Pdx-1 Cre Research Articles

Abstract PR-01: Systemic targeting of therapeutic RNA to pancreatic ductal adenocarcinoma via a novel, cell-penetrating, and nucleic acid-binding monoclonal antibody

Abstract There is intense interest in developing nucleic acid ligands for immune stimulation of the tumor microenvironment via pattern recognition receptors (PRRs), particularly for treating “cold” tumors like pancreatic ductal adenocarcinoma (PDAC). However, selective delivery of intact and functional RNAs after intravenous administration in a tumor-specific manner to avoid systemic toxicity has been challenging. Many current efforts rely on the direct intra-tumoral injection of RNAs or other stimulatory immune ligands, which is therapeutically sub-optimal, especially for pancreatic tumors and metastases. Here, we present a groundbreaking and unique cancer treatment approach using a lupus-derived, cell-penetrating antibody to deliver nucleic acids to PDAC tumors in vivo. This antibody, a modified version of the initially reported 3E10-D31N, designated TMAB3, forms non-covalent complexes with RNAs. We tested our delivery platform in genetically engineered mouse models (Pdx-1 Cre KrasG12D) and flank and orthoptic allographs of pancreatic cancer (Pdx-1 Cre KrasG12D, p53R172H/- KPC cells) in immunocompetent models. Using 3p-hpRNA, an agonist of the pattern recognition receptor retinoic acid-inducible gene-I (RIG-I), we observed robust anti-tumor efficacy of systemically administered TMAB3/3p-hpRNA complexes in an orthotopic pancreatic cancer model. Treatment tripled animal survival and decreased tumor growth, specifically targeting the malignant cells, with a 1500-fold difference in RNA delivery into tumor cells versus non-malignant cells within the tumor mass. While an immunosuppressive microenvironment typically characterizes PDAC, we found that RIG-I stimulation via TMAB3/3p-hpRNA treatment elicited a potent anti-tumoral immune response characterized by induction of apoptosis in tumor cells and infiltration of CD8+ T cells using single-cell RNASeq and confirming using FACS and immunocompromised mice. In vitro and in vivo studies demonstrated that cancer-cell delivery was mediated by nucleoside transporter ENT2, which is highly expressed in PDAC compared to healthy and benign tissues. These results were recapitulated in orthogonal models of immunologically cold tumors, including melanoma and medulloblastoma. Together, these studies demonstrate that the TMAB3 antibody can 1) precisely localize and enter pancreatic tumor cells, 2) promote CD8+ T cell infiltrates, and 3) rapidly deliver RNA payloads specifically to tumors. Given the known stromal barrier in pancreatic ductal adenocarcinoma, TMAB3 provides a novel targeted delivery platform for a broad range of therapeutics for these difficult-to-treat tumors. Citation Format: Diana Martinez-Saucedo, Elias Quijano, Zaira Ianniello, Madison Rackear, Yanfeng Liu, Denise Hegan, Haoting Chen, Xinning Shan, Robert Tseng, Deanne Yugawa, Natasha Pinto-Medici, Sumedha Chowdhury, Ranjit S Bindra, Marie E Robert, W. Mark Saltzman, Luisa F Escobar-Hoyos, Peter M Glazer. Systemic targeting of therapeutic RNA to pancreatic ductal adenocarcinoma via a novel, cell-penetrating, and nucleic acid-binding monoclonal antibody [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr PR-01.

UPF1 deficiency enhances mitochondrial ROS which promotes an immunosuppressive microenvironment in pancreatic ductal adenocarcinoma

Upstream frameshift 1 (UPF1) is an RNA helicase involved in a number of mRNA regulatory processes including nonsense-mediated decay. Mutations in the UPF1 locus that reduce its expression have been associated with adenosquamous carcinoma of the pancreas, a particularly aggressive form of the disease. To determine the effect of Upf1 suppression in a murine model of pancreatic adenocarcinoma, we silenced with shRNA Upf1 in cells derived from an autochthonous tumor in an LSL-Kras G12D/+ ; Trp53 R172H/+ ; Pdx-1 Cre/+ mouse (KPC) and orthotopically implanted these cells in the pancreas of C57BL/6 mice. Tumors derived from Upf1-deficient cells were markedly larger than those derived from control cells, a difference observed only in immunocompetent mice. The immune infiltrate of Upf1-deficient tumors was enriched in myeloid-derived suppressor cells (MDSCs) and depleted of CD8 + cells compared to control KPC tumors. Upf1-deficient KPC cells secreted inflammatory cytokines including G-CSF and CXCL2, known to recruit MDSCs. Cytokine secretion from Upf1-deficient KPC cells was induced by increased levels of mitochondrial reactive oxygen species (ROS), which in turn were due to an increase in complex I activity in the electron transport chain. Thus, Upf1 helicase deficiency leads to increased mitochondrial complex I activity which produces ROS that signals for cytokine release that drives immune suppression and enhanced tumor growth.

PAF1 cooperates with YAP1 in metaplastic ducts to promote pancreatic cancer

Acinar-to-ductal metaplasia (ADM) is a precursor lesion of pancreatic ductal adenocarcinoma (PDAC); however, the regulators of the ADM-mediated PDAC development and its targeting are poorly understood. RNA polymerase II-associated factor 1 (PAF1) maintains cancer stem cells leading to the aggressiveness of PDAC. In this study, we investigated whether PAF1 is required for the YAP1-mediated PDAC development and whether CA3 and verteporfin, small molecule inhibitors of YAP1/TEAD transcriptional activity, diminish pancreatic cancer (PC) cell growth by targeting the PAF1/YAP1 axis. Here, we demonstrated that PAF1 co-expresses and interacts with YAP1 specifically in metaplastic ducts of mouse cerulein- or KrasG12D-induced ADM and human PDAC but not in the normal pancreas. PAF1 knockdown (KD) reduced SOX9 in PC cells, and the PC cells showed elevated PAF1/YAP1 complex recruitment to the promoter of SOX9. The PAF1 KD reduced the 8xTEAD and SOX9 promoter-luciferase reporter activities in the mouse KC (KrasG12D; Pdx-1 Cre) cells and human PC cells, indicating that the PAF1 is required for the YAP1-mediated development of ADM and PC. Moreover, treatment with CA3 or verteporfin reduced the expressions of PAF1, YAP1, TEAD4, and SOX9 and decreased colony formation and stemness in KC and PC cells. CA3 treatment also reduced the viability and proliferation of PC cells and diminished the duct-like structures in KC acinar explants. CA3 or verteporfin treatment decreased the recruitment of the PAF1/YAP1 complex to the SOX9 promoter in PC cells and reduced the 8xTEAD and SOX9 promoter-luciferase reporter activities in KC and PC cells. Overall, PAF1 cooperates with YAP1 during ADM and PC development, and verteporfin and CA3 inhibit ADM and PC cell growth by targeting the PAF1/YAP1/SOX9 axis in vitro and ex vivo models. This study identified a regulatory axis of PDAC initiation and its targeting, paving the way for developing targeted therapeutic strategies for pancreatic cancer patients.

Exposure to perfluorooctanoic acid leads to promotion of pancreatic cancer.

Pancreatic cancer is the fourth leading cause of cancer deaths in the United States. Perfluorooctanoic acid (PFOA), a persistent environmental pollutant, has been shown to induce pancreatic acinar cell tumors in rats. Human epidemiologic studies have linked PFOA exposure to adverse chronic health effects including several types of cancer. Previously, we demonstrated that PFOA induces oxidative stress and focal ductal hyperplasia in the mouse pancreas. Here, we evaluated whether PFOA promotes pancreatic cancer using the LSL-KRasG12D;Pdx-1 Cre (KC) mouse model of pancreatic cancer. KC mice were exposed to 5ppm PFOA in drinking water starting at 8 weeks of age and analyzed at 6 and 9 months of age. At the 6-month time point, PFOA exposure increased pancreatic intraepithelial neoplasia (PanIN) area by 58%, accompanied by a 2-fold increase in lesion number. Although PanIN area increased at 9 months, relative to 6 months, no treatment effect was observed. Collagen deposition was enhanced by PFOA at both the 6- and 9-month time points. PFOA also induced oxidative stress in the pancreas evidenced by elevated antioxidant activity of superoxide dismutase (Sod), catalase and thioredoxin reductase, and a ~3-fold increase in Sod1 mRNA and protein levels at 6 months. Although antioxidant activity was not enhanced by PFOA exposure at the 9-month time point, increased pancreatic oxidative damage was observed. Collectively, these results show that PFOA elicited temporal increases in PanIN lesion area and desmoplasia concomitant with the induction of oxidative stress, demonstrating that it functions to promote pancreatic cancer progression.

Abstract PR-009: Targeting the sterol regulatory element-binding protein pathway in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a very aggressive tumor with limited diagnostic and therapeutic options. Due to its proliferative nature and thick, desmoplastic stroma, PDAC tumor cells are challenged with meeting a high demand for lipids in a hypoxic, lipid-poor environment. Cancer cells respond to lipid conditions through sterol regulatory element-binding proteins (SREBPs). SREBPs are master transcriptional regulators of lipid homeostasis and require SREBP cleavage activating protein (SCAP) during signaling. While the SREBP pathway has been implicated in several cancers, its role in PDAC has not been examined. In this study, we assess the requirement of this pathway using both in vitro and in vivo model systems. Furthermore, we target the SREBP pathway using a novel combination therapy of two FDA-approved compounds: dipyridamole and statins. Methods: We acquired four patient-derived pancreatic adenocarcinoma cell lines containing mutations in both Kras and p53 and knocked out SCAP in each cell line followed by gene-based rescue. Functional growth assays were performed in both lipid-poor and lipid-rich media conditions. Subcutaneous and pancreatic orthotopic xenografts were performed in nude mice using these same cell lines. A previously established mouse line, LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1 Cre (KPC) on a C57Bl/6 background, was utilized as a PDAC model. KPC mice lacking Scap in one or both alleles (Sfl/+ or Sfl/fl) were generated. Cohort survival and histologic analysis were performed for all mice, and results plotted on a Kaplan-Meier survival curve. In each patient cell line, the following drugs were applied individually and in combination: Dipyridamole (DP), tetramethyl-DP (TMDP), Fluvastatin, and Simvastatin. Cell viability post-treatment was assessed, and synergy calculated for each combination using SynergyFinder. Results: In lipid-poor conditions, SCAP knockout cells showed significantly reduced growth when compared to wild type or SCAP rescued cells. In tumor xenograft models, SCAP knockout cells exhibited reduced tumor growth and tumor volume when compared to wild-type cells. In the genetically engineered mouse models, KPC mice exhibited a median survival time of 289 days, while KPCSfl/+ mice show a significantly increased median survival time of 425 days. Additionally, KPCSfl/fl mice did not develop invasive PDAC. Cells treated individually with DP, TMDP and both statins exhibit lipid-dependent growth defects in all cell lines tested. In combination, DP with either statin as well as TMDP with either statin demonstrate synergy in lipid-poor conditions. Conclusions: Our results demonstrate that loss of SCAP in PDAC tumor cells alters the growth capability both in vitro and using mouse xenograft models. Additionally, heterozygous loss of Scap in the KPC mouse model significantly increased survival. Finally, dipyridamole works in synergy with statins to alter growth of PDAC tumor cells. These findings suggest that targeting the SREBP pathway has significant therapeutic potential in PDAC. Citation Format: Stephanie Myers, Meredith McGuire, Wei Shao, Chine Liu, Theodore Ewachiw, Zeshaan Rasheed, William Matsui, Toni Sepalla, Richard Burkhart, Peter Espenshade. Targeting the sterol regulatory element-binding protein pathway in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PR-009.

PGC1α-Mediated Metabolic Reprogramming Drives the Stemness of Pancreatic Precursor Lesions.

Metabolic reprogramming and cancer stem cells drive the aggressiveness of pancreatic ductal adenocarcinoma (PDAC). However, the metabolic and stemness programs of pancreatic precursor lesions (PPL), considered early PDAC development events, have not been thoroughly explored. Meta-analyses using gene expression profile data from NCBI Gene Expression Omnibus and IHC on tissue microarrays (TMA) were performed. The following animal and cellular models were used: cerulean-induced KrasG12D; Pdx1 Cre (KC) acinar-to-ductal metaplasia (ADM) mice, KrasG12D; Smad4Loss; Pdx-1 Cre (KCSmad4-) intraductal papillary mucinous neoplasm (IPMN) mice, LGKC1 cell line derived from the doxycycline-inducible Gnas IPMN model, and human IPMN organoids. Flow cytometry, Seahorse extracellular flux analyzer, qRT-PCR, and sphere assay were used to analyze metabolic and stemness features. SR18292 was used to inhibit PGC1α, and short hairpin RNA was used to knockdown (KD) PGC1α. The meta-analysis revealed a significant upregulation of specific stemness genes in ADM-mediated pancreatic intraepithelial neoplasms (PanIN) and IPMN. Meta- and TMA analyses followed by in vitro and in vivo validation revealed that ADM/PanIN exhibit increased PGC1α and oxidative phosphorylation (OXPhos) but reduced CPT1A. IPMN showed elevated PGC1α, fatty acid β-oxidation (FAO) gene expression, and FAO-OXPhos. PGC1α was co-overexpressed with its coactivator NRF1 in ADM/PanINs and with PPARγ in IPMN. PGC1α KD or SR18292 inhibited the specific metabolic and stemness features of PPLs and repressed IPMN organoid growth. ADM/PanINs and IPMNs show specific stemness signatures with unique metabolisms. Inhibition of PGC1α using SR18292 diminishes the specific stemness by targeting FAO-independent and FAO-dependent OXPhos of ADM/PanINs and IPMNs, respectively.

Secretory Mucin 5AC Promotes Neoplastic Progression by Augmenting KLF4-Mediated Pancreatic Cancer Cell Stemness.

Secreted mucin 5AC (MUC5AC) is the most abundantly overexpressed member of the mucin family during early pancreatic intraepithelial neoplasia stage I (PanIN-I) of pancreatic cancer. To comprehend the contribution of Muc5ac in pancreatic cancer pathology, we genetically ablated it in an autochthonous murine model (KrasG12D; Pdx-1cre, KC), which mirrors the early stages of pancreatic cancer development. Neoplastic onset and the PanIN lesion progression were significantly delayed in Muc5ac knockout (KrasG12D; Pdx-1 cre; Muc5ac-/-, KCM) animals with a 50% reduction in PanIN-2 and 70% reduction in PanIN-3 lesions compared with KC at 50 weeks of age. High-throughput RNA-sequencing analysis from pancreatic tissues of KCM animals revealed a significant decrease in cancer stem cell (CSC) markers Aldh1a1, Klf4, EpCAM, and CD133. Furthermore, the silencing of MUC5AC in human pancreatic cancer cells reduced their tumorigenic propensity, as indicated by a significant decline in tumor formation frequency by limiting dilution assay upon subcutaneous administration. The contribution of MUC5AC in CSC maintenance was corroborated by a significant decrease in tumor burden upon orthotopic implantation of MUC5AC-depleted pancreatic cancer cells. Mechanistically, MUC5AC potentiated oncogenic signaling through integrin αvβ5, pSrc (Y416), and pSTAT3 (Y705). Phosphorylated STAT3, in turn, upregulated Klf4 expression, thereby enriching the self-renewing CSC population. A strong positive correlation of Muc5ac with Klf4 and pSTAT3 in the PanIN lesions of KC mouse pancreas reinforces the crucial involvement of MUC5AC in bolstering the CSC-associated tumorigenic properties of Kras-induced metaplastic cells, which leads to pancreatic cancer onset and progression. SIGNIFICANCE: This study elucidates that de novo expression of MUC5AC promotes cancer cell stemness during Kras-driven pancreatic tumorigenesis and can be targeted for development of a novel therapeutic regimen.

Selective inhibition of stemness through EGFR/FOXA2/SOX9 axis reduces pancreatic cancer metastasis.

Pancreatic cancer (PC) is difficult to defeat due to mechanism (s) driving metastasis and drug resistance. Cancer stemness is a major challenging phenomenon associated with PC metastasis and limiting therapy efficacy. In this study, we evaluated the pre-clinical and clinical significance of eradicating pancreatic cancer stem cells (PCSC) and its components using a pan-EGFR inhibitor afatinib in combination with gemcitabine. Afatinib in combination with gemcitabine, significantly reduced KrasG12D/+; Pdx-1 Cre (KC) (P<0.01) and KrasG12D/+; p53R172H/+; Pdx-1 Cre (KPC) (P<0.05) derived mouse tumoroids and KPC-derived murine syngeneic cell line growth compared to gemcitabine/afatinib alone treatment. The drug combination also reduced PC xenograft tumor burden (P<0.05) and the incidence of metastasis by affecting key stemness markers, as confirmed by co-localization studies. Moreover, the drug combination significantly decreases the growth of various PC patient-derived organoids (P<0.001). We found that SOX9 is significantly overexpressed in high-grade PC tumors (P<0.05) and in chemotherapy-treated patients compared to chemo-naïve patients (P<0.05). These results were further validated using publicly available datasets. Moreover, afatinib alone or in combination with gemcitabine decreased stemness and tumorspheres by reducing phosphorylation of EGFR family proteins, ERK, FAK, and CSC markers. Mechanistically, afatinib treatment decreased CSC markers by downregulating SOX9 via FOXA2. Indeed, EGFR and FOXA2 depletion reduced SOX9 expression in PCSCs. Taken together, pan EGFR inhibition by afatinib impedes PCSCs growth and metastasis via the EGFR/ERK/FOXA2/SOX9 axis. This novel mechanism of panEGFR inhibitor and its ability to eradicate CSC may serve as a tailor-made approach to enhance chemotherapeutic benefits in other cancer types.

Nanoscale platform for delivery of active IRINOX to combat pancreatic cancer

Due to its late diagnosis and dismal prognosis, pancreatic ductal adenocarcinoma (PDAC) is one of the most devastating solid malignancies, with only 9% of patients surviving after being diagnosed. A multidrug chemotherapeutic regimen FOL-F-IRIN-OX (combination of 5-fluorouracil, leucovorin, irinotecan, and oxaliplatin) offers survival benefits superior to that of gemcitabine single agent, but the treatment-related side effects are also severe. To overcome this therapeutic barrier, we developed polymeric micelles bearing active formats of irinotecan and oxaliplatin, SN38 and 1,2-diaminocyclohexane‑platinum (II), DACHPt. Crosslinked micelles were prepared using amphiphilic PEG-b-poly(L-glutamic acid)/SN38 conjugates and subsequently loaded with DACHPt. The dual drug-loaded micelles exhibited improved colloidal stability, prolonged drug release and remarkable cytotoxicity in human pancreatic cancer cell lines and KrasG12D; Trp52R172H/+; Pdx-1 Cre murine tumor organoids models. In vivo, (SN38 + DACHPt)-loaded micelles displayed superior antitumor and antimetastatic activities without impairing safety. Our results suggest that nanomedicine mimicking irinotecan and oxaliplatin as parts of FOLFIRINOX regimen may further improve the feasibility of this multidrug treatment for patients with advanced pancreatic cancer.

Metabolic programming of distinct cancer stem cells promotes metastasis of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) metastasizes to distant organs, which is the primary cause of mortality; however, specific features mediating organ-specific metastasis remain unexplored. Emerging evidence demonstrates that cancer stem cells (CSCs) and cellular metabolism play a pivotal role in metastasis. Here we investigated the role of distinct subtypes of pancreatic CSCs and their metabolomic signatures in organ-specific metastatic colonization. We found that PDAC consists of ALDH+/CD133+ and drug-resistant (MDR1+) subtypes of CSCs with specific metabolic and stemness signatures. Human PDAC tissues with gemcitabine treatment, autochthonous mouse tumors from KrasG12D; Pdx1-Cre (KC) and KrasG12D; Trp53R172H; Pdx-1 Cre (KPC) mice, and KPC- Liver/Lung metastatic cells were used to evaluate the CSC, EMT (epithelial-to-mesenchymal transition), and metabolic profiles. A strong association was observed between distinct CSC subtypes and organ-specific colonization. The liver metastasis showed drug-resistant CSC- and EMT-like phenotype with aerobic glycolysis and fatty acid β-oxidation-mediated oxidative (glyco-oxidative) metabolism. On the contrary, lung metastasis displayed ALDH+/CD133+ and MET-like phenotype with oxidative metabolism. These results were obtained by evaluating FACS-based side population (SP), autofluorescence (AF+) and Alde-red assays for CSCs, and Seahorse-based oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and fatty acid β-oxidation (FAO)-mediated OCR assays for metabolic features along with specific gene signatures. Further, we developed in vitro human liver and lung PDAC metastasis models by using a combination of liver or lung decellularized scaffolds, a co-culture, and a sphere culture methods. PDAC cells grown in the liver-mimicking model showed the enrichment of MDR1+ and CPT1A+ populations, whereas the PDAC cells grown in the lung-mimicking environment showed the enrichment of ALDH+/CD133+ populations. In addition, we observed significantly elevated expression of ALDH1 in lung metastasis and MDR1/LDH-A expression in liver metastasis compared to human primary PDAC tumors. Our studies elucidate that distinct CSCs adapt unique metabolic signatures for organotropic metastasis, which will pave the way for the development of targeted therapy for PDAC metastasis.

Abstract 3811: Circadian disruption enhances development of pancreatic inflammation and pancreatic cancer precursors

Abstract Introduction: Emerging data suggest a role for circadian disruption in tumor formation and progression. Yet little is known regarding the effects of circadian disruption on the oncogenesis of pancreatic ductal adenocarcinoma (PDAC). We sought to determine if circadian disruption enhances PDAC development through a genetically engineered mouse model, and evaluate possible mechanisms with transcriptomic analysis. Methods: KrasG12D/+ mice (K) were crossed with Pdx-1 Cre (C) mice on a C57BL/6 background to generate KC mice. At 4 weeks, mice were subjected to standard lighting conditions (KC normal circadian [KCNC]) or a chronic jet-lag protocol known to induce circadian disruption (KCCD), with light/dark cycles shifted 8 hours every 2-3 days. Mice were sacrificed at 9 months for histologic analysis, examining for PDAC and its precursor lesions (PanINs). Comparisons were made with Fischer's Exact Test. For pancreatic transcriptomic profiling, 144 4-week-old wild-type (WT) C57BL/6 mice underwent disruption (WTCD, n = 72) or standard lighting conditions (WTNC, n = 72) for 4 weeks to identify cycling genes over a 48 hour period. Four weeks was chosen to understand the effects of disruption on gene expression impacting tumor initiation. Standard gene cycling analysis was performed with meta2d utilizing a false discovery rate of q = 0.1, and comparisons were made with the Audic-Claverie Distribution tool. Results: All KC mice (27 KCNC &amp; 7 KCCD) exhibited chronic pancreatitis and PanIN-1 (p = 1). However, KCCD mice developed higher rates of acute pancreatitis (86% vs 4%; p &amp;lt; 0.01) and PanIN-2 (43% vs 7%; p = 0.04) versus KCNC mice. PanIN-3 (14% vs 7% p = 0.48) and PDAC (29% vs 16% p = 0.6) were also increased but did not reach statistical significance. Given this phenotype, we sought a mechanism through transcriptomic analysis. In total, we found 12.8% of the protein-coding pancreatic transcriptome was cycling in either WTNC or WTCD. Pathway enrichment analysis demonstrated genes important for circadian rhythm were cycling in WTNC, while those important for catabolism and protein localization were cycling in WTCD. Only 129 genes, shared between male and female mice, exclusively cycled following circadian disruption. Of those, roughly 15% have been implicated in PDAC development, reported as prognostic biomarkers in PDAC or possibly contribute to chemotherapeutic resistance in PDAC. Conclusion: Little is known about the effects of circadian disruption on PDAC. We have demonstrated histopathologic evidence of an increased incidence of pancreatic inflammation and pathogenic lesions following circadian disruption. Transcriptomic analysis of wild-type pancreas revealed an increased number of cycling genes involved in energy production following disruption. Future work will investigate the candidate genes identified to ascertain how circadian disruption leads to increased development of pancreatic lesions. Citation Format: Patrick Beard Schwartz, Morgan Walcheck, Noah D. Carrillo, Kristina A. Matkowskyj, Christopher A. Bradfield, Sean Ronnekleiv-Kelly. Circadian disruption enhances development of pancreatic inflammation and pancreatic cancer precursors [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3811.

Predicting Gemcitabine Delivery by 18F-FAC PET in Murine Models of Pancreatic Cancer.

18F-FAC (2'-deoxy-2'-18F-fluoro-β-d-arabinofuranosylcytosine) has close structural similarity to gemcitabine and thus offers the potential to image drug delivery to tumors. We compared tumor 18F-FAC PET images with 14C-gemcitabine levels, established ex vivo, in 3 mouse models of pancreatic cancer. We further modified tumor gemcitabine levels with injectable PEGylated recombinant human hyaluronidase (PEGPH20) to test whether changes in gemcitabine would be tracked by 18F-FAC. Methods:18F-FAC was synthesized as described previously. Three patient-derived xenograft (PDX) models were grown in the flanks of NSG mice. Mice were given PEGPH20 or vehicle intravenously 24 h before coinjection of 18F-FAC and 14C-gemcitabine. Animals were euthanized and imaged 1 h after tracer administration. Tumor and muscle uptake of both 18F-FAC and 14C-gemcitabine was obtained ex vivo. The efficacy of PEPGPH20 was validated through staining with hyaluronic acid binding protein. Additionally, an organoid culture, initiated from a KPC (Pdx-1 Cre LSL-KrasG12D LSL-p53R172H) tumor, was used to generate orthotopically growing tumors in C57BL/6J mice, and these tumors were then serially transplanted. Animals were injected with PEGPH20 and 14C-gemcitabine as described above to validate increased drug uptake by ex vivo assay. PET/MR images were obtained using a PET insert on a 7-T MR scanner. Animals were imaged immediately before injection with PEGPH20 and again 24 h later. Results: Tumor-to-muscle ratios of 14C-gemcitabine and 18F-FAC correlated well across all PDX models and treatments (R2 = 0.78). There was a significant increase in the tumor PET signal in PEGPH20-treated PDX animals, and this signal was matched in ex vivo counts for 2 of 3 models. In KPC-derived tumors, PEGPH20 raised 14C-gemcitabine levels (tumor-to-muscle ratio of 1.9 vs. 2.4, control vs. treated, P = 0.013). PET/MR 18F-FAC images showed a 12% increase in tumor 18F-FAC uptake after PEGPH20 treatment (P = 0.023). PEGPH20-treated animals uniformly displayed clear reductions in hyaluronic acid staining. Conclusion:18F-FAC PET was shown to be a good surrogate for gemcitabine uptake and, when combined with MR, to successfully determine drug uptake in tumors growing in the pancreas. PEGPH20 had moderate effects on tumor uptake of gemcitabine.

Abstract A12: Promotion of pancreatic cancer by perfluorooctanoic acid (PFOA)

Abstract Pancreatic cancer is the fourth leading cause of cancer deaths and one of the most lethal forms of cancer diagnosed in the United States. While the exact cause of pancreatic cancer is unknown, established risk factors for pancreatic cancer include smoking, alcohol consumption, and pancreatitis, all of which share the ability to generate oxidative stress—a condition known to promote cancer progression. Perfluorooctanoic acid (PFOA), a chemical widely used in consumer and industrial applications, has been shown to induce pancreatic acinar cell tumors in rodents through a yet to be determined mechanism. In humans, epidemiologic studies have linked PFOA exposure to adverse chronic health effects including several types of cancer. PFOA has been detected in essentially all of the American population, with mean serum levels close to 4 ng/ml and a predicted half-life of ~4 yrs. We have previously shown that exposure of mice to PFOA for 7 days triggered oxidative stress in the pancreas, which was associated with focal ductal hyperplasia and inflammation. The purpose of this study was to determine if PFOA exposure promotes the progression of pancreatic cancer in a well-characterized mouse model of pancreatic cancer, the LSL-KRasG12D;Pdx-1 Cre (KC) model. For our studies, KC mice received either tap water or tap water containing 5 ppm PFOA, starting at 2 months of age. Mice were sacrificed at 6 and 9 months of age, corresponding to 4 and 7 months of PFOA treatment, at which time serum and tissues were collected. Pancreata were processed for histologic examination (H&amp;E stain), Alcian Blue (AB) staining, used as a marker to identify neoplastic lesion area, and RNA isolation for gene expression. Amylase, lipase, PFOA, and inflammatory cytokines were quantified in serum. Our results show that PFOA accumulates in the serum and pancreas of treated KC mice. In 6-month-old KC mice, exposure to PFOA increased the total AB+ lesion area from 4.87% ± 1.65% to 7.71% ± 3.01%. While the average lesion size was similar between control and PFOA-treated groups, the number of lesions per mm2 increased from 8.73 ± 3.19 to 16.73 ± 6.31, demonstrating that PFOA exposure leads to increased lesion development. qPCR analysis of pancreata verified increased expression of lesion markers CK19 and Sox9 in PFOA-treated mice. Analysis of mRNA expression in the pancreas revealed an increase in oxidative stress, evidenced by elevated Sod1 expression, and an increase in inflammatory markers (Tnfα and IL1α). Collectively, these results demonstrate that PFOA leads to expansion of the pancreatic lesion area in the LSL-KRasG12D;Pdx-1 Cre mouse model, which is accompanied by oxidative stress and an inflammatory response. These results, coupled with the widespread human exposure to PFOA and its biologic persistence, suggest that it may participate in promoting pancreatic cancer progression. Citation Format: Barbara A. Hocevar, Lisa M. Kamendulis. Promotion of pancreatic cancer by perfluorooctanoic acid (PFOA) [abstract]. In: Proceedings of the AACR Special Conference on Environmental Carcinogenesis: Potential Pathway to Cancer Prevention; 2019 Jun 22-24; Charlotte, NC. Philadelphia (PA): AACR; Can Prev Res 2020;13(7 Suppl): Abstract nr A12.

1789-P: Inactivation of Acyl-Protein Thioesterase 1, a Major Mediator of Palmitoylation Cycling, Promotes Insulin Secretion and ß-Cell Failure

Palmitoylation cycling involves successive addition and removal of lipid from proteins to regulate cell structure and membrane interactions. Cycling requires depalmitoylation, mediated mostly by the enzyme acyl-protein thioesterase 1 (APT1). To test the hypothesis that depalmitoylation is critical for normal glucose metabolism, we generated global and islet-specific (using tamoxifen-inducible Pdx1 Cre) APT1 knockout mice. Both models on a chow diet had enhanced glucose tolerance due to increased insulin secretion. Pancreatic insulin content was unaffected by APT1 deficiency. Isolated islets from APT1-deficient models demonstrated increased glucose-stimulated insulin secretion under static and dynamic conditions. Quantitative histomorphometry demonstrated β cell hypertrophy in islets from global APT1 KO mice. Calcium dynamics in isolated islets were unaffected by APT1 deficiency, suggesting that increased insulin secretion is mediated downstream of depolarization-dependent calcium influx. APT1 knockdown in replicates of human islets from two donors showed increased insulin secretion. At 4 weeks of age, global APT1 KO db/db mice had improved glucose tolerance due to increased insulin secretion compared to littermate db/db mice. However, by 16 weeks of age APT1-deficient db/db mice had severe glucose intolerance, hypoinsulinemia, and β cell atrophy compared to littermate db/db mice. Islet palmitoylation proteomics identified putative APT1 substrates including Scamp1, -2 and -3. Scamp1 was validated as a palmitoylated protein, APT1 deficiency would be predicted to impair Scamp1 function, and Scamp1 knockdown in insulinoma cells increased insulin secretion. These results suggest that deficiency of APT1, an enzyme susceptible to post-translational regulation in the setting of insulin resistance, causes hypersecretion of insulin that leads to β cell failure, modeling the clinical evolution of human type 2 diabetes. Disclosure G. Dong: None. X. Wei: None. L. Yin: None. C. Feng: None. S. Adak: None. Z.A. Shyr: None. Q. Zhang: None. S. Morikawa: None. R. Asada: None. F. Urano: Stock/Shareholder; Self; CytRx Corporation. Other Relationship; Self; AETAS Pharma, Amylyx Pharmaceuticals, Mitochon, National Center for Advancing Translational Sciences, Novus Biologicals?. M.S. Remedi: None. C.F. Semenkovich: None.

Irreversible and sustained upregulation of endothelin axis during oncogene-associated pancreatic inflammation and cancer

Endothelin-1 (ET-1) and its two receptors, endothelin receptor A (ETAR) and endothelin receptor B (ETBR) exhibit deregulated overexprerssion in pancreatic ductal adenocarcinoma (PDAC) and pancreatitis. We examined the expression pattern of endothelin (ET) axis components in the murine models of chronic and acute inflammation in the presence or absence of oncogenic K-ras. While the expression of endothelin converting enzyme-1 (ECE-1), ET-1, ETAR and ETBR in the normal pancreas is restricted predominantly to the islet cells, progressive increase of ET receptors in ductal cells and stromal compartment is observed in the KC model (Pdx-1 Cre; K-rasG12D) of PDAC. In the murine pancreas harboring K-rasG12D mutation (KC mice), following acute inflammation induced by cerulein, increased ETAR and ETBR expression is observed in the amylase and CK19 double positive cells that represent cells undergoing pancreatic acinar to ductal metaplasia (ADM). As compared to the wild type (WT) mice, cerulein treatment in KC mice resulted in significantly higher levels of ECE-1, ET-1, ETAR and ETBR, transcripts in the pancreas. Similarly, in response to cigarette smoke-induced chronic inflammation, the expression of ET axis components is significantly upregulated in the pancreas of KC mice as compared to the WT mice. In addition to the expression in the precursor pancreatic intraepithelial neoplasm (PanIN lesions) in cigarette smoke-exposure model and metaplastic ducts in cerulein-treatment model, ETAR and ETBR expression is also observed in infiltrating F4/80 positive macrophages and α-SMA positive fibroblasts and high co-localization was seen in the presence of oncogenic K-ras. In conclusion, both chronic and acute pancreatic inflammation in the presence of oncogenic K-ras contribute to sustained upregulation of ET axis components in the ductal and stromal cells suggesting a potential role of ET axis in the initiation and progression of PDAC.

Abstract C33: Effects of curcumin on pancreatic ductal adenocarcinoma

Abstract Without an early method of diagnosis or effective treatment plan, the prognosis for pancreatic ductal adenocarcinoma (PDAC) is grim, which begs for the development of additional treatment options. The plant-derived chemical, curcumin, has recently shown some promise as a potential therapy when tested in xenograft models of PDAC, glioblastoma, lung, breast, cervical, and colon tumors and is currently being tested in early clinical trials of certain cancers. However, efficacy studies against PDAC have not included preclinical mouse models with an intact immune system. To address the potential complication of a fully functional immune system on curcumin effectiveness, we assessed the effects of curcumin on murine PDAC cell lines both in vitro and in vivo. Murine PDAC lines were previously derived from genetically engineered KrasG12D; Trp53R172H; Pdx-1 Cre (KPC) mice, which spontaneously produce metastatic PDAC that closely mimics human disease. The dose- and time-dependent toxicity of curcumin was assessed using various cell lines in vitro, and our findings support previously published results. To assess effectiveness in vivo, tumor cells were implanted subcutaneously into syngeneic, C57Bl/6J mice. Once tumors were established, curcumin (or vehicle alone) was administered via intraperitoneal (i.p.) or intratumoral injection. Treatment efficacy was assessed by measuring tumor growth and the overall survival of mice. We found that curcumin treatment had no impact on tumor growth or overall survival of immunocompetent mice. To circumvent potential solubility issues common to curcumin and illustrate the importance of the immune system in treatment resistance, we repeated our in vivo studies using curcumin encapsulated in liposomes. In addition, we used T cell-depleting antibodies for a subset of mice to mimic athymic mice used in previously published xenograft models. T-cell depletion was verified prior to tumor cell implantation by flow cytometry. Once tumors were established, liposomal curcumin or mock treatment was administered via i.p. injection. For T cell-depleted mice, we found that treatment with liposomal curcumin stalled tumor growth and increased overall survival compared to mock treated mice. For T cell-replete mice, we found that treatment with liposomal curcumin had no impact on tumor growth or overall survival compared to mock treated mice, which agrees with our findings. Taken together, our data suggest that the presence of T cells causes a difference in responsiveness to curcumin in vivo. This finding warrants additional studies in order to understand this mechanism and enhance its potential success in the clinic. Citation Format: Adrianna M. Vaskas, Joseph A. Fraietta, Kristen B. Long. Effects of curcumin on pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C33.

Ultrasound-Guided Orthotopic Implantation of Murine Pancreatic Ductal Adenocarcinoma.

The recent success of immune checkpoint blockade in melanoma and lung adenocarcinoma has galvanized the field of immuno-oncology as well as revealed the limitations of current treatments, as the majority of patients do not respond to immunotherapy. Development of accurate preclinical models to quickly identify novel and effective therapeutic combinations are critical to address this unmet clinical need. Pancreatic ductal adenocarcinoma (PDA) is a canonical example of an immune checkpoint blockade resistant tumor with only 2% of patients responding to immunotherapy. The genetically engineered KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mouse model of PDA recapitulates human disease and is a valuable tool for assessing therapies for immunotherapy resistant in the preclinical setting, but time to tumor onset is highly variable. Surgical orthotopic tumor implantation models of PDA maintain the immunobiological hallmarks of the KPC tissue-specific tumor microenvironment (TME) but require a time-intensive procedure and introduce aberrant inflammation. Here, we use an ultrasound-guided orthotopic tumor implantation model (UG-OTIM) to non-invasively inject KPC-derived PDA cell lines directly into the mouse pancreas. UG-OTIM tumors grow in the endogenous tissue site, faithfully recapitulate histological features of the PDA TME, and reach enrollment-sized tumors for preclinical studies by four weeks after injection with minimal seeding on the peritoneal wall. The UG-OTIM system described here is a rapid and reproducible tumor model that may allow for high throughput analysis of novel therapeutic combinations in the murine PDA TME.

Abstract 115: A CD40 agonist potentiates the efficacy and immune-stimulatory capacity of chemotherapy in combination with a focal adhesion kinase inhibitor in a mouse model of pancreatic ductal adenocarcinoma

Abstract INTRODUCTION: Pancreatic ductal adenocarcinoma (PDAC) has proved to be remarkably resistant to immune based therapies. This resistance may be in part due to the PDAC microenvironment that is characterized by an abundance of myeloid cells, a scarcity of effector T cells and a dense fibrotic stroma. Activation of focal adhesion kinase (FAK) signaling promotes the formation of an immune suppressive microenvironment in PDAC, and FAK inhibition (FAKi) can reduce stromal density and improve the sensitivity of tumors to chemotherapy. Likewise, CD40-dependent innate immune responses can disrupt the stromal matrix that surrounds the tumor and sensitize tumors to the cytotoxic and immune-stimulatory effects of chemotherapy. Here, we hypothesize that FAK signaling may limit the efficacy of CD40 agonists and thus, FAK and CD40 may be non-redundant pathways that can be targeted for improving outcomes to cytotoxic chemotherapy. METHODS: For our studies, we used polyclonal pancreatic cancer cells derived from the KrasG21D/+; Trp53R172H/+; Pdx-1 Cre (KPC) genetic mouse model of PDAC, which recapitulates many of the features of human disease, including a paucity of T cells and a robust infiltration of myeloid cells. KPC cell lines were subcutaneously implanted into syngeneic C57BL/6 mice. Approximately 2 weeks after tumor implantation, mice were block randomized to receive treatment with a CD40 agonist (FGK45) or PBS administered prior to (5 days prior) or after (2 days post) gemcitabine (gem) chemotherapy with or without FAKi. RESULTS: We first examined the impact of chemotherapy in combination with FAKi to enhance the immune stimulatory capacity of a CD40 agonist. We found that the triple combination (FAKi+gem→FGK45), in which FGK45 is given after FAKi and gem, significantly reduced tumor growth, increased tumor necrosis, and improved survival compared to FAKi alone or gem→FGK45. In this treatment setting, both gem→FGK45 and the triple combination significantly increased the CD8+/Foxp3+ ratio and the proliferation of CD8+ T cells in tumors. We next examined the capacity of a CD40 agonist to enhance the efficacy of chemotherapy, with or without FAKi. The triple combination (FGK45→FAKi+gem), in which FGK45 is given before FAKi and gem, significantly inhibited tumor growth and improved survival compared to all other groups. In this treatment setting, both FGK45→FAKi+gem and FGK45→gem increased the number and proliferation of CD3+ T cells in tumors. Importantly, the triple combination significantly increased the CD8+/Foxp3+ ratio compared to FGK45→Gem, which may account in part for the improvement in tumor response. CONCLUSION: FAK and CD40 signaling are non-redundant pathways that can be targeted to reverse immune suppressive features in the tumor microenvironment and improve PDAC sensitivity to chemotherapy. Citation Format: Meredith L. Stone, Kathleen Graham, Daniel Aldridge, Kanika Jain, Xiaoqing Pan, Jonathan Pachter, Gregory Beatty. A CD40 agonist potentiates the efficacy and immune-stimulatory capacity of chemotherapy in combination with a focal adhesion kinase inhibitor in a mouse model of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 115.

Abstract A054: Tumor cell intrinsic factors dictate immune cell infiltration and response to immunotherapy

Abstract The establishment of immune heterogeneity in the tumor microenvironment (TME) is poorly understood, despite recent data that the success of immunotherapies is dictated by the immune environment of the tumor site. Pancreatic ductal adenocarcinoma (PDA) is characteristically devoid of CD8 T-cells and resistant to therapeutic intervention. However, a small subset of patients (15%) have tumors highly infiltrated by CD8 T-cells, correlating with improved overall survival. To better elucidate the determinants of immune heterogeneity in the PDA TME, we generated clones from spontaneous tumors harvested from KrasG12D+/-;Trp53R172H+/-;Pdx-1 Cre (KPC) mice, a genetically engineered mouse model of PDA. Using a panel of 17 tumor clones, we found the clones segregated in to two groups with differential immune cell infiltration upon implantation in congenic C57BL/6 mice. 7/17 tumor clones were categorized as “T-cell high,” with an immune infiltrate comprising CD8 T-cells and CD103+ dendritic cells (DCs). In contrast, the remaining 10 tumor clones were categorized as “T-cell low” lines, with the TME dominated by myeloid cells and macrophages, especially granulocytic myeloid-derived suppressor cells. Hypothesizing that increased T-cell infiltrate would render PDA sensitive to therapy, we treated two T-cell high and two T-cell low tumor clones with combination immunotherapy. Mice bearing T-cell high clones responded to therapy (7/7 and 4/7 mice cured) and formed protective memory responses against secondary tumor challenge, while none of the mice bearing T-cell low tumors responded to treatment (0/7 and 0/7 mice cured). At baseline, T-cell high tumors had similar proportions of functional CD8 T-cells as in T-cell low tumors. However, the proportion of activated CD44hiPD-1+ CD8 T-cells was significantly increased in T-cell high tumors (62.2% vs. 35.1% in T-cell low clones, p&amp;lt;0.0001), and predicted response to therapy across the panel of tumor clones. CD44hiPD-1+ CD8 T-cells trafficked in to the tumor site independently of CXCR3 (53.8% in control vs. 50.0% in anti-CXCR3 treated tumors, p=0.8), in contrast to the bulk CD8 T-cell population which required CXCR3 to enter the TME (7.5% in control vs 0.53% in anti-CXCR3 treated tumors, p&amp;lt;0.01). Furthermore, in Batf3 KO mice, the T-cell high TME lacked CD8 T-cells and resembled the T-cell low phenotype, including resistance to combined immunotherapy with 0/7 mice responding to treatment, indicating the requirement for CD103+ DCs in the TME for T-cell infiltration and tumor regression. Co-mixing experiments revealed that the T-cell low phenotype was dominant in the local – but not systemic – setting, suggesting a secreted protein was involved in establishing the T-cell low TME, and CXCL1 was identified as a top candidate after transcriptomic and epigenetic profiling (padj = 0.088, basemean = 772.79). Overexpression of CXCL1 in T-cell high tumors converted the TME to a T-cell low phenotype, while ablation of CXCL1 in T-cell low tumors converted the TME to a T-cell high phenotype and rendered T-cell low sensitive to combined immunotherapy (16/16 responders or 7/16 responders in two independent CXCL1 KO T-cell low clones). These data reveal tumor cell intrinsic factors as major determinants of immune heterogeneity in the TME and highlight the use of this panel of clonal tumor lines as a tool to probe TME heterogeneity to develop optimized, patient-specific therapies. Citation Format: Katelyn T. Byrne, Jinyang Li, Robert H. Vonderheide, Ben Stanger. Tumor cell intrinsic factors dictate immune cell infiltration and response to immunotherapy [abstract]. In: Proceedings of the Fourth CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; Sept 30-Oct 3, 2018; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2019;7(2 Suppl):Abstract nr A054.

Trefoil factor 1 inhibits epithelial-mesenchymal transition of pancreatic intraepithelial neoplasm.

The tumor-suppressive role of trefoil factor family (TFF) members in gastric carcinogenesis has been suggested, but their significance and mechanisms in other digestive diseases remain elusive. To clarify the role of TFF1 in pancreatic carcinogenesis, we performed IHC on human samples, transfected siRNA against TFF1 into pancreatic cancer cell lines, and employed mouse models in which PanIN development and loss of TFF1 occur simultaneously. In human samples, the expression of TFF1 was specifically observed in pancreatic intraepithelial neoplasm (PanIN), but was frequently lost in the invasive component of pancreatic ductal adenocarcinoma (PDAC). When the expression of TFF1 was suppressed in vitro, pancreatic cancer cell lines showed enhanced invasive ability and features of epithelial-mesenchymal transition (EMT), including upregulated Snail expression. TFF1 expression was also observed in PanIN lesions of Pdx-1 Cre; LSL-KRASG12D (KC) mice, a model of pancreatic cancer, and loss of TFF1 in these mice resulted in the expansion of PanIN lesions, an EMT phenotype in PanIN cells, and an accumulation of cancer-associated fibroblasts (CAFs), eventually resulting in the development of invasive adenocarcinoma. This study indicates that the acquisition of TFF1 expression is an early event in pancreatic carcinogenesis and that TFF1 might act as a tumor suppressor to prevent EMT and the invasive transformation of PanIN.