Mechanisms and targeted prevention of chronic pancreatitis-acinar to ductal metaplasia caused by a low concentration of di-(2-ethylhexyl)-phthalate.

Impaired Autophagy Triggers Chronic Pancreatitis: Lessons From Pancreas-Specific Atg5 Knockout Mice

Aberrant glycosylation is one of the earliest known hallmarks of cancer. Amongst the predominate changes in tumor cell glycosylation is an increase in α2‐6 sialylation on N‐glycans, a modification elaborated by the ST6Gal1 sialyltransferase. ST6Gal1 activity dramatically alters cellular function via the sialylation of various surface receptors such as EGFR and TNFR1, resulting in altered signal transduction and gene expression. ST6Gal1 expression is upregulated in multiple cancers, including pancreatic ductal adenocarcinoma (PDAC). Recent studies from our group suggest that the upregulation of ST6Gal1 in pancreatic acinar cells may promote PDAC initiation by facilitating acinar to ductal metaplasia (ADM), a process in which acinar cells de‐differentiate into ductal‐like, progenitor cells. In the nonmalignant pancreas, ADM is induced primarily by pancreatitis, a known risk factor for PDAC. Pancreatitis causes acinar cell apoptosis, however some cells undergo ADM and acquire regenerative capabilities essential for tissue healing. Notably, ADM‐like cells are particularly susceptible to oncogenic transformation. In the current study we determined that ST6Gal1 was strikingly upregulated in pancreatic tissues from chronic pancreatitis patients and mice with experimental pancreatitis. Furthermore, ST6Gal1 was selectively expressed in the ADM‐like cells, as identified both morphologically and by staining for ADM‐specific markers. ST6Gal1 activity was associated with a proliferative phenotype. More than 50% of cells with upregulated ST6Gal1 co‐expressed the proliferative marker, Ki67, whereas activated caspase 8 was undetectable in ST6Gal1‐positive cells. To interrogate a functional role for ST6Gal1 in ADM, we developed genetically‐engineered mice with ST6Gal1 expression in the pancreas (“SC” mice). Importantly, acinar cells from SC mice exhibited ADM‐like characteristics, indicated by increased expression of classic stem and ductal genes. RNASeq analyses comparing SC and WT pancreata indicated that SC cells had an enrichment in: stem cell pathways; a pancreatic ductal cell program; and gene networks associated with pancreatic cancer. Additionally, organoids derived from SC pancreata displayed increased expression of stem and ductal genes relative to WT organoids, as well as greater organoid‐forming potential and growth. To evaluate inflammation‐induced ADM, SC and WT mice were injected with cerulein to induce pancreatitis, and the ADM‐like cells were quantified by examining surface ADM markers by flow cytometry. Following induction of pancreatitis, more ADM‐like cells were present in SC versus WT pancreata. Finally, we crossbred SC mice to the “KC” PDAC model, which expresses oncogenic KRas (KrasG12D) in the pancreas. Mice with dual expression of ST6Gal1 and KrasG12D had greatly accelerated PDAC initiation, progression and mortality as compared with KC mice. Together these data suggest that the upregulation of ST6Gal1 during chronic pancreatitis may prime acinar cells for neoplastic transformation by facilitating ADM.

Almost all reported cases of pancreatic ductal adenocarcinoma, the most common form of pancreatic cancer, harbor an oncogenic Kras mutation. Recently, targeting oncogenic Kras to specific cell types in the adult pancreas has shown the acinar compartment to be more susceptible to neoplastic transformation than ductal cells. Acinar to ductal metaplasia (ADM) is a phenomenon where acinar cells undergo a morphological change and begin expressing markers normally found only in ductal cells while losing markers typical of acinar cells. ADM is highly proliferative with the potential to develop into pancreatic intraepithelial neoplasias, a precursor lesion to pancreatic cancer. Acinar cells harvested from mice expressing mutant Kras undergo spontaneous ADM when embedded in 3D collagen cultures, however, we and others have found the epidermal growth factor receptor (EGFR) is required for ADM induced by mutant Kras expression in vitro. Activation of EGFR by treatment of WT collagen embedded acinar cell explants with the EGFR ligand TGF-α has also been shown to induce ADM in a manner requiring Notch pathway activation. Furthermore, it has been demonstrated that expression of Notch2 intracellular domain (N2ICD) is sufficient to induce ADM in WT acinar cells. To directly investigate the relationship of the EGFR, Kras, and Notch signaling pathways in ADM, we examined the effect of Notch signaling in acinar cells harvested from EGFRf/f; Ptf1a+/Cre (EKO) mice, which harbor a pancreas specific deletion of EGFR. Surprisingly, we found that expression of N2ICD failed to induce ADM in acinar cells from EKO mice. We also observed that co-expression of a constitutively active p110α mutant or mutant Kras with N2ICD was able to rescue ADM in acinar cells from EKO mice, though neither was sufficient to drive ADM in the absence of N2ICD. Additionally, treatment of acinar cells from WT mice with either the p110α inhibitor Pik75 or the expression of a dominant negative mutant of the PI3K downstream target Rac1 reduced Erk1/2 phosphorylation, whereas expression of constitutively active p110α increased the level of Erk1/2 phosphorylation. These results demonstrate the importance of EGFR signaling in ADM, and identify the need to determine the mechanism of crosstalk between the PI3K and MAPK pathways in acinar cells. Citation Format: Christopher J. Halbrook, Howard C. Crawford. Notch signaling couples with Kras or PI3K to drive EGFR independent pancreatic acinar to ductal metaplasia. [abstract]. In: Proceedings of the AACR Special Conference on RAS Oncogenes: From Biology to Therapy; Feb 24-27, 2014; Lake Buena Vista, FL. Philadelphia (PA): AACR; Mol Cancer Res 2014;12(12 Suppl):Abstract nr A09. doi: 10.1158/1557-3125.RASONC14-A09

Multiple animal studies suggest that pancreatitis-induced acinar-to-ductal metaplasia (ADM) is a key event for the initiation of pancreatic ductal adenocarcinoma (PDAC) tumorigenesis. However, until now, there has not been an adequate system to explore the mechanisms of ADM induction in humans. To address this problem, we have developed a flow cytometry-based, high resolution lineage tracing method to analyse ADM in primary human cells. Using a set of cell surface markers, we have successfully defined and purified human acinar, ductal and acinar-to-ductal transdifferentiated (AD) cells. A Matrigel-based 3D culture system which suppresses spontaneous ADM was developed to identify ADM inducers of primary human acinar cells. In contrast to the effect in mouse acinar cells, TGF-β1 efficiently converted human acinar cells to AD cells in a SMAD-dependent manner. Consistently, phospho-SMAD3 (p-SMAD3) expression was significantly increased in acinar cells from human pancreatitis samples, supporting an important role of the TGF-β/SMAD axis for human ADM in vivo. Furthermore, all known inducers of ADM in mice did not promote ADM in human cells, highlighting fundamental differences between the species. Capitalizing on our ability to separate different cell populations for functional analysis, we found that AD cells gained transient proliferative capacity. More interestingly, while oncogenic KRAS did not induce acinar cell proliferation, it did sustain proliferation of AD cells, suggesting that oncogenic KRAS requires ADM-associated-changes to promote PDAC initiation. This ADM model provides a novel platform to explore the mechanisms involved in the development of human pancreatic diseases. Grant Support: Support received from the Cancer Prevention and Research Institute of Texas (P. Wang). Jun Liu received support by way of the training grant (RP140105) from the Cancer Prevention and Research Institute of Texas. Citation Format: Jun Liu, Naoki Akanuma, Luzhe Sun, Pei Wang.{Authors}. Human pancreatic cells have distinct aspects in induction of acinar to ductal metaplasia. [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2016 May 12-15; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(24 Suppl):Abstract nr A63.

Ready, Set, Go: The EGF Receptor at the Pancreatic Cancer Starting Line

264 METAPLASIA-INDUCED EPITHELIAL HETEROGENEITY DIRECTS PANCREATIC TISSUE INJURY AND RECOVERY

Introduction: Pancreatic acinar cells can de-differentiate after acute injury (acute pancreatitis) to a progenitor-like cell type with ductal characteristics in a process termed acinar-to-ductal metaplasia (ADM). In the absence of oncogenic mutation, the ADM lesions resolve and reform the acinar compartment (adaptive ADM). However, in the presence of oncogenic Kras mutations (oncogenic ADM), ADM lesions can evolve to pancreatic intraepithelial neoplasia (PanIN) and pancreatic ductal adenocarcinoma (PDAC). Our comprehensive and unbiased approach previously identified the Paired-Related homebox1 (Prrx1) as the most up-regulated transcription factor during pancreatic development, regeneration and evolution of PanIN. We showed that Prrx1 expression is upregulated in both ADM and PanIN lesion. Here, we explore the role of Prrx1 in ADM and PanIN formation using novel mouse models, ex vivo acinar culture systems, and human pancreatitis tissue microarrays (TMA). Methods: We generated novel Ptf1acre-ERT;Prrx1fl/fl;Rosa26YFP/YFP (Prrx1 KO) mice, in which Prrx1 is deleted and YFP is expressed exclusively in adult pancreatic acinar cells upon tamoxifen induction of Cre recombinase activity. Intraperitoneal caerulein injection was administered to induce acute pancreatitis. Human pancreatitis TMAs were utilized for the comparison of Prrx1 expression between pancreatitis and normal human pancreas. We also generated Pdx1Cre;LSL-KrasG12D/+; Prrx1fl/fl;Rosa26YFP/YFP (KCYPrrx1KO) mice to evaluate the function of Prrx1 in oncogenic Kras mutation induced pancreatic PanIN. Quantification of ADM regions was performed through histological examination by a pathologist (blinded to the genetic basis of the tissues) and automated cell-counting of immunofluorescence staining (IF). Dissociated acinar cell culture in collagen was utilized for the evaluation of ADM under ex vivo conditions. We also established a novel method for the induction of Prrx1 overexpression in dissociated acinar cells via nucleofection. Results: IF staining revealed that Prrx1 expression is efficiently deleted in ADM cells of Prrx1 KO mice 3 days post-caerulein treatment, a timepoint at which we see peak ADM region formation. Additionally, areas of ADM lesions and amylase loss were significantly reduced in Prrx1 KO mice compared with Prrx1 WT mice at day 3. We also found that Prrx1 overexpression promotes ADM formation in ex vivo acinar cell cultures. In human TMAs, pancreatitis tissues had higher expression of Prrx1 than in normal pancreas. In the presence of oncogenic Kras mutation, loss of Prrx1 significantly attenuates ADM formation in ex vivo culture. Moreover, 5-month-old KCYPrrx1KO mice have very limited ADM/PanIN region compared to 5-month-old KCY mice, which have a pancreas that has predominantly replaced the ADM/PanIN. Conclusions: Prrx1 promotes ADM formation in both adaptive ADM and oncogenic ADM. Our preliminary data suggest that Prrx1 facilitates PDAC progression through PanIN formation. Citation Format: Kensuke Suzuki, Alina Li, Jason R. Pitarresi, Anna M. Chiarella, Gizem Efe, Kensuke Sugiura, Rohit Chandwani, Anil K. Rustgi. The elucidation of the role of Prrx1 for acinar to ductal metaplasia in response to acute injury of pancreas in the novel mouse models [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-075.

Background: Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer deaths with a 5-year survival rate of approximately 7%. PDAC may originate from acinar cell trans-differentiation into ductal-like cells, termed acinar-to-ductal metaplasia (ADM), triggered by chronic pancreatitis and/or mutations in K-Ras. The progression to PDAC is associated with a dense fibrotic stroma, including cancer-associated fibroblasts (CAFs). YAP is a tension-stimulated CAF activator that promotes ECM stiffening, creating a permissive microenvironment for cancer progression. We hypothesize that the Hippo pathway may coordinate fibroinflammatory signals emanating from the stromal compartment during regenerative responses to acinar cell injury and progression towards PDAC. Methods: To resolve the transcriptional changes occurring during the transition to ADM and PDAC, we mapped the in situ expression of over 1800 RNA targets in patient-derived tissues using NanoString Technologies’ Digital Spatial Profiling (DSP) technology. We also performed immune-profiling and evaluated Yap expression in human ADM by immunohistochemistry. To study the in vivo role of Hippo signaling in stromal cells, we conditionally deleted Yap/Taz in Collagen1a2-producing cells in a murine model of caerulein-induced pancreatitis, which recapitulates many of the features associated with human ADM. I will analyze the resulting phenotype by immunostaining for metaplastic, proliferative, immune and stromal markers. Results: DSP analysis revealed genes implicated in fibroblast activation, epithelial-to-mesenchymal transition (EMT), neutrophil activation and IFNγ signaling as potential key drivers of ADM. I will further evaluate the expression of candidate genes and survey Yap expression at the single cell level in human ADM tissue by multiplexed RNAscope in situ hybridization. We found up-regulation of CD4+ and CD8+ T cells in ADM, and an increasing trend of neutrophil and macrophage accumulation in the progression from normal parenchyma to ADM to PDAC. Conclusions: This work will provide an in-depth understanding of epithelial-stroma crosstalk in ADM and a foundation for the development of new therapeutic strategies for treating non-invasive precursor lesions like ADM, thereby preventing pancreatic cancer progression. Source of Funding: This research is supported by the Fonds de Recherce du Quebec – Santé (FRQS), Canadian Institutes of Health Research (CIHR) and the Research Institute of the McGill University Health Centre (RI-MUHC). Citation Format: Julia Messina-Pacheco, Yasser Riazalhosseini, Zu-hua Gao, Alex Gregorieff. The role of Hippo signaling in stromal-epithelial interactions in acinar-to-ductal metaplasia and pancreatic cancer initiation [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-117.

Acinar to ductal metaplasia (ADM) is the process by which acinar cells transition to a duct-like state acquiring ductal markers and morphology. This process can be driven by pancreas inflammation or injury, such as pancreatitis, where it is thought to be an adaptive response to help mitigate damage and facilitate repair. ADM is also observed following acinar-specific oncogenic KRAS mutations and known to precede PDAC precancerous lesions termed pancreatic intraepithelial neoplasias (PanINs). While ADM is well characterized histologically, cellular and molecular details of ADM and PanIN formation remain less well understood, in part due to high levels of digestive enzyme production in acinar cells that hamper characterization. Leveraging our recently described DSP-fixation method, FixNCut, to improve capture and transcriptome quality of acinar and ADM populations in the pancreas, we characterize ADM arising in three contexts: acute pancreatitis, recurrent acute pancreatitis, and acute pancreatitis with an oncogenic KRAS mutation known to drive the formation of PanINs. FixNCut and single-cell RNA-sequencing enabled us to elucidate the transcriptomic landscape of ADM and define changes to the microenvironment in unprecedented detail. Our findings indicate that the cellular and transcriptional landscape of recurrent acute pancreatitis closely mirrors that of acute pancreatitis for up to 6 weeks (or 6 bouts of 2-day acute pancreatitis), which was the longest timepoint tested. In contrast, acute pancreatitis in the presence of an acinar-specific oncogenic KRAS mutation results in a distinct transcriptional profile for both ADM and the surrounding microenvironment compared to pancreatitis alone. Additionally, we use FixNCut to preserve the epigenomes of acinar cells, enabling us to sort acinar cells and profile their histone modifications in various contexts of ADM. We benchmark and validate our results against existing mouse and human datasets of precancer and cancer. Through this work, we aim to enhance our understanding of ADM in various contexts, providing insights into acinar cell plasticity and early PDAC precursor formation that may inform strategies for early detection and interception. Citation Format: Katherine Aney, Woo-Jeong Jeong, Pal Koak, Isabel Kim, Sahar Nissim. Exploring acinar to ductal metaplasia in pancreatitis and precancerous contexts using FixNCut [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 C079.

Acinar cells, which synthesize digestive enzymes, undergo acinar-to-ductal metaplasia (ADM) upon injury, a process implicated in early pancreatic tumorigenesis. Previous studies suggest that ADM involves metabolic rewiring, and that mitochondrial fission is critical for established tumor cells survival. Emerging evidence suggests that interactions between mitochondria and the endoplasmic reticulum (ER) at ER-mitochondria contact sites (ERMCS) may regulate these early events. However, the interplay between metabolism, organelle dynamics, and ADM remains poorly understood. We hypothesize that disruption of ERMCS will impair mitochondrial fission and hinder ADM progression toward malignancy. Using ex vivo organoid models derived from acinar cells of our newly developed split-GFP based contact site sensor (SPLICS) reporter mouse – a cutting-edge tool engineered to express a whole-body, tamoxifen-inducible SPLICS reporter which allows real-time quantification of ERMCS – we investigated the effects of Fedratinib, an ERMCS inducer recently characterized by our research group. Fedratinib demonstrated a potent ability to activate ERMCS and ADM in acinar cells derived from SPLICS mice ex vivo. Treatment with Fedratinib resulted in the formation of ductal-like structures in quantities comparable to those induced by our positive control, which involves KRAS-MAPK pathway activation through TGF-α signaling, although these structures were marginally smaller in size than those formed with TGF-α alone. Importantly, detection of GFP signal further confirmed the enhanced proximity of ER and mitochondria, supporting Fedratinib’s efficacy as an ERMCS activator. Our recent studies have demonstrated that Fedratinib exerts its effects via inhibition of BRD4, a key regulator of gene transcription during cell proliferation and differentiation. Furthermore, the inhibition of BRD4 has been previously linked to accelerated ADM and impaired tissue regeneration post-injury. Our findings establish Fedratinib as a potent activator of ERMCS and ADM in ex vivo pancreatic models. This supports a novel link between ER-mitochondrial dynamics and cell fate transitions during pancreatic injury. These results provide a platform for further investigation of mitochondrial network alterations and the potential for targeting ERMCS in prevention and early detection of pancreatic cancer. Citation Format: Mariana Tannús Ruckert, Megan D. Radyk, Brandon Chen, Yatrik Shah, Costas A. Lyssiotis. ERMCS induction drives ADM in ex vivo models [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research—Emerging Science Driving Transformative Solutions; Boston, MA; 2025 Sep 28-Oct 1; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2025;85(18_Suppl_3):Abstract nr B060.

Introduction: Acinar cells in the adult pancreas demonstrate cellular plasticity and undergo de-differentiation to a progenitor-like cell type with ductal characteristics after injury. This process, termed acinar-to-ductal metaplasia (ADM), is an important feature facilitating pancreas regeneration after injury. In the absence of oncogenic mutations, the ADM lesions resolve and reform the acinar compartment (adaptive ADM). However, in the presence of oncogenic Kras mutations (oncogenic ADM), acinar cells undergo neoplastic transformation after ADM and evolve to pancreatic intraepithelial neoplasia (PanIN), a well-known precursor of pancreatic ductal adenocarcinoma (PDAC). We have characterized the role of Paired-Related Homeobox1 (PRRX1) in adaptive ADM. We demonstrated through our novel conditional Prrx1 knock-out mouse model that loss of Prrx1 abrogated ADM formation. Here we explore the relationship between Prrx1 and mutant Kras on promoting ADM and a pro-ADM microenvironment. Methods: We generated novel Pdx1-Cre;LSLKrasG12D/+;Prrx1fl/fl;Rosa26YFP/YFP (KCY Prrx1 KO) mice, in which mutant Kras is efficiently expressed and Prrx1 is deleted in a pancreas-specific manner. KCY Prrx1 WT and KO mice were sacrificed at 3 months and 5 months for histological analysis. Immunofluorescence (IF) staining for CK19, characterized for the rate of ADM formation and evaluated for F4/80 and SMA. Quantification of ADM regions, F4/80 and SMA was performed through automated cell-counting of immunofluorescence staining (IF). Dissociated acinar cell culture in collagen was utilized for the evaluation of ADM under ex vivo conditions. Results: IF staining revealed that KCY Prrx1 KO mice had fewer ADM lesions compared to Prrx1 WT mice at 3 months. This difference became dramatically apparent at the 5 month timepoint. Additionally, lower areas of fibrosis were identified via H&E staining in KCY Prrx1 KO mice, which was accompanied with lower F4/80 and SMA positivity at both 3 months and 5 months. Ex vivo cultures also demonstrated significant reduction in ADM formation in the context of oncogenic Kras and loss of Prrx1. Conclusions: PRRX1 can influence ADM formation in both a cell-intrinsic and cell-extrinsic manner in the presence of oncogenic KRAS. Our preliminary data suggest Prrx1 facilitates PDAC progression through PanIN formation. We will continue to investigate the mechanisms driving Prrx1-dependent ADM formation. Citation Format: Alina L. Li, Kensuke Sugiura, Kensuke Suzuki, Jason R. Pitarresi, Anna M. Chiarella, Gizem Efe, Rohit Chandwani, Anil K. Rustgi. Prrx1 regulates acinar cell plasticity in Kras-driven pancreatic acinar-to-ductal metaplasia [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 788.

Pancreatic acinar cells possess very high protein synthetic rates as they need to produce and secrete large amounts of digestive enzymes. Acinar cell damage and dysfunction cause malnutrition and pancreatitis, and inflammation of the exocrine pancreas that promotes development of pancreatic ductal adenocarcinoma (PDAC), a deadly pancreatic neoplasm. The cellular and molecular mechanisms that maintain acinar cell function and whose dysregulation can lead to tissue damage and chronic pancreatitis are poorly understood. It was suggested that autophagy, the principal cellular degradative pathway, is impaired in pancreatitis, but it is unknown whether impaired autophagy is a cause or a consequence of pancreatitis. To address this question, we generated Atg7(Δpan) mice that lack the essential autophagy-related protein 7 (ATG7) in pancreatic epithelial cells. Atg7(Δpan) mice exhibit severe acinar cell degeneration, leading to pancreatic inflammation and extensive fibrosis. Whereas ATG7 loss leads to the expected decrease in autophagic flux, it also results in endoplasmic reticulum (ER) stress, accumulation of dysfunctional mitochondria, oxidative stress, activation of AMPK, and a marked decrease in protein synthetic capacity that is accompanied by loss of rough ER. Atg7(Δpan) mice also exhibit spontaneous activation of regenerative mechanisms that initiate acinar-to-ductal metaplasia (ADM), a process that replaces damaged acinar cells with duct-like structures.

Activating mutations in KRAS reprogram cellular metabolism to support continuous growth, proliferation, and survival in pancreatic cancer. However, there is little information on how KRAS-dependent alterations in metabolism change precancerous states and cancer initiation. Acinar cells can give rise to pancreatic tumors through acinar-to-ductal metaplasia (ADM), a reversible cell state that persists and progresses to neoplasia and cancer with oncogenic KRAS. During ADM, acinar cells transdifferentiate to duct-like cells, a process we hypothesized was mediated by metabolic rewiring. We performed transcriptomic analysis on acinar cells undergoing ADM and found metabolic programs are globally enhanced. Indeed, we and others have demonstrated how inhibiting metabolic pathways necessary for ADM can prevent transdifferentiation and tumorigenesis. A detailed analysis of our data revealed that the transcripts for NADPH-producing enzymes, Glucose-6-phosphate dehydrogenase (G6pd) and Malic enzyme 1 (Me1), were significant enriched during ADM. Following these results, we obtained G6pd-mutant mice and generated Me1flox/flox mice and bred them into the KC (KrasLSL-G12D/+;Ptf1aCre/+) line, resulting in KCG (KrasLSL-G12D/+;Ptf1aCre/+;G6pdmut/mut) and KCM (KrasLSL-G12D/+;Ptf1aCre/+;Me1flox/flox) mice. Interestingly, KCG and KCM mice develop ADM and neoplasia faster than their KC counterparts. We also observe that acinar cells and ADM in KCG cells have higher levels of lipid peroxidation and reactive oxygen species (ROS) both in vivo and using primary ex vivo acinar cell cultures. Thus, increased ROS levels may underlie ADM acceleration in this system. In primary acinar cell culture, we can rescue the accelerated ADM phenotype in KCG cells with the addition of antioxidants. Furthermore, we find that treatment of wild type acinar cells with buthionine sulphoximine (BSO) to deplete glutathione levels and increase ROS also accelerates ADM in culture. These results suggest that NADPH and glutathione pools are important for regulating redox levels during ADM, and that reducing these pools leads to accelerated tumorigenesis. Together, this work demonstrates new metabolic pathways driving pancreatic cancer initiation. Citation Format: Megan D. Radyk, Barbara S. Nelson, Christopher J. Halbrook, Mengrou Shan, Jonathan Alektiar, Howard C. Crawford, Yatrik M. Shah, Costas A. Lyssiotis. NADPH production and redox homeostasis regulate acinar to ductal metaplasia [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 2176.

Background and AimsDespite years of research, mechanisms of pancreatic injury and healing remain poorly understood. Acute pancreatitis is a painful and debilitating condition; chronic pancreatitis may be asymptomatic, but greatly enhances the risk of pancreatic ductal adenocarcinoma (PDAC). Acinar to ductal metaplasia (ADM), or the transdifferentiation of digestive enzyme producing acinar cells to ductal cells, is an early event in both conditions. While ADM is thought to function in healing and regeneration, it also represents a first step in tumorigenesis, demonstrating the duplicitous nature of this inherent plasticity. The goal of these studies was to define the populations arising in ADM, associated transcriptional changes, and their role in disease progression.MethodsAcinar cells were lineage traced to follow their fate upon injury. Transcripts of more than 13,000 EYFP+ cells were determined using single cell RNA sequencing (scRNA‐seq). Developmental trajectories were generated using several computational biology approaches that rely on non‐overlapping assumptions. Data were compared to scRNA‐seq studies of gastric metaplasia, oncogenic KrasG12D‐induced ADM, and human pancreatitis. Results were confirmed using immunostaining and electron microscopy. Tuft and enteroendocrine cell (EEC) populations were quantified throughout tumorigenesis. KrasG12D was expressed in injury‐induced ADM populations using several inducible Cre drivers.ResultsscRNA‐seq of ADM from chronically injured pancreata revealed emergence of a mucin/ductal population that resembles gastric pyloric metaplasia. Developmental trajectories suggest that some pyloric metaplasia cells generate tuft or EEC populations as distinct lineages. Comparison to KrasG12D‐induced ADM reveals populations associated with disease progression. Immunostaining demonstrates that tuft and EEC formation is an early event in tumorigenesis. Activation of KrasG12D in ADM populations results in neoplastic transformation and the formation of MUC5AC+ pit cells. Human pancreatitis samples reflect a pyloric metaplasia phenotype as well as the formation of tuft and EEC populations.ConclusionsADM under conditions of chronic injury results in the formation of a pyloric‐type metaplasia which seeds disparate tuft and EEC lineages. This carefully orchestrated plasticity generates myriad epithelial cell types which likely mitigate injury, providing protection from the formation of pancreatitis and PDAC. KrasG12D expression is sufficient to drive neoplasia when targeted to injury‐induced ADM populations offering an alternative origin for tumorigenesis. This program is conserved in human pancreatitis and provides insight into early events in pancreas diseases.

Tumor cells are well‐known to have elevated levels of sialylated surface glycoproteins. The addition of sialic acid (a negatively‐charged sugar) to select surface receptors modulates the structure and function of such receptors, leading to changes in intracellular signaling and gene expression. Increased tumor cell sialylation occurs, in part, through the upregulation of sialyltransferases such as ST6GAL1, an enzyme that adds an α2‐6 linked sialic acid to N‐glycosylated proteins. ST6GAL1 is overexpressed in numerous malignancies, including pancreatic ductal adenocarcinoma (PDAC), and high expression correlates with a poor prognosis. Prior work from our group revealed that ST6GAL1 confers a cancer stem cell phenotype, typified by invasiveness and apoptosis‐resistance, through the sialylation‐dependent activation of receptors such as EGFR. In the current investigation we used human Suit2 PDAC cells, as well as two metastatic Suit2 subclones, to show that ST6GAL1 activity promotes tumor progression and metastasis in tumor xenograft models. Moreover, high ST6GAL1 expression in this isogenic cell series induces EGFR activation and epithelial to mesenchymal transition. To complement tumor xenograft experiments, we developed a genetically‐engineered mouse (GEM) model with conditional ST6GAL1 expression in the pancreas (termed, “SC” mice) and crossed this line to the “KC” PDAC model, which expresses oncogenic Kras (KrasG12D) in the pancreas. Mice with dual expression of ST6GAL1 and KrasG12D (”KSC” mice) exhibit greatly accelerated PDAC initiation, progression, and mortality when compared with KC mice. In light of ST6GAL1's known role in conferring stem/progenitor properties, we hypothesized that ST6GAL1 activity contributes to PDAC initiation by fostering acinar to ductal metaplasia (ADM). During ADM, pancreatic acinar cells de‐differentiate into ductal‐like, progenitor cells and acquire greater proliferative potential as well as apoptosis resistance. Cells undergoing ADM are particularly vulnerable to oncogenic transformation. To interrogate whether ST6GAL1 promotes ADM, we compared SC mice with control, wild‐type littermates. Using a variety of analyses and model systems including RNA‐Seq of GEM pancreata, organoid lines derived from GEM pancreata, the canonical 266‐6 ADM cell model, and in vivo model of inflammation‐induced ADM, we determined that high expression of ST6GAL1 in acinar cells imparts an ADM‐like phenotype, as evidenced by the upregulation of stem and ductal genes and downregulation of genes associated with differentiated acinar cells. Furthermore, GEM tissues and organoids with ectopic ST6GAL1 expression display strikingly elevated levels of activated EGFR. Given that activation of EGFR is one the major drivers of ADM, these data suggest a potential mechanism by which ST6GAL1‐mediated sialylation facilitates ADM. Collectively these results point to a critical role for ST6GAL1 in both early and late stages of pancreatic malignancy.