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1. Activation of the integrated stress response (ISR) pathways in response to Ref-1 inhibition in human pancreatic cancer and its tumor microenvironment

   https://doi.org/10.3389/fmed.2023.1146115  

   Mijit, Mahmut; Boner, Megan; Cordova, Ricardo A; Gampala, Silpa; Kpenu, Eyram; Klunk, Angela J; Zhang, Chi; Kelley, MarK R; Staschke, Kirk A; Fishel, Melissa L (April 2023, Frontiers in Medicine)

   Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment. 

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2. Targeting APE1 endonuclease activity impairs metastasis and enhances genotoxic therapy response in pancreatic cancer

   https://doi.org/10.1186/s13046-026-03705-7  

   Kpenu, Eyram K; Mijit, Mahmut; Gampala, Silpa; Liu, Sheng; Wan, Jun; Wireman, Randall S; Peil, Jacqueline; Mitchell, Dana K; Haiaty, Sanya; Sardar, Rajesh; et al (December 2026, Journal of Experimental & Clinical Cancer Research)

   Pancreatic ductal adenocarcinoma (PDAC) is a highly deadly cancer with limited treatment options. The base excision repair (BER) pathway, crucial for fixing DNA abasic sites, is driven by apurinic/apyrimidinic endonuclease 1 (APE1). While APE1 redox function in PDAC has been extensively studied, its endonuclease activity in PDAC homeostasis and therapeutic response remains poorly understood. We created stable, homozygous APE1 endonuclease-reduced PDAC cell lines to examine the effects of impaired BER activity on pancreatic cancer growth and response to treatment. MethodsCRISPR/Cas9-mediated editing was used to introduce an E96A mutation into the Pa03C PDAC cell line, generating three clonal mutant cell lines: E96A B1, E96A B4, E96A G8. APE1 expression and activity were verified in vitro through biochemical assays. Cellular responses to genotoxic stress were examined using cytotoxicity, colony formation, and DNA damage assays. Transcriptomic changes were evaluated via RNA sequencing. In vivo tumor growth and metastatic dissemination were studied in orthotopic PDAC mouse models, with and without treatment. ResultsThe E96A mutant cell lines exhibited significantly decreased endonuclease activity but showed no changes in redox signaling or APE1 protein expression. Short-term cytotoxic assays revealed no enhancement in acute sensitivity; however, long-term assessment demonstrated a proliferative defect and a vulnerability to genotoxic stress. Quantitation of nuclear and mitochondrial DNA damage showed mutant cells accumulated significantly more damage to both genomes compared to controls. Transcriptomic analysis revealed that the mutant cell lines maintain a stressed phenotype at baseline, which becomes more pronounced following DNA damage. In vivo, E96A mutants had notably lower tumor burden and metastasis without treatment, and the mutation potentiated the effect of the alkylating drug temozolomide, which inhibited tumor growth and metastasis in a dose-dependent manner. ConclusionWe established the first stable human PDAC cell models deficient in APE1 endonuclease activity. Our findings demonstrate that selective impairment of APE1’s DNA repair function expands therapeutic options by lowering the threshold for effective processing of DNA damage, validating combination treatments with targeted inhibitors and DNA-damaging agents. Targeting APE1 endonuclease activity represents a promising therapeutic strategy for PDAC, capable of suppressing metastatic spread and enhancing tumor responsiveness to genotoxic therapies. 

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3. Dual-hit strategy for therapeutic targeting of pancreatic cancer in patient-derived xenograft tumors

   https://doi.org/10.1158/1078-0432.CCR-23-0131  

   Roy Chaudhuri, Tista; Lin, Qingxiang; Stachowiak, Ewa K.; Rosario, Spencer R.; Spernyak, Joseph A.; Ma, Wen Wee; Stachowiak, Michal K.; Greene, Michelle K.; Quinn, Gerard P.; McDade, Simon S.; et al (January 2024, Clinical Cancer Research)

   Abstract Purpose: Paracrine activation of pro-fibrotic hedgehog (HH) signaling in pancreatic ductal adenocarcinoma (PDAC) results in stromal amplification that compromises tumor drug delivery, efficacy, and patient survival. Interdiction of HH-mediated tumor-stroma crosstalk with smoothened (SMO) inhibitors (SHHi) ‘primes’ PDAC patient-derived xenograft (PDX) tumors for increased drug delivery by transiently increasing vascular patency/permeability, and thereby macromolecule delivery. However, patient tumor isolates vary in their responsiveness, and responders show co-induction of epithelial-mesenchymal transition (EMT). We aimed to identify the signal derangements responsible for EMT induction and reverse them, and devise approaches to stratify SHHi-responsive tumors non-invasively based on clinically-quantifiable parameters. Experimental design: Animals underwent diffusion-weighted magnetic resonance (DW-MR) imaging for measurement of intra-tumor diffusivity. In parallel, tissue-level deposition of nanoparticle probes was quantified as a marker of vascular permeability/perfusion. Transcriptomic and bioinformatic analysis was employed to investigate SHHi-induced gene reprogramming and identify key ‘nodes’ responsible for EMT induction. Results: multiple patient tumor isolates responded to short-term SHH inhibitor exposure with increased vascular patency and permeability, with proportionate increases in tumor diffusivity. Non-responding PDXs did not. SHHi-treated tumors showed elevated FGF drive and distinctly higher nuclear localization of fibroblast growth factor receptor (FGFR1) in EMT-polarized tumor cells. Pan-FGFR inhibitor NVP-BGJ398 (Infigratinib) reversed the SHHi-induced EMT marker expression and nuclear FGFR1 accumulation without compromising the enhanced permeability effect. Conclusion: This dual-hit strategy of SMO and FGFR inhibition provides a clinically-translatable approach to compromise the profound impermeability of PDAC tumors. Furthermore, clinical deployment of DW-MR imaging could fulfill the essential clinical-translational requirement for patient stratification. 

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4. Intracellular Salmonella delivery of an exogenous immunization antigen refocuses CD8 T cells against cancer cells, eliminates pancreatic tumors and forms antitumor immunity

   https://doi.org/10.3389/fimmu.2023.1228532  

   Raman, Vishnu; Howell, Lars M.; Bloom, Shoshana M.; Hall, Christopher L.; Wetherby, Victoria E.; Minter, Lisa M.; Kulkarni, Ashish A.; Forbes, Neil S. (October 2023, Frontiers in Immunology)

   IntroductionImmunotherapies have shown great promise, but are not effective for all tumors types and are effective in less than 3% of patients with pancreatic ductal adenocarcinomas (PDAC). To make an immune treatment that is effective for more cancer patients and those with PDAC specifically, we genetically engineered Salmonella to deliver exogenous antigens directly into the cytoplasm of tumor cells. We hypothesized that intracellular delivery of an exogenous immunization antigen would activate antigen-specific CD8 T cells and reduce tumors in immunized mice. MethodsTo test this hypothesis, we administered intracellular delivering (ID) Salmonella that deliver ovalbumin as a model antigen into tumor-bearing, ovalbumin-vaccinated mice. ID Salmonella delivers antigens by autonomously lysing in cells after the induction of cell invasion. ResultsWe showed that the delivered ovalbumin disperses throughout the cytoplasm of cells in culture and in tumors. This delivery into the cytoplasm is essential for antigen cross-presentation. We showed that co-culture of ovalbumin-recipient cancer cells with ovalbumin-specific CD8 T cells triggered a cytotoxic T cell response. After the adoptive transfer of OT-I CD8 T cells, intracellular delivery of ovalbumin reduced tumor growth and eliminated tumors. This effect was dependent on the presence of the ovalbumin-specific T cells. Following vaccination with the exogenous antigen in mice, intracellular delivery of the antigen cleared 43% of established KPC pancreatic tumors, increased survival, and prevented tumor re-implantation. DiscussionThis response in the immunosuppressive KPC model demonstrates the potential to treat tumors that do not respond to checkpoint inhibitors, and the response to re-challenge indicates that new immunity was established against intrinsic tumor antigens. In the clinic, ID Salmonella could be used to deliver a protein antigen from a childhood immunization to refocus pre-existing T cell immunity against tumors. As an off-the-shelf immunotherapy, this bacterial system has the potential to be effective in a broad range of cancer patients. 

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5. Nanoparticles for Delivery of Encapsulated Drugs to Hypoxic Pancreatic Ductal Adenocarcinoma Cell Spheroids

   https://doi.org/10.1021/acs.molpharmaceut.5c01395  

   Ozoude, Chukwuebuka; Edvall, Connor; Ambhore, Shubhashri; Kale, Narendra; S_Ramakrishnan, Yogaraj; Hossain, Rayat; Van_Horsen, Karl; Tani, Sakurako; Choi, Yongki; Sathish, Venkatachalem; et al (February 2026, Molecular Pharmaceutics)

   Tumor hypoxia promotes angiogenesis, dysfunctional vascular formation, and the epithelial-to-mesenchymal transition phenotype. We propose that a CXCR4 inhibitor (AMD3100) could decrease cancer stemness and improve the efficacy of the anticancer drug gemcitabine (GEM) in targeting hypoxic pancreatic ductal adenocarcinoma (PDAC) cells. In this study, we synthesized a hypoxia-responsive polymer containing poly(lactic acid)-diazobenzene-poly(ethylene glycol) to produce hypoxia-responsive polymersomes. Cell viability experiments showed that AMD3100 enhanced the cytotoxicity of GEM-encapsulated polymersomes under hypoxic conditions compared to normoxia. The combined treatment significantly elevated pro-apoptotic BAX mRNA levels and reduced anti-apoptotic BCL2 mRNA levels. Additionally, the combination therapy decreased the size of cancer cell spheroids from PANC-1 and patient-derived cells. 

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