Pancreatic Intraepithelial Neoplasia Progression Research Articles

From precursor to cancer: decoding the intrinsic and extrinsic pathways of pancreatic intraepithelial neoplasia progression.

This review explores the progression of pancreatic intraepithelial neoplasia (PanIN) to pancreatic ductal adenocarcinoma through a dual lens of intrinsic molecular alterations and extrinsic microenvironmental influences. PanIN development begins with Kirsten rat sarcoma viral oncogene (KRAS) mutations driving PanIN initiation. Key additional mutations in cyclin-dependent kinase inhibitor 2A (CDKN2A), tumor protein p53 (TP53), and mothers against decapentaplegic homolog 4 (SMAD4) disrupt cell cycle control and genomic stability, crucial for PanIN progression from low-grade to high-grade dysplasia. Additional molecular alterations in neoplastic cells, including epigenetic modifications and chromosomal alterations, can further contribute to neoplastic progression. In parallel with these alterations in neoplastic cells, the microenvironment, including fibroblast activation, extracellular matrix remodeling, and immune modulation, plays a pivotal role in PanIN initiation and progression. Crosstalk between neoplastic and stromal cells influences nutrient support and immune evasion, contributing to tumor development, growth, and survival. This review underscores the intricate interplay between cell-intrinsic molecular drivers and cell-extrinsic microenvironmental factors, shaping PanIN predisposition, initiation, and progression. Future research aims to unravel these interactions to develop targeted therapeutic strategies and early detection techniques, aiming to alleviate the severe impact of pancreatic cancer by addressing both genetic predispositions and environmental influences.

Abstract A057: Investigating Fibroblast-Epithelial crosstalk in pre-malignant and cancerous microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) originates from precursor lesions, the most common of which are known as pancreatic intraepithelial neoplasia (PanIN). PanIN formation and progression is accompanied by accumulation of a precursor microenvironment (PME) composed of desmoplastic stroma that plays an important role in promoting tumor growth and immune evasion. The crosstalk between tumor cells and stromal cells is an area of active investigation. While PanIN and the PME have been extensively studied in mouse models, their nature in humans have remained elusive as there is no clinical indication to sample healthy pancreas. Through a unique collaboration with the Gift of Life Organ and Tissue donation program, we performed transcriptomic analysis on normal pancreata declined for transplant. We noted that epithelial cells in the PanIN and cancer microenvironment were transcriptomically similar; however, their surrounding stromal fibroblasts were markedly distinct. Given that most precursor lesions remain quiescent while some progress to tumor, we sought out to elucidate how the transcriptional programs in epithelial cells influence fibroblast cell fate. To do so, we developed a 3D ex-vivo organoid-fibroblast coculture system using primary organoid and fibroblast cultures successfully derived from Gift of Life pancreata or PDAC patients undergoing surgical resection. Our coculture system retains histological features of heterogeneity observed in vivo. Using single cell RNA-sequencing analysis of matched fibroblast-organoid cocultures we identified factors that were highly expressed in fibroblasts in tumor cocultures, but not in normal cocultures. We are currently interrogating candidate ligand/receptor interactions in our cocultures to determine the mediators of this epithelial-mediated fibroblast polarization. This model will allow us to gain an understanding about how components of the microenvironment restrain or promote malignant transformation and serve as a great tool to study therapies targeting tumor and stromal cells in a patient specific manner. Citation Format: Padma Kadiyala, Ahmed Elhossiny, Jianhua Liu, Alexander Bray, Katelyn Donahue, Arvind Rao, Jiaqi Shi, Yaqing Zhang, Filip Bednar, Timothy Frankel, Eileen Carpenter, Marina Pasca Di Magliano. Investigating Fibroblast-Epithelial crosstalk in pre-malignant and cancerous microenvironment [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 A057.

Preventive Treatment with a CD73 Small Molecule Inhibitor Enhances Immune Surveillance in K-Ras Mutant Pancreatic Intraepithelial Neoplasia.

Immunoprevention is an emerging consideration for solid tumors, including pancreatic ductal adenocarcinoma (PDAC). We and others have shown that Kras mutations in genetic models of spontaneous pancreatic intraepithelial neoplasia (PanIN), which is a precursor to PDAC, results in CD73 expression in the neoplastic epithelium and some populations of infiltrating immune cells, including macrophages and CD8 T cells. CD73 is an ecto-enzyme that converts extracellular adenosine monophosphate to adenosine, a critical immune inhibitory molecule in PDAC. We hypothesized inhibition of CD73 would reduce the incidence of PanIN formation and alter the immune microenvironment. To test our hypothesis, we used the KrasG12D; PdxCre1 (KC) genetically engineered mouse model and tested the utility of AB-680, a small molecule inhibitor targeting CD73, to inhibit PanIN progression. AB-680, or vehicle control, was administered using oral gavage delivery 3 days/week at 10 mg/kg, beginning when the mice were 2 months old and lasting 3 months. We euthanized the mice at 5 months old. In the KC model, we quantified significantly less pancreatitis, early and advanced PanIN, and quantified a significant increase in M1 macrophages in AB-680-treated mice. Single-cell RNA sequencing (scRNA-seq) of pancreata of AB-680-treated mice revealed increased infiltration of CD4+ T cells, CD8+ T cells, and mature B cells. The scRNA-seq analysis showed that CD73 inhibition reduced M2 macrophages, acinar, and PanIN cell populations. CD73 inhibition enhanced immune surveillance and expanded unique clonotypes of TCR and BCR, indicating that inhibition of CD73 augments adaptive immunity early in the neoplastic microenvironment. Prevention Relevance: Previous studies found PanIN lesions in healthy pancreata. Not all progress to PDAC, suggesting a window for enhanced antitumor immunity through immunoprevention therapy. CD73 inhibition in our study prevents PanIN progression, reduces immune-suppressive macrophages and expands TCR and BCR unique clonotypes, highlighting an encouraging therapeutic avenue for high-risk individuals.

Stromal Rigidity Stress Accelerates Pancreatic Intraepithelial Neoplasia Progression and Chromosomal Instability via Nuclear Protein Tyrosine Kinase 2 Localization

Since the mechanotransduction by stromal stiffness stimulates the rupture and repair of the nuclear envelope in pancreatic progenitor cells, accumulated genomic aberrations are under selection in the tumor microenvironment. Analysis of cell growth, micronuclei, and γH2AX foci links to mechanotransduction pressure in vivo during serial orthotopic passages of mouse KrasLSL-G12D/+;Trp53flox/flox;Pdx1-Cre (KPC) cancer cells in the tumor and in migrating through the size-restricted 3μm micropores. To search for pancreatic cancer cell-of-origin, analysis of single-cell datasets revealed that the ECM shapes an alternate route of acinar-ductal transdifferentiation of acinar cells into a central hub of elegantly restrained TOP2A-overexpressing cancer cells that spread out as unique cancer clusters with copy number amplifications in MYC-PTK2 locus and PIK3CA. High-PTK2 expression is associated with 171 differentially methylated CpG loci, 319 differentially expressed genes, and poor overall survival in TCGA-PAAD patients. Abolished RGD-integrin signaling by disintegrin KG blocked the PTK2 phosphorylation, increased cancer apoptosis, decreased VAV1 expression, and prolonged overall survival in the KPC mice. Decreases of αSMA deposition in the CD248 knockout KPC mice remodel the tissue stroma and downregulated TOP2A expression in the epithelium. In summary, stromal stiffness induces the onset of cells-of-origin of cancer by ectopic TOP2A expression, and the genomic amplification of MYC-PTK2 locus via alternative transdifferentiation of pancreatic progenitor cells is the vulnerability useful for disintegrin KG treatment against cells-of-origin cancer.

Oral bacteria accelerate pancreatic cancer development in mice

ObjectiveEpidemiological studies highlight an association between pancreatic ductal adenocarcinoma (PDAC) and oral carriage of the anaerobic bacterium Porphyromonas gingivalis, a species highly linked to periodontal disease. We analysed the potential...

Cholesterol Metabolism in Pancreatic Cancer.

Pancreatic cancer's substantial impact on cancer-related mortality, responsible for 8% of cancer deaths and ranking fourth in the US, persists despite advancements, with a five-year relative survival rate of only 11%. Forecasts predict a 70% surge in new cases and a 72% increase in global pancreatic cancer-related deaths by 2040. This review explores the intrinsic metabolic reprogramming of pancreatic cancer, focusing on the mevalonate pathway, including cholesterol biosynthesis, transportation, targeting strategies, and clinical studies. The mevalonate pathway, central to cellular metabolism, significantly shapes pancreatic cancer progression. Acetyl coenzyme A (Acetyl-CoA) serves a dual role in fatty acid and cholesterol biosynthesis, fueling acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) development. Enzymes, including acetoacetyl-CoA thiolase, 3-hydroxy-3methylglutaryl-CoA (HMG-CoA) synthase, and HMG-CoA reductase, are key enzymes in pancreatic cancer. Inhibiting HMG-CoA reductase, e.g., by using statins, shows promise in delaying PanIN progression and impeding pancreatic cancer. Dysregulation of cholesterol modification, uptake, and transport significantly impacts tumor progression, with Sterol O-acyltransferase 1 (SOAT1) driving cholesterol ester (CE) accumulation and disrupted low-density lipoprotein receptor (LDLR) expression contributing to cancer recurrence. Apolipoprotein E (ApoE) expression in tumor stroma influences immune suppression. Clinical trials targeting cholesterol metabolism, including statins and SOAT1 inhibitors, exhibit potential anti-tumor effects, and combination therapies enhance efficacy. This review provides insights into cholesterol metabolism's convergence with pancreatic cancer, shedding light on therapeutic avenues and ongoing clinical investigations.

Farnesoid X receptor activation inhibits pancreatic carcinogenesis

Farnesoid X receptor (FXR), a member of the nuclear receptor superfamily that controls bile acid (BA) homeostasis, has also been proposed as a tumor suppressor for breast and liver cancer. However, its role in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis remains controversial. We recently found that FXR attenuates acinar cell autophagy in chronic pancreatitis resulting in reduced autophagy and promotion of pancreatic carcinogenesis. Feeding Kras-p48-Cre (KC) mice with the BA chenodeoxycholic acid (CDCA), an FXR agonist, attenuated pancreatic intraepithelial neoplasia (PanIN) progression, reduced cell proliferation, neoplastic cells and autophagic activity, and increased acinar cells, elevated pro-inflammatory cytokines and chemokines, with a compensatory increase in the anti-inflammatory response. Surprisingly, FXR-deficient KC mice did not show any response to CDCA, suggesting that CDCA attenuates PanIN progression and decelerate tumorigenesis in KC mice through activating pancreatic FXR. FXR is activated in pancreatic cancer cell lines in response to CDCA in vitro. FXR levels were highly increased in adjuvant and neoadjuvant PDAC tissue compared to healthy pancreatic tissue, indicating that FXR is expressed and potentially activated in human PDAC. These results suggest that BA exposure activates inflammation and suppresses autophagy in KC mice, resulting in reduced PanIN lesion progression. These data suggest that activation of pancreatic FXR has a protective role by reducing the growth of pre-cancerous PDAC lesions in response to CDCA and possibly other FXR agonists.

Hematopoietic progenitor kinase 1 inhibits the development and progression of pancreatic intraepithelial neoplasia.

Ras plays an essential role in the development of acinar-to-ductal metaplasia (ADM) and pancreatic ductal adenocarcinoma (PDAC). However, mutant Kras is an inefficient driver for PDAC development. The mechanisms of the switching from low Ras activity to high Ras activity that are required for development and progression of pancreatic intraepithelial neoplasias (PanINs) are unclear. In this study, we found that hematopoietic progenitor kinase 1 (HPK1) was upregulated during pancreatic injury and ADM. HPK1 interacted with the SH3 domain and phosphorylated Ras GTPase-activating protein (RasGAP) and upregulated RasGAP activity. Using transgenic mouse models of HPK1 or M46, a kinase-dead mutant of HPK1, we showed that HPK1 inhibited Ras activity and its downstream signaling and regulated acinar cell plasticity. M46 promoted the development of ADM and PanINs. Expression of M46 in KrasG12D Bac mice promoted the infiltration of myeloid-derived suppressor cells and macrophages, inhibited the infiltration of T cells, and accelerated the progression of PanINs to invasive and metastatic PDAC, while HPK1 attenuated mutant Kras-driven PanIN progression. Our results showed that HPK1 plays an important role in ADM and the progression of PanINs by regulating Ras signaling. Loss of HPK1 kinase activity promotes an immunosuppressive tumor microenvironment and accelerates the progression of PanINs to PDAC.

The role of Smad4/TGFB signaling in tumorigenesis of pancreatic ductal adenocarcinoma (PDAC).

748 Background: Pancreatic cancer ranks 4th among cancers as a cause of death in the United States and 85- 90% of pancreatic cancer are pancreatic ductal adenocarcinoma (PDAC). Previous studies have demonstrated 2 major types of precursors to PDAC including pancreatic intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasia (IPMN); the mechanisms by which IPMNs give rise to PDAC are incompletely defined. We hypothesize that SMAD4 signaling is specifically involved in IPMN pathway with PanINs and IPMN having partially overlapping signaling pathways and molecular markers. Methods: We performed immunohistochemistry staining on different precursor lesions of mouse pancreatic tissue (PanIN precursor: Ptf1aCre LSL-KrasG12D; IPMN precursor: Ptf1aCreERT LSL- KrasG12D Smad4fl/fl treated with tamoxifen) using markers of different IPMN subtypes and signaling pathways known to be involved in PDAC including, MUC1, MUC2, MUC5AC, SMAD4, ERK1/2, SOX9, and YAP. We treated IPMN cells lines derived from Ptf1aCreERTM LSL-KrasG12D GnasR201C mice with TGF-β, BMP-2, or vehicle control and quantified RNA expression of Smad7, Serpine1, Id2, and normalized to Pmm1 expression. We performed a western blot to detect the protein expression of phospho-SMAD1, phospho-SMAD2, SMAD1, and SMAD2. Results: Both PanIN and IPMN mouse models exhibited strong expression of MUC5AC and MUC1, reflecting the pancreatobiliary subtype. The tumor cells in both models expressed phosphorylated substrates of protein kinase A, phosphorylated ERK1/2, and SOX9. Regional differences were noted for SOX9 signaling with IPMN demonstrating a greater expression around the outer edges of the lesion, compared to the center. PanIN model demonstrated a more prominent nuclear YAP staining. Treatment of BMP2 and TGF-β in pancreatic IPMN cells from Ptf1aCreERTM LSL-KrasG12D GnasR201C cell line significantly increased RNA levels of Serpine1 (2.4 and 199.6, respectively; n = 4; p < 0.05) and Smad7 (2.9 and 6.3, respectively; n = 4; p < 0.05). BMP2 treatment significantly increased RNA levels of Id2 (1.9; n = 4; p < 0.05) while TGF-β significantly decreased Id2 level (0.3; n = 4; p < 0.05). Phosphorylated-SMAD2 was detected only with TGF-β treatment while phosphorylated-SMAD1 was detected with vehicle, TGF-β, and BMP2 treatment with the highest level with BMP2 treatment. Conclusions: Knocking out Smad4, concurrent with activating KrasG12D mutation, induces a specific IPMN progression model that shares similar molecular markers as the PanIN progression model, shown in Kras mutation alone. SMAD4 signaling remains intact in IPMN lesions that arise from activating the KrasG12D GnasR201C mutation, possibly suggesting that Smad4 mutation induces the IPMN-PDAC progression model that differs from Gas-cAMP-PAK-YAP1 cascade found in GNAS signaling.

Abstract A014: Targeting TAK1 for intercepting PanIN progression to ductal adenocarcinoma in an inducible KC mouse model

Abstract Despite advances in our knowledge of human pancreatic ductal adenocarcinoma (PDAC), targeted therapies have not yet significantly translated to an improved overall survival for patients. Pancreatic tumor microenvironment enriched by infiltrating immune cells and consisting of dense fibrotic stroma is characterized by desmoplasia, the major contributors of tumor-associated inflammation. Transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) is widely accepted as a key player in the TNF-α and TGF-β -induced signaling, and principal contributor of tumor fibrosis, inflammation and cell proliferation. Here, we show that pancreatic tumors over- express TAK1, and are associated with fibrotic-stroma and infiltrating immune cells. Also, p-TAK1(ser412) expression is correlated with progression of pancreatic intraepithelial neoplasia (PanIN) lesions to PDAC. Thus, we hypothesized TAK1 as an important target for inhibition of the tumor-associated fibrosis, inflammation and PanIN progression to PDAC. To prove above hypothesis, we tested a selective TAK1 inhibitor, Takinib for its toxicity and efficacy against PanIN progression in the inducible Ptf1CreERT.LSL-KrasG12D (KC) mouse model. Takinib was synthesized and fed to wild type C57BL/6J mice (n=6) at 250 ppm and 500ppm in diet for 6 weeks to determine toxicity. Bodyweight gain, organ weights and serum enzyme analysis did not indicate any toxicity at the tested doses. For efficacy study, LSL-KrasG12D mice were bred with Ptf1-CreERTmice. Pups were genotyped and randomized to groups (n=12). PanINs -PDAC was induced in the KC mice by tamoxifen (oral gavage) followed by 250ppm Takinib administration in diet for 20 weeks. After termination, pancreas tissue sections were evaluated for PanIN multiplicity and PDAC incidence/spread. Administration of Takinib led to significant reduction in PanIN 1 by 54% (p<0.02), PanIN 2 by 77% (p<0.001) and PanIN 3 by 80% (p<0.02) in the KC mice compared to untreated mice. PDAC incidence was also reduced with an increase in normal pancreas in Takinib administered KC mice. Taken together our results suggest that TAK1 is a valuable target for PDAC, and Takinib possesses efficacy against the PanIN and PDAC progression in the KC mouse model warranting further studies. (Supported by PHF Team Science grant and Kerley-Cade Endowed Chair). Citation Format: Venkateshwar Madka, Gopal Pathuri, Gaurav Kuma, Anil Singh, Nicole Stratton, Courtney Houchen, Altaf Mohammed, Chinthalapally V. Rao. Targeting TAK1 for intercepting PanIN progression to ductal adenocarcinoma in an inducible KC mouse model [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr A014.

Abstract IA013: Preclinical testing of CD73 inhibitors for pancreatic cancer immunoprevention

Abstract Introduction: The all stages combined five-year survival rate for pancreatic adenocarcinoma (PDA) is 11%; however, the five-year survival rate for localized PDA is 42%. These statistics highlight the importance of early prevention strategies to prevent disease progression and metastatic dissemination. Through the NCI PREVENT program, this research program explores immunoprevention strategies for PDA by targeting CD73, a gatekeeper ectoenzyme responsible for production of extracellular adenosine. We have recently shown aggressive subtypes of pancreatic intraepithelial neoplasia (PanIN) and PDA arising in ductal pancreatic epithelium have elevated CD73 and intrapancreatic adenosine indicating adenosine generation may be an early trigger of immunosuppression. We hypothesize inhibition of CD73 and adenosine generation will promote a more robust anti-tumor immune response and prevent PanIN and PDA progression. Methods: We tested three small molecule CD73 inhibitors (APCP, OP-5244, and AB680) in a syngeneic PDA mouse model by injecting 100-200k murine PDA cells derived from KrasG12D;Trp53R172H/+;Pdx:Cre (KPC) mice in the flanks of C57BL/6 female mice. Tumor sizes were measured weekly and tumor volume and mass were recorded at time of death. Dosage: APCP oral gavage (3x/week at 20mg/kg) and intraperitoneal (IP) (3x/week at 20 mg/kg). OP-5244 oral (3x/week at 25mg/kg and 10mg/kg). AB680 oral gavage (3x/week at 10mg/kg). HPLC analysis was performed for each inhibitor to quantify adenosine levels. Results: IP delivery of APCP significantly reduced tumor growth and intratumoral adenosine levels; however oral gavage delivery did not reduce tumor growth. Similarly, oral gavage delivery of OP-5244 did not reduce tumor growth. AB680 significantly reduced tumor volume and intratumoral adenosine levels and CyTOF immunoprofiling showed activated CD8+ T cells, dendritic cells, and macrophages were significantly increased in the tumors from AB680 treated mice. Conclusion: APCP IP delivery is more effective than oral gavage delivery and OP-5244 oral gavage delivery does not significantly decrease tumor growth. AB680 oral gavage delivery significantly decreases tumor growth and tumor adenosine concentrations. We observed a significant increase in infiltration of activated CD8+ T cells. AB680 shows high translational potential for preclinical testing in spontaneous GEM models. Citation Format: Lincoln Ballew, Kanchan Singh, Vidhi Chandra, Tingting Mills, Erika Y. Faraoni, Victoria Mota, Trent Clark, Lana Vornik, Michelle I. Savage, Shizuko Sei, Altaf Mohammed, Holger K. Eltzschig, Powel H. Brown, Florencia McAllister, Jennifer M. Bailey-Lundberg. Preclinical testing of CD73 inhibitors for pancreatic cancer immunoprevention [abstract]. In: Proceedings of the Second Biennial NCI Meeting: Translational Advances in Cancer Prevention Agent Development (TACPAD); 2022 Sep 7-9. Philadelphia (PA): AACR; Can Prev Res 2022;15(12 Suppl_2): Abstract nr IA013.

Abstract 816: Cyclin c suppresses pancreatic intraepithelial neoplasm progression and neuroendocrine neoplasm development in a murine Kras model

Abstract Cyclin c is a component of the Cdk8 kinase module that plays both a positive and negative role in transcription. In addition, cellular damage induces cyclin c re-localization to the mitochondria where it stimulates fission and recruits Bax in preparation for apoptotic initiation. Previous studies revealed that cyclin c suppresses thyroid hyperplasia in a Pten knockout mouse model. In the present study, a role for cyclin c in suppressing pancreatic cancer progression was investigated in the Pdx1-Cre LSL-KrasG12D murine pancreatic cancer model. Within eight weeks, a 6-8 fold increase in the appearance of pancreatic intraepithelial neoplasia (PanIN) and acinar ductal metaplasia (ADM) lesions was observed in the KrasG12D+Ccnc-/- animals compared to KrasG12D alone. Surprisingly, high-grade neuroendocrine neoplasms (NEN) were also observed. These aggressive NEN showed mixed morphology with areas of acinar-like differentiation. High grade NEN exhibited pronounced proliferative index (Ki67), high EZH2 expression, and elevated Notch1, suggesting a connection between aberrant Notch signaling and NEN formation. Our previous studies identified a positive role for cyclin c in autophagy gene transcription. Similarly, cell lines derived from a KrasG12D+Ccnc-/- pancreas exhibited a reduction in autophagy compared to KrasG12D+Ccnc+/+ cells. Autophagic deficiency induces pancreatic cell stress and is associated with apoptosis. Consistent with this model, high caspase 3 expression was observed in early KrasG12D+Ccnc-/- NEN but not in KrasG12D+Ccnc+/+ tissues. Taken together, these results demonstrate a role for cyclin c in suppressing both progression of early PanIN and the development of high-grade NEN and suggest a model that cyclin c loss suppresses autophagy and increases apoptosis. Acknowledgements: Support was provided by NCI CCSG grant CA06927 for the FCCC Histopathology and Cell Culture Facilities, the PA Department of Health CURE fund (KSC), the Boye Foundation (RS), and the New Jersey Health Foundation (RS). Citation Format: Kathy Q. Cai, Sara E. Hanley, Andres Klein-Szanto, Kerry S. Campbell, Randy Strich. Cyclin c suppresses pancreatic intraepithelial neoplasm progression and neuroendocrine neoplasm development in a murine Kras model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 816.

Rapid acceleration of KRAS-mutant pancreatic carcinogenesis via remodeling of tumor immune microenvironment by PPARδ

Pancreatic intraepithelial neoplasia (PanIN) is a precursor of pancreatic ductal adenocarcinoma (PDAC), which commonly occurs in the general populations with aging. Although most PanIN lesions (PanINs) harbor oncogenic KRAS mutations that initiate pancreatic tumorigenesis; PanINs rarely progress to PDAC. Critical factors that promote this progression, especially targetable ones, remain poorly defined. We show that peroxisome proliferator-activated receptor-delta (PPARδ), a lipid nuclear receptor, is upregulated in PanINs in humans and mice. Furthermore, PPARδ ligand activation by a high-fat diet or GW501516 (a highly selective, synthetic PPARδ ligand) in mutant KRASG12D (KRASmu) pancreatic epithelial cells strongly accelerates PanIN progression to PDAC. This PPARδ activation induces KRASmu pancreatic epithelial cells to secrete CCL2, which recruits immunosuppressive macrophages and myeloid-derived suppressor cells into pancreas via the CCL2/CCR2 axis to orchestrate an immunosuppressive tumor microenvironment and subsequently drive PanIN progression to PDAC. Our data identify PPARδ signaling as a potential molecular target to prevent PDAC development in subjects harboring PanINs.

Abstract PO-060: N-terminal RHAMM cooperates with dysfunctional p53 to accelerate the progression of pancreatic cancer

Abstract Pancreatic cancer has the lowest survival rate in all types of cancer. Pancreatic cancer patients are often diagnosed at advanced stages. A better therapeutic development for this devastating disease is urgently needed. Receptor for hyaluronan-mediated motility (RHAMM), not expressed in adult normal pancreas, has been suggested as a prognostic factor and a potential therapeutic target for pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumor (PNET). In this study, we initially sought to determine whether genetic deletion of RHAMM would slow down pancreatic cancer progression using Rhamm−/− mice. However, we found that Rhamm−/− mice expressed a N-terminal RHAMM protein. While N-terminal RHAMM did not enable malignant progression of pancreatic intraepithelial neoplasia in p48-Cre; LSL-KRASG12D mice, it accelerated the formation of invasive PDAC and shortened the survival of p48-Cre; LSL-KRASG12D mice with heterozygous p53 knockout. KrasG12D PDAC mice with homozygous p53 knockout mice died around 10 weeks, and the effect of N-terminal RHAMM was not apparent in these mice with short life span. In addition, N-terminal RHAMM shortened the survival of PNET-bearing RIP-Tag mice, which had inactivated p53. In our analysis of TCGA dataset, pancreatic cancer patients with mutant TP53 or loss of one copy of TP53 had higher RHAMM expression, which, combined, predicted worse outcomes. Taken together, by collaborating with dysfunctional p53, N-terminal RHAMM that lacks the centrosome targeting domain and degrons for interaction with the Anaphase-Promoting Complex (APC) accelerated pancreatic cancer progression. Citation Format: Anthony Lin, Jennifer Feng, Xiang Chen, Dunrui Wang, Megan Wong, George Zhang, Joseph Na, Tiantian Zhang, Zhengming Chen, Yao-Tseng Chen, Yi-Chieh Nancy Du. N-terminal RHAMM cooperates with dysfunctional p53 to accelerate the progression of pancreatic cancer [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 PO-060.

Abstract PR-008: Kdm6 demethylases are critical regulators of pancreatic cancer initiation, progression and subtype specification

Abstract Aberrant epigenetic regulation is a hallmark of pancreatic cancer. Since changes in histone methylation are potentially reversible, they present an attractive target for therapeutic intervention. Kdm6a and Kdm6b belong to a family of histone demethylases specific for lysine 27 of histone 3 and have been implicated in multiple types of cancer, including pancreatic ductal adenocarcinoma (PDA). We have gathered data from the extensive investigation of a mouse model of PDA in which either Kdm6b or both Kdm6a and Kdm6b have been deleted with concomitant activation of oncogenic KrasG12D restricted to adult acinar cells. While prior work had demonstrated an important role for Kdm6a in PDA formation, our data now show that the closely related Kdm6b is implicated in PDA tumorigenesis as well. Partial or complete loss of Kdm6b results in accelerated acinar-to-ductal metaplasia (ADM) and formation of pancreatic intraepithelial neoplasia (PanIN) with increased inflammation and fibrosis. Accelerated ADM formation was also observed ex vivo upon culturing acinar cells in the absence of oncogenic Kras, indicating that Kdm6b activity is critical for the maintenance of acinar identity and suppresses the early stages of pancreatic neoplasia development. However, only mice heterozygous for Kdm6b continue to exhibit a reduced overall median survival compared to KrasG12D alone, while complete loss of Kdm6b results in a similar overall median survival compared to KrasG12D. This suggests a requirement for oncogenic Kdm6b function during later progression of PanIN to invasive cancer. Conversely, concomitant loss of Kdm6a and Kdm6b protects against ADM and PanIN formation in vivo and ex vivo and double mutants display a trend towards an increased overall median survival compared to mice carrying only the KrasG12D mutation. Strikingly, the resulting tumors of Kdm6a/Kdm6b deficient mice exhibit features of an extremely rare subtype of sarcomatoid pancreatic cancer found in human patients. Taken together, this suggests that Kdm6 demethylase function is required during the initiation and progression of PDA. Furthermore, targeting Kdm6a/Kdm6b with specific inhibitors at distinct stages might provide opportunities for novel treatment avenues for pancreatic cancer. Citation Format: Laura Leonhardt, Lucia Y. Li, David I. Berrios, Sudipta Ashe, Audrey M. Hendley, Grace E. Kim, Matthias Hebrok. Kdm6 demethylases are critical regulators of pancreatic cancer initiation, progression and subtype specification [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-008.

Abstract 2668: Pancreatic cancer cell extracellular vesicles drive the unfolded protein response in recipient normal pancreas cells

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is projected to become the second leading cause of cancer-related deaths by 2030. The 5-year survival rate remains low, hovering ~9%. A key driver of poor patient outcomes is late-stage diagnosis of disease. Currently, no diagnostics exist to identify indolent disease. A better understanding of the complex tumor microenvironment in PDAC is needed for discerning disease etiology and progression. Extracellular vesicles (EVs) are nanometer sized lipid-bilayer particles released from cells of all tissue types, which carry an array of biomolecules. EVs mediate a myriad of cancer progression events including tumor proliferation, angiogenesis, immune escape, and metastasis. Their role in PDAC tumorigenesis, however, is largely unknown. In this study we report on the effects of PDAC cell derived EVs on normal pancreatic epithelial cells. Results: We treated normal pancreas cells with pancreas cancer cell derived EVs (cEVs). Global gene expression analysis identified 376 differentially expressed genes (DEGs) enriched for genes in the aminoacyl-tRNA biosynthesis, tRNA aminoacylation, ATF4 and ATF6 signaling pathways. Gene ontology analysis identified enrichment in biological processes associated with endoplasmic reticulum (ER) stress, NF-kB signaling and DNA damage response. Lipid synthesis was severely altered, with decreased expression in several cholesterol, sterol, and acetyl-CoA pathways. Transcription factor analysis identified enrichment of CHOP/DDIT3 target motifs, as well as targets of XBP1, ATF6 and ATF4. Unfolded protein response (UPR) and ER stress genes in our dataset were significantly upregulated, including DDIT3, NFYB, ATF6 and ATF4. We validated upregulation of UPR/ER stress in these cells using qPCR and luciferase activity assays. Multi-omics characterization of EVs isolated from PDAC and normal cell lines showed distinct biochemical profiles. This analysis identified potential mediators of UPR/ER stress enriched in cEVs, including proteins which regulate protein trafficking and ubiquitination, lipid species esterified to palmitic acid and metabolites which regulate tRNA charging. Conclusions: We show that cEVs induce significant gene expression changes within recipient normal pancreatic cells and likely induce ER stress, leading to an UPR. Long-term UPR/ER stress can impact a variety of cancer hallmarks, including angiogenesis, genome stability, inflammation, metastasis, and drug resistance. ER stress has been shown to mediate progression of pancreatic intraepithelial neoplasia (PanIN) to PDAC. These results highlight a potential role of EVs in mediating cellular messages from pancreatic cancer cells to alter the function of recipient normal cells, aiding disease onset and progression. Further studies are ongoing to identify key mediators within cEVs for the induction of UPR/ER stress. Citation Format: Charles Phillip Hinzman, Shivani Bansal, Yaoxiang Li, Anton Iliuk, Jose Trevino, Partha Banerjee, Amrita Cheema. Pancreatic cancer cell extracellular vesicles drive the unfolded protein response in recipient normal pancreas cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2668.

High levels of truncated RHAMM cooperate with dysfunctional p53 to accelerate the progression of pancreatic cancer.

Pancreatic cancer has the lowest survival rate out of all types of cancer. Pancreatic cancer patients are often diagnosed at advanced stages, hence an urgent need for a better therapeutic development of this devastating disease. Receptor for hyaluronan-mediated motility (RHAMM), not expressed in adult normal pancreas, has been suggested as a prognostic factor and a potential therapeutic target for pancreatic ductal adenocarcinoma (PDAC) and pancreatic neuroendocrine tumor (PNET). In this study, we initially sought to determine whether genetic deletion of RHAMM would slow down pancreatic cancer progression using Rhamm-/- mice. However, we found that Rhamm-/- mice expressed a truncated HMMRΔexon8-16 protein at higher abundance levels than wild-type RHAMM. While HMMRΔexon8-16 did not enable malignant progression of pancreatic intraepithelial neoplasia in p48-Cre; LSL-KRASG12D mice, it accelerated the formation of invasive PDAC and shortened the survival of p48-Cre; LSL-KRASG12D mice with heterozygous p53 knockout. KrasG12D PDAC mice with homozygous p53 knockout mice died around 10 weeks, and the effect of HMMRΔexon8-16 was not apparent in these short lifespan mice. In addition, HMMRΔexon8-16 shortened the survival of PNET-bearing RIP-Tag mice, which had inactivated p53. In our analysis of TCGA dataset, pancreatic cancer patients with mutant TP53 or loss of one copy of TP53 had higher RHAMM expression, which, combined, predicted worse outcomes. Taken together, by collaborating with dysfunctional p53, high levels of HMMRΔexon8-16 , which lacks the centrosome targeting domain and degrons for interaction with the Anaphase-Promoting Complex (APC), accelerated pancreatic cancer progression.

Single-PanIN-seq unveils that ARID1A deficiency promotes pancreatic tumorigenesis by attenuating KRAS-induced senescence.

ARID1A is one of the most frequently mutated epigenetic regulators in a wide spectrum of cancers. Recent studies have shown that ARID1A deficiency induces global changes in the epigenetic landscape of enhancers and promoters. These broad and complex effects make it challenging to identify the driving mechanisms of ARID1A deficiency in promoting cancer progression. Here, we identified the anti-senescence effect of Arid1a deficiency in the progression of pancreatic intraepithelial neoplasia (PanIN) by profiling the transcriptome of individual PanINs in a mouse model. In a human cell line model, we found that ARID1A deficiency upregulates the expression of aldehyde dehydrogenase 1 family member A1 (ALDH1A1), which plays an essential role in attenuating the senescence induced by oncogenic KRAS through scavenging reactive oxygen species. As a subunit of the SWI/SNF chromatin remodeling complex, our ATAC sequencing data showed that ARID1A deficiency increases the accessibility of the enhancer region of ALDH1A1. This study provides the first evidence that ARID1A deficiency promotes pancreatic tumorigenesis by attenuating KRAS-induced senescence through the upregulation of ALDH1A1 expression.

Downregulation of ZNF395 Drives Progression of Pancreatic Ductal Adenocarcinoma through Enhancement of Growth Potential

Background: Progression of pancreatic intraepithelial neoplasia (PanIN) to invasive carcinoma is a critical factor impacting the prognosis of patients with pancreatic tumors. However, the molecular mechanisms involved are not fully understood. We have reported that the process frequently involves loss of chromosome 8p, causing downregulation of DUSP4, thus conferring invasive ability on cancer cells. Here, we focus on ZNF395, whose expression was also found to be decreased by 8p loss and was predicted to be a growth suppressor gene. Methods: Pancreatic cancer cell lines inducibly expressing ZNF395 were established to assess the functional significance of ZNF395 in pancreatic carcinogenesis. Immunohistochemistry was also performed to analyze the expression levels of ZNF395 in pancreatic cancer tissues. Results: Induction of ZNF395 in pancreatic cancer cells resulted in marked activation of JNK and suppression of their proliferation through a delay in cell cycle progression. Immunohistochemistry revealed that ZNF395 was expressed ubiquitously in both normal pancreatic ducts and PanINs but was significantly reduced in invasive cancers, especially those showing poor differentiation. Conclusion: ZNF395 acts as a novel tumor suppressor gene. Its downregulation caused by 8p loss in intraepithelial cells accelerates their proliferation through dysregulation of the cell cycle, leading to progression to invasive cancer.

68477 Pancreatic cancer cell extracellular vesicles drive the unfolded protein response in recipient normal pancreatic cells.

ABSTRACT IMPACT: This study advances our understanding of potentially key drivers in the early formation of pancreatic cancer, a disease with few treatment options and poor patient outcomes. OBJECTIVES/GOALS: Patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) have a 5-year survival rate of ˜9%. A key driver of poor patient outcomes is late-stage diagnosis. A better understanding of PDAC onset is needed. This study was developed to understand how extracellular vesicles may be involved in the early formation of PDAC. METHODS/STUDY POPULATION: Extracellular vesicles (EVs) were isolated from several human PDAC and normal pancreatic cell lines, using ultracentrifugation with filtration or size exclusion chromatography. We next treated normal pancreatic cell lines with cancer cell EVs (cEVs). Next generation sequencing was used to measure global gene expression changes after treatment. Validations were performed using qPCR and luciferase activity assays. Multi-omics characterization of EVs was accomplished using mass spectrometry based proteomics, metabolomics and lipidomics analysis. RESULTS/ANTICIPATED RESULTS: We found that normal cells upregulated a variety of stress response pathways in response to cEVs. Lipid synthesis was also severely downregulated in these cells. We further validated activation of the unfolded protein response (UPR) in normal cells treated with cEVs. Multi-omics characterization of cEVs identified several enriched proteins, lipids and metabolites which may play a role in the activation of the UPR. DISCUSSION/SIGNIFICANCE OF FINDINGS: Our results indicate that cEVs induce stress, and in particular the UPR, in normal pancreatic cells. Long-term UPR can impact a variety of cancer hallmarks. The UPR can mediate progression of pancreatic intraepithelial neoplasia (PanIN) to PDAC. Our results highlight a potential role for cEVs to alter the function of normal cells, aiding disease onset.