumvent this challenge, we utilized single‐cell RNA sequencing to identify differentially expressed genes (DEGs) in relation to APE1 protein levels within the cell. Using a straightforward yet novel statistical design, we identified 2837 genes whose expression is significantly changed following APE1 knockdown. Using this gene expression profile, we identified multiple new pathways not previously linked to APE1, including the EIF2 signaling and mechanistic target of Rapamycin pathways and a number of mitochondrial‐related pathways. We demonstrate that APE1 has an effect on modifying gene expression up to a threshold of APE1 expression, demonstrating that it is not necessary to completely knockout APE1 in cells to accurately study APE1 function. We validated the findings using a selection of the DEGs along with siRNA knockdown and qRT‐PCR. Testing additional patient‐derived pancreatic cancer cells reveals particular genes (ITGA1, TNFAIP2, COMMD7, RAB3D) that respond to APE1 knockdown similarly across all the cell lines. Furthermore, we verified that the redox function of APE1 was responsible for driving gene expression of mitochondrial genes such as PRDX5 and genes that are important for proliferation such as SIPA1 and RAB3D by treating with APE1 redox‐specific inhibitor, APX3330. Our study identifies several novel genes and pathways affected by APE1, as well as tumor subtype specificity. These findings will allow for hypothesis‐driven approaches to generate combination therapies using, for example, APE1 inhibitor APX3330 with other approved FDA drugs in an innovative manner for pancreatic and other cancer treatments.

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.

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.