PDAC Development Research Articles

SREBP-Dependent Regulation of Lipid Homeostasis Is Required for Progression and Growth of Pancreatic Ductal Adenocarcinoma.

Our findings demonstrate that SREBP pathway activation is a critical part of the metabolic reprogramming that occurs in PDAC development and progression. Therefore, targeting the SREBP pathway has significant therapeutic potential.

NEMO/NF-κB signaling functions as a double-edged sword in PanIN formation versus progression to pancreatic cancer

BackgroundPancreatic ductal adenocarcinoma (PDAC) is marked by a dismal survival rate, lacking effective therapeutics due to its aggressive growth, late-stage diagnosis, and chemotherapy resistance. Despite debates on NF-κB targeting for PDAC treatment, no successful approach has emerged.MethodsTo elucidate the role of NF-κB, we ablated NF-κB essential modulator (NEMO), critical for conventional NF-κB signaling, in the pancreata of mice that develop precancerous lesions (KC mouse model). Secretagogue-induced pancreatitis by cerulein injections was utilized to promote inflammation and accelerate PDAC development.ResultsNEMO deletion reduced fibrosis and inflammation in young KC mice, resulting in fewer pancreatic intraepithelial neoplasias (PanINs) at later stages. Paradoxically, however, NEMO deletion accelerated the progression of these fewer PanINs to PDAC and reduced median lifespan. Further, analysis of tissue microarrays from human PDAC sections highlighted the correlation between reduced NEMO expression in neoplastic cells and poorer prognosis, supporting our observation in mice. Mechanistically, NEMO deletion impeded oncogene-induced senescence (OIS), which is normally active in low-grade PanINs. This blockage resulted in fewer senescence-associated secretory phenotype (SASP) factors, reducing inflammation. However, blocked OIS fostered replication stress and DNA damage accumulation which accelerated PanIN progression to PDAC. Finally, treatment with the DNA damage-inducing reagent etoposide resulted in elevated cell death in NEMO-ablated PDAC cells compared to their NEMO-competent counterparts, indicative of a synthetic lethality paradigm.ConclusionsNEMO exhibited both oncogenic and tumor-suppressive properties during PDAC development. Caution is suggested in therapeutic interventions targeting NF-κB, which may be detrimental during PanIN progression but beneficial post-PDAC development.Graphical

BCAA metabolism in pancreatic cancer affects lipid balance by regulating fatty acid import into mitochondria

BackgroundPancreatic ductal adenocarcinoma (PDAC) has been associated with the host dysmetabolism of branched-chain amino acids (BCAAs), however, the implications for the role of BCAA metabolism in PDAC development or progression are not clear. The mitochondrial catabolism of valine, leucine, and isoleucine is a multistep process leading to the production of short-chain R-CoA species. They can be subsequently exported from mitochondria as short-chain carnitines (SC-CARs), utilized in anabolic pathways, or released from the cells.MethodsWe examined the specificities of BCAA catabolism and cellular adaptation strategies to BCAA starvation in PDAC cells in vitro. We used metabolomics and lipidomics to quantify major metabolic changes in response to BCAA withdrawal. Using confocal microscopy and flow cytometry we quantified the fluorescence of BODIPY probe and the level of lipid droplets (LDs). We used BODIPY-conjugated palmitate to evaluate transport of fatty acids (FAs) into mitochondria. Also, we have developed a protocol for quantification of SC-CARs, BCAA-derived metabolites.ResultsUsing metabolic profiling, we found that BCAA starvation leads to massive triglyceride (TG) synthesis and LD accumulation. This was associated with the suppression of activated FA transport into the mitochondrial matrix. The suppression of FA import into mitochondria was rescued with the inhibitor of the acetyl-CoA carboxylase (ACC) and the activator of AMP kinase (AMPK), which both regulate carnitine palmitoyltransferase 1A (CPT1) activation status.ConclusionsOur data suggest that BCAA catabolism is required for the import of long chain carnitines (LC-CARs) into mitochondria, whereas the disruption of this link results in the redirection of activated FAs into TG synthesis and its deposition into LDs. We propose that this mechanism protects cells against mitochondrial overload with LC-CARs and it might be part of the universal reaction to amino acid perturbations during cancer growth, regulating FA handling and storage.

Abstract 6533: Pivotal role of specific LYVE1+ tissue resident macrophages in PDAC progression & immunotherapy resistance

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by dense fibrotic stroma and abundant myeloid cell infiltration, which render it resistant to any therapeutic interventions, including immunocheckpoint blockade (ICB) therapy. Understanding the composition and function of myeloid cells within the PDAC microenvironment may help identify promising targets for PDAC immunotherapy. Tissue resident macrophages (TRMs) contribute to organogenesis and tissue regeneration and metabolism. In this study, we discovered a distinct population of pancreas TRMs that highly express lymphatic vessel hyaluronan receptor 1 (LYVE-1). Single cell RNAseq data reveal that LYVE-1+ TRM cluster in the pancreas expresses high levels of complement activation genes and transcription factors such as c-Maf and Stab1. RNAseq data also confirm that LYVE-1+ TRMs express enriched genes related to mannose binding, endocytosis, and glutathione metabolic process, while LYVE-1 negative macrophages express enriched genes related to antigen processing and presentation, T cell activation, and phagocytosis. Multiparameter flow cytometric analysis demonstrates that LYVE-1+ TRMs express M2-like markers, including increased CD163, CD206 and decreased MHC Class II molecule. During PDAC development and progression, LYVE-1+ TRMs expand and localize around the tumor border, promoting fibrotic stroma formation. Imaging mass cytometry (IMC) also reveals abundant infiltration of LYVE-1+CD14+CD68+ macrophages in human PDAC tumors and colocalized with collagen deposition. This subset of macrophages also highly express c-Maf. Through the use of Lys-Cre-c-Maf f/f mice, we demonstrate that depletion of c-Maf from myeloid cells significantly reduces LYVE-1+ TRMs and polarizes them to resemble an M1-like phenotype. In conclusion, our findings suggest that LYVE-1+ macrophages are a unique subset of myeloid cells within the pancreas and likely play a critical role in PDAC progression and immunotherapeutic resistance. Future work will focus on targeting LYVE1+ TRMs using various approaches, such as conditional knockout mice and small molecule inhibitors, to determine their therapeutic potential for PDAC treatment. Presented by Dr. Hong Li Citation Format: Hong Li, Jun Yan. Pivotal role of specific LYVE1+ tissue resident macrophages in PDAC progression & immunotherapy resistance [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6533.

Abstract 4460: Investigating a non-canonical role for SHP2 in pancreatic ductal adenocarcinoma metabolism and response to dietary modifications

Abstract Pancreatic ductal adenocarcinoma (PDAC) is predicted to be the second leading cause of cancer death in the US by 2025, with rising incidence. Mutant KRAS in >90% of PDAC cases promotes activation of the MAPK pathway, yet targeting the MAPK pathway clinically has failed to improve patient outcomes. SHP2 canonically promotes KRAS activation and is essential for PDAC development in mouse models. Because targeting MAPK is insufficient, yet SHP2 loss blocks PDAC development, we hypothesized SHP2 has both MAPK dependent and independent functions. Initial studies revealed that SHP2 inhibition with SHP099 in murine KPC [K8484: KRASG12D/+, p53R172H/+, Pdx1Cre/+] cells required concentrations exceeding 50 μM to elicit a minimal ERK inhibition. This led us to predict that there may be a tumor suppressive mechanism of SHP2 inhibition that is KRAS/MAPK independent. We utilized available RNAseq data (NCBI GEO: PRJNA558508) of PDAC MiaPaCa2 cells treated with the MEK inhibitor, Trametinib, or SHP099 to compare the direct effects of inhibition on transcriptional outcomes. We identified inositol phosphate signaling as a candidate target, whose downstream consequence is Ca2+ flux. We measured IL-1β induced Ca2+ flux in a panel of human PDAC cells exposed to MEK or SHP2 inhibition. Trametinib treated cells showed no effect on Ca2+ flux, however, SHP099 led to a drastic depletion of free Ca2+ and response to stimulus. We then validated the Ca2+ response to IL-1β stimulus in Crispr-Cas9 SHP2-knockout K8484 cells. While we did not observe a difference in basal free calcium, we still found a reduction in response to IL-1β stimulus. Because Ca2+ is an important co-factor for mitochondrial metabolism, we then interrogated metabolic effects. We tested mitochondrial function using a Seahorse Mito-Stress test and found that while MEK inhibition failed to affect mitochondrial metabolism, SHP2 inhibition ablated it. A Glycolysis-Stress Test revealed that SHP2 inhibition led to glucose consumption primarily through anaerobic respiration. TMT-labeled phospho-proteomic analysis comparing SHP2KO and MEK inhibited K8484s revealed SHP2 dependent deficiencies in mitochondrial proteins, among others, involved in mitochondrial fission and lipid metabolism. Western diets have been shown to promote tumor progression in GEM models of PDAC, so we used KRASG12D/+, SHP2fl/fl or wt./wt, Ptf1aCre/+ (SKC and KC) mice to demonstrate that while diet-induced obesity affects KC tumor burden, the lack of SHP2 in SKCs prevents the diet associated discrepancy in tumor progression. Collectively, these data led us to conclude that while SHP2 has been studied as a contributor to KRAS signaling in PDAC, the outcomes of its role specifically on mitochondrial metabolism have yet to be appreciated or thoroughly investigated, opening the path for a new approach to its contribution to tumor progression. Citation Format: R. McKinnon Walsh, Jarrid L. Jack, Austin E. Eades, Bailey A. Bye, Mariana T. Ruckert, Joseph Ambrose, Michael N. VanSaun. Investigating a non-canonical role for SHP2 in pancreatic ductal adenocarcinoma metabolism and response to dietary modifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4460.

Abstract 3154: The role of nuclear kinases in pancreatic carcinogenesis

Abstract Pancreatic ductal adenocarcinoma (PDAC), the most common histological subtype of pancreatic cancer, is a devastating disease predicted to be the second leading cause of cancer deaths in the next 15 years. Thus, there is an urgent need to develop new treatment approaches based on a better understanding of cellular and molecular mechanisms controlling this dismal progression. PDAC development is marked by nuclear morphological changes likely resulting in changes in chromatin organization and gene expression. Oncogenic KRAS, present in more than 90% of tumors, plays a key role in disease initiation and progression and is sufficient to induce these nuclear morphology changes. Work from our lab has identified the nuclear lamina protein Emerin as a mediator of KRAS-induced nuclear morphology changes. Further, a BioID experiment identified a KRAS-induced interaction of Emerin with a group of nuclear kinases belonging to the ribosomal S6 kinase (RSK) and mitogen- and stress- activated protein kinase (MSK) families. In silico analysis identified candidate MSK and RSK phosphorylation sites in Emerin. Thus, we hypothesize that the nuclear kinase families RSK and MSK can act as effectors of the KRAS-Emerin axis to regulate PDAC nuclear morphology. Further studies will be done to confirm if Emerin can be phosphorylated by RSK and MSK, and to validate that this relationship is dependent on oncogenic KRAS signaling and if its nuclear morphological changes are regulated by this GTPase. Overall, this work aims to define a novel signaling axis for RSK and MSK in controlling nuclear dynamics that play a role in disease progression of PDAC, which can lead to the identification of new therapeutic targets. Citation Format: Kayla LaRue, Ashley Sigafoos, Luis Flores, Brooke Tader, Martin Fernandez-Zapico. The role of nuclear kinases in pancreatic carcinogenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3154.

Abstract 5283: Polyamine blockade therapy: A strategy to block immunosuppression in pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is lethal in 88% of patients and is characterized by a dense tumor microenvironment (TME) which impedes tumor entry of immune cells and delivery of therapeutics. Immunosuppressive myeloid populations of PDAC, including myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs), are known to not only support PDAC development but also impede the anti-tumor immune response elicited by therapeutic agents. To improve prognosis while maintaining tolerability, this study aims to target polyamine metabolism that is upregulated in the immunosuppressive myeloid populations and cancer cells to disrupt their contribution to PDAC progression and the immunosuppressive TME. We previously demonstrated that polyamine blockade therapy (PBT), via blocking biosynthesis with difluoromethylornithine (DFMO) and transport with a novel inhibitor (Trimer44NMe), elicits an anti-tumor response and improves median survival in PDAC engrafted immunocompetent mice. In the current study, PBT was found to lead to a selective reduction of the dominant MDSC subtype in PDAC, polymorphonuclear (PMN) MDSCs. Upon co-culturing of MDSCs with CD8+ T cells, PBT dampened MDSC-mediated suppression of T cell activity. These findings were corroborated by tracking Arginase 1, a driver of MDSC-mediated immunosuppression, which exhibited a significant decrease in response to PBT at both the activity and gene expression level. Ex-vivo generated M2 macrophages and TAMs treated with PBT also demonstrated a shift from the M2 pro-tumor phenotype towards the anti-tumor M1 macrophage phenotype. In summary, these findings support that PBT is effective at reducing myeloid-mediated immunosuppression through subtype specific modulation and supports the use of PBT for treatment of PDAC in combination with other immunomodulatory therapeutics. Citation Format: Joseph A. Goode. Polyamine blockade therapy: A strategy to block immunosuppression in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5283.

Alteration of chromosome structure impacts gene expressions implicated in pancreatic ductal adenocarcinoma cells

BackgroundPancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with a five-year survival rate of approximately 10%. Genetic mutations are pivotal drivers in PDAC pathogenesis, but recent investigations also revealed the involvement of non-genetic alterations in the disease development. In this study, we undertook a multi-omics approach, encompassing ATAC-seq, RNA-seq, ChIP-seq, and Hi-C methodologies, to dissect gene expression alterations arising from changes in chromosome accessibility and chromatin three-dimensional interactions in PDAC.ResultsOur findings indicate that chromosomal structural alterations can lead to abnormal expressions on key genes during PDAC development. Notably, overexpression of oncogenes FGFR2, FOXA2, CYP2R1, and CPOX can be attributed to the augmentation of promoter accessibility, coupled with long-range interactions with distal elements. Additionally, our findings indicate that chromosomal structural alterations caused by genomic instability can lead to abnormal expressions in PDACs. As an example, by analyzing chromosomal changes, we identified a putative oncogenic gene, LPAR1, which shows upregulated expression in both PDAC cell lines and clinical samples. The overexpression is correlated with alterations in LPAR1-associated 3D genome structure and chromatin state. We further demonstrated that high LPAR1 activity is required for enhanced PDAC cell migration in vitro.ConclusionsCollectively, our findings reveal that the chromosomal conformational alterations, in addition to the well-known genetic mutations, are critical for PDAC tumorigenesis.

Abstract B098: Oncogenic KRAS relies on β1 integrin expression to drive pancreatic neoplasia and PDAC development

Abstract Oncogenic Kras drives cancer progression through cooperation with multiple cellular factors, many of which are yet to be determined. The β1 integrins, a family of extracellular matrix receptors, are strong candidates; they are implicated in many aspects of cancer progression including cell growth and survival signaling, cell migration and metastasis. Additionally, multiple studies have identified β1 integrins within the interactome of oncogenic Kras. We are investigating the potential cooperation if β1 integrins in Kras-mediated oncogenesis using genetic perturbations within the tamoxifen-inducible and conditional Ptf1a-CreERTM; LSL- KrasG12D (p48-KCER) model of pancreatic cancer. Conditional deletion of the β1 integrins from the adult pancreas produced minimal tissue abnormalities; there were no obvious tissue defects at 6 months post induction, although there was some evidence of pancreatitis at 1 year post induction. However, deletion of the β1 integrins within the KCER model of pancreatic cancer (KCER-Δβ1) resulted in a profound suppression of pancreatic disease progression. There was no detectable neoplasias at 3 months post-induction and minimally detectable neoplasias at 6 months post-induction, compared to prominent neoplasia development and stromal expansion in the control KCER mice at these time-points. Moderate stromal expansion was observed in KCER-Δβ1 mice at 6 months post-induction despite the lack of neoplasia development. To investigate the mechanisms of this integrin-KRAS cooperation, we conditionally deleted the expression of two canonical integrin signaling molecules, the integrin-linked kinase (ILK) and the focal adhesion kinase (FAK). Neither the ILK knockout (KCER-ΔILK) nor the FAK knockout (KCER-ΔFAK) phenocopied the KCER-Δβ1 mice, as both developed neoplasias and stromal expansion at 3 months post-induction and beyond. To explore the role of integrin activation, we next deleted expression of the integrin-cytoskeleton linker molecule Talin-1 in the KCER model. Surprisingly, even though Talin-1 is key to integrin activation, the KCER-ΔTalin-1 mice also failed to phenocopy the KCER-Δβ1, showing ample neoplasia development at 3 and 6 months post-induction, and trending towards faster disease progression at later stages. These combined results demonstrate crucial cooperation between oncogenic Kras and β1 integrins to drive pancreatic cancer progression, and point to non-canonical β1 integrin functions in mediating this cooperation. Citation Format: John L. Muschler, Ge Huang, Alexander C. Smith, Rosalie C. Sears. Oncogenic KRAS relies on β1 integrin expression to drive pancreatic neoplasia and PDAC development [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B098.

Abstract B103: Development and characterisation of KRASmulti inhibitors for the treatment of KRAS mutant pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is predicted to become the second most common cause of cancer related death within the next decade. Late detection and limited therapeutic options drive the dismal prognosis for PDAC patients. Activating mutations within the KRAS gene occur within the vast majority (>85%) of PDAC tumors. KRAS mutations occur at an early stage within PDAC development but remain necessary for continued tumor growth. Clinical responses observed in patients with KRASG12C mutant PDAC treated with selective KRASG12C inhibitors have driven drug development programmes that can address other, more frequently occurring KRAS alleles. Here we report the characterization of a KRASmulti inhibitor, BI-2493, that potently inhibits the proliferation of mutant KRAS PDAC cancer cell lines in vitro. The DMPK properties of the compound have been optimized so that oral administration resulted in regression of KRAS mutant PDAC xenograft models. These data support the continued development of compounds capable of addressing the most prevalent KRAS mutant alleles in PDAC with the potential to provide a therapeutic option to patients in an indication with a high unmet medical need. Citation Format: Antonio Tedeschi, Otmar Schaaf, Michael Gmachl, Lorenz Herdeis, Francesca Rocchetti, Johannes Popow, Fiorella Schischlik, Daniel Gerlach, Jesse Lipp, Joachim Bröker, Andreas Mantoulidis, Chris Smethurst, Dirk Kessler, Jark Boettcher, Tobias Wunberg, Valeria Santoro, Alex Waterson, Jason Phan, Andrew Little, Jason Abbott, Qi Sun, Stephen Fesik, Darryl Mcconnell, Mark A. Pearson, Norbert Kraut, Dorothea Rudolph. Development and characterisation of KRASmulti inhibitors for the treatment of KRAS mutant pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B103.

Abstract C028: DKK3 in pancreatic cancer – Elucidating the roles of a double-edged sword

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a highly challenging disease with a poor prognosis due to high tumor heterogeneity, lack of diagnostic biomarkers and limited treatment options. Therefore, it is crucial to meticulously examine the intricate molecular mechanisms involved in the interplay between different cellular compartments in PDAC. We previously identified DKK3 (Dickkopf-3) as a novel factor promoting organ regeneration during pancreatitis and established a previously unknown link between DKK3 and the Wnt and Hedgehog signaling. Given that pancreatitis is a significant risk factor for pancreatic cancer, we were motivated to investigate the potential role of DKK3 in PDAC development. We utilized PDAC mouse models driven by LSL-KrasG12D/+; Ptf1aCre/+ with or without homozygous/heterozygous DKK3 deletion in both epithelial and stromal compartment and employed various in vitro and ex vivo systems to examine the stage and compartment-specific roles of DKK3 during PDAC progression. Through a comprehensive and time-resolved analysis of tumor characteristics, we discovered that the loss of DKK3 led to rapid oncogenic transformation, marked by a significant increase in metaplasia and desmoplasia starting at an early stage (10 weeks). Ex vivo acinar-to-ductal metaplasia assay and rescue experiments with wildtype and DKK3-null acinar cells revealed that secreted DKK3 operates as a tumor suppressive roadblock in PDAC-initiating steps. This advanced preneoplastic stage translated into accelerated progression toward invasive tumors at 36 weeks, shortened overall survival, and a higher liver metastasis incidence in the DKK3-null animals at the endpoint. Further characterization of isolated tumor cells underpinned our observations, revealing prominent mesenchymal features and enhanced migratory capacities upon loss of DKK3. Interestingly, DKK3-null PDACs exhibited a pronounced stroma remodeling with a myofibroblastic cancer-associated fibroblast-enriched environment, as well as an immunosuppressive stroma with abundant regulatory T cells and cytotoxic T cell depletion, as substantiated by coculture experiments. Intriguingly, while displaying similar phenotypic features to DKK3-null tumors, Dkk3+/- heterozygotes exhibited the most aggressive outcome. To further dissect this finding, we conducted a set of in silico approaches and immunostainings revealing persistent stromal DKK3 expression in DKK3-proficient PDACs. This suggested a possible dual role of DKK3 that could operate either as a friend or a foe depending on its spatiotemporal expression in PDAC. In line, in vitro systems demonstrated a distinct oncogenic effect of DKK3 on endpoint tumor cells, particularly promoting malignant cell migration. In summary, we propose that epithelial DKK3 exerts cell-autonomous tumor suppressive functions during PDAC onset, while stromal DKK3 acts as an oncogenic cue driving cancer progression. These findings highlight DKK3 as a promising biomarker and a novel therapeutic target for addressing the oncogenic dialog within PDAC tumors. Citation Format: Dharini Srinivasan, Frank Arnold, Elodie Roger, Anna Härle, Michael Melzer, Patrick Hermann, Lukas Perkhofer, Johann Gout, Alexander Kleger. DKK3 in pancreatic cancer – Elucidating the roles of a double-edged sword [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C028.

Abstract B040: Neural control of cancer-associated fibroblasts in PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) poses a formidable challenge to public health, with a dismal 5-year survival rate and a projected rise as the second leading cause of cancer-related deaths by 2030. The pathogenesis of PDAC involves a robust desmoplastic reaction and a complex tumor microenvironment (TME), constituting up to 80% of the tumor volume. The TME is a heterogeneous mixture of cells, including cancer-associated fibroblasts (CAFs), immune cells, endothelial cells, and neurons, which all interact and contribute to disease progression. However, the precise interplay between the tumor-associated nerves and PDAC development remains unclear. It is known that neural remodeling within the tumor microenvironment are closely associated with intense neuropathic pain, metastasis, and shortened patient survival. The prevalence of nerve fibers present in the dense desmoplastic stroma of PDAC tumors suggest a potential interaction between neurons and stromal cells, particularly CAFs, through various paracrine signaling mechanisms, influencing CAF behavior and facilitating neural remodeling, ultimately driving PDAC progression. Our investigation revealed that the neurotransmitter noradrenaline, induces calcium signaling and prompt contraction in pancreatic stellate cells (PSCs). Preliminary in vitro findings indicate that this calcium signaling is mediated by alpha 1 adrenergic receptors. Whole-mount 3D imaging mouse PDAC tissues, indicates a spatial organization of adrenergic neurons in immediate proximity to neoplastic cells and cancer-associated fibroblasts. Moreover, our study demonstrated that noradrenaline and alpha 1 agonists modulate extracellular matrix (ECM) production and cytokine secretion in PSCs and CAFs. Finally, we discovered that chemical ablation of the sympathetic nervous system significantly suppresses tumor growth while promoting metastasis, underscoring the critical role of the noradrenergic system in tumor development. Collectively, our findings propose that noradrenergic neurons possess the capacity to influence CAF behavior, opening new avenues of investigation to unravel the intricate crosstalk between neural regulation and tumor progression in PDAC. Citation Format: Jeremy Nigri, Wenjun Lan, Sangeeta S Chavan, Youngkyu Park, Jeremy Borniger, Kevin J Tracey, David A Tuveson. Neural control of cancer-associated fibroblasts in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B040.

Abstract B032: Spatial proteomics and transcriptomics reveal early immune cell organization in human pancreatic intraepithelial neoplasia

Abstract A common precursor of pancreatic adenocarcinoma (PDAC) are microscopic lesions, termed pancreatic intraepithelial neoplasia (or PanIN). These lesions are initiated at least a decade before overt PDAC forms, thus providing a large window of opportunity to modulate the immune microenvironment before significant pro-carcinogenic signals prevail. To best inform strategies for interception of these precursor lesions, it is critical to understand the quantity and spatial localization of the lymphoid compartment recruited to PanINs compared to PDAC. Here, we report the use of spatial–omics (proteomic and transcriptomics) to evaluate the recruitment of lymphoid cells to PanIN lesions compared to immune–enriched regions of PDAC in a unique cohort of 5 treatment–naïve PDAC patients who underwent resection of their primary pancreas tumor. Using imaging mass cytometry, we found lymphoid populations recruited preferentially to low–grade PanINs compared with normal tissue. This population of predominantly CD4+ T cells in low–grade PanINs was markedly different from the increased Treg and exhausted CD4+ T cell populations found infiltrating in the tumor. Interestingly, our approach to evaluate the spatial localization of these recruited lymphoid populations uncovered their capacity to organize into tertiary lymphoid structures (TLSs). Structurally and functionally immature TLSs were associated with PanIN lesions, while mature TLSs were found adjacent to the invasive tumor border of PDAC. Mature TLSs restricted to the outside of the tumor had markers of proliferation indicating functional capabilities; however, these aggregates also contained immunosuppressive lymphoid subtypes. Signatures of immature PanIN associated TLSs and mature PDAC associated TLSs were recapitulated by the spatial transcriptomics analysis performed on this same cohort. Additionally, the proteomic analysis identified that the immune population restricted to the periphery of the tumor was substantially denser and enriched with greater numbers of naïve CD4+ T cells and central memory T cells than the intra–tumoral population. Together, these data demonstrate that PanINs are capable of recruiting a substantial lymphoid population that can organize into immature TLSs. The quality and quantity of lymphoid cells recruited to PanINs compared to regions inside and surrounding PDAC provides evidence of the dynamic immune microenvironment in response to PanIN–to–PDAC progression. These data provide insight into the immunological landscape of PDAC development and will inform the next generation of strategies to prevent PanIN lesions from progressing to overt PDAC. Citation Format: Melissa R. Lyman, Jacob T. Mitchell, Luciane T. Kagohara, Amanda L. Huff, Daniel Haldar, Sarah Shin, Samantha Guinn, Benjamin Barrett, Gabriella Longway, Alexei Hernandez, Erin Coyne, Xuan Yuan, Lalitya Andaloori, Jiaying Lai, YunZhou Liu, Rachel Karchin, Anuj Gupta, Ashley Kiemen, André Forjaz, Denis Wirtz, Pei-Hsun Wu, Atul Deshpande, Todd Armstrong, Nilofer S. Azad, Elizabeth Thompson, Robert Anders, Won Jin Ho, Elizabeth Jaffee, Elana Fertig, Neeha Zaidi. Spatial proteomics and transcriptomics reveal early immune cell organization in human pancreatic intraepithelial neoplasia [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B032.

Abstract C056: The initiation and progression of pancreatic ductal adenocarcinoma through a p53 lens

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a 5-year survival rate of only 11%. Slated to be the 2nd most deadly cancer by 2026, pancreatic cancer survival rates have not improved substantially over the years. With a lack of early detection methods and a highly immunosuppressive tumor microenvironment (TME), PDAC tumors are highly resistant to treatment. Oncogenic mutations in KRAS are present in >90% of cases and mutations in the tumor suppressor gene TP53 are present in ~72% of cases. However, why p53 mutations are so prevalent and how they enhance PDAC development is not well understood. To elucidate how exactly p53 loss promotes PDAC, we are using genetically engineered mouse models combined with single-cell and spatial transcriptomic technologies. In this mouse model, PDAC is driven from adult acinar cells with a tamoxifen-inducible Ptf1aCreER allele by oncogenic Kras (KrasG12D) along with either p53 wild type (p53wt) or p53 deficiency (p53flox). In this model, acinar cells can give rise to PDAC likely through a transdifferentiation process called acinar-to-ductal metaplasia (ADM), which leads to pancreatic intraepithelial neoplasias (PanINs) and then PDAC. The initial cell fate transition, ADM, can be activated by growth factors, inflammation, and injury. We have conducted single-cell and spatial transcriptomics to compare early stages of tumor development in the presence and absence of p53, and we will present our latest analyses. Providing insight into the early events of PDAC will lead to new approaches for improving clinical intervention in human PDAC. Citation Format: Kathryn J. Hanson, Allegra C. Minor, Brittany M. Flowers, Alyssa M. Kaiser, Nicholas Hughes, Hannes Vogel, Le Cong, Laura D. Attardi. The initiation and progression of pancreatic ductal adenocarcinoma through a p53 lens [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C056.

Cytoplasmic-delivery of polyinosine-polycytidylic acid inhibits pancreatic cancer progression increasing survival by activating Stat1-CCL2-mediated immunity

BackgroundPancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer without effective therapies and with poor prognosis, causing 7% of all cancer-related fatalities in the USA. Considering the lack of effective therapies...

B-233 Chromatin Accessibility Uncovers KRAS-driven FOSL2 Promoting Pancreatic Ductal Adenocarcinoma Progression Via Upregulating CCL28

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal human cancer with 5-year survival rate less than 10%. Pancreatic cancer remains a malignancy of poor prognosis and effective therapies are lacking, therefore, studying the pathogenesis and identifying more therapeutic targets are urgently needed for PDAC treatment. The epigenetic mechanisms of PDAC progression are poorly understood. This study aims to identify key transcription factor (TF) by multiomics sequencing to explore the molecular mechanisms of TF with critical roles in PDAC. Methods we used ATAC-seq, H3K27ac ChIP-seq, and RNA-seq to characterized the epigenetic landscape by genetically engineered mouse models (GEMMs) of PDAC with or without KRAS and/or TP53 mutations. Survival of FOSL2 was assessed using Kaplan-Meier method and multivariate Cox regression analysis for patients with PDAC. We performed Cleavage Under Targets and Tagmentation (CUT&Tag) to study the potential targets of FOSL2. CCK8, transwell migration and invasion, RT-qPCR, western blot, IHC, ChIP-qPCR, dual-luciferase reporter, and xenograft model assays were used to explore the functions and underlying mechanisms of FOSL2 in PDAC progression. Results We found that the epigenetic changes were involved in some immuno-suppressed signaling with PDAC progression. Also, we identified a critical regulator FOSL2 which showed enriched open chromatin, H3K27ac signal, and higher RNA expression in KC and KPC than WT. The high expression of FOSL2 was associated with poor prognosis of patients with PDAC. Single-cell RNA-seq data showed that FOSL2 expression was very low in T, B, and acinar cells, and enriched in endothelial, ductal cells and upregulated in cancerous ductal cells compared with normal pancreatic ductal cells. functionally, FOSL2 can not only promote the proliferation, migration and invasion of pancreatic cancer, but also inhibit tumor immunity such as reducing the infiltration of CD8+T cells and increasing the infiltration of regulatory T (Treg) cells.In terms of mechanism, we found that CCL28 was a key target gene of FOSL2 by CUT&Tag sequencing. We confirmed that FOSL2 can regulate the expression of CCL28 by knockdown or overexpression of FOSL2 in vitro. ChIP-qPCR and dual luciferase reporter assays confirmed that FOSL2 activated the transcription of CCL28 by binding to the upstream of CCL28. In vivo mouse assay showed that FOSL2 promoted tumor growth and Treg cell recruitment through CCL28. We also explored the mechanism by which FOSL2 was upregulated in pancreatic cancer and found that KRAS mutation mediated FOSL2 transcription and subsequent CCL28 expression through the MAPK/ERK pathway, which implicated an immuno-suppressed regulatory axis of KRAS/MAPK-FOSL2-CCL28-Treg cells in PDAC development. Conclusion We described the changes of epigenetic landscape during PDAC progression and found that KRAS mutation promoted FOSL2 expression, which recruited more Treg cells by upregulating CCL28 and ultimately led to immunosuppression in pancreatic cancer. Targeting CCL28 with CCL28 antibody can reduce the invasion of Treg cells, which provides a new way to overcome the Treg cell-mediated tumor immune escape in patients with PDAC.

Genetic ablation of ketohexokinase C isoform impairs pancreatic cancer development

Although dietary fructose is associated with an elevated risk for pancreatic cancer, the underlying mechanisms remain elusive. Here, we report that ketohexokinase (KHK), the rate-limiting enzyme of fructose metabolism, is a driver of PDAC development. We demonstrate that fructose triggers KHK and induces fructolytic gene expression in mouse and human PDAC. Genetic inactivation of KhkC enhances the survival of KPC-driven PDAC even in the absence of high fructose diet. Furthermore, it decreases the viability, migratory capability, and growth of KPC cells in a cell autonomous manner. Mechanistically, we demonstrate that genetic ablation of KHKC strongly impairs the activation of KRAS-MAPK pathway and of rpS6, a downstream target of mTORC signaling. Moreover, overexpression of KHKC in KPC cells enhances the downstream KRAS pathway and cell viability. Our data provide new insights into the role of KHK in PDAC progression and imply that inhibiting KHK could have profound implications for pancreatic cancer therapy.

Abstract 1344: myCAFs and iCAFs have similar lineage

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense stroma primarily composed of cancer associated fibroblasts (CAFs). These fibroblasts are a diverse population of cells; some groups of fibroblasts have been shown to play tumor supporting roles while others have been shown to be tumor suppressing. Two prolific and well-characterized CAF subtypes—myofibroblasts (myCAFs) and inflammatory CAFs (iCAFs)—could potentially have different regulatory effects on tumor growth. Understanding the tissue origin of these two groups of CAFs could assist in understanding the complex and heterogeneous roles they might play in PDAC development. Previous studies in our group showed that the splanchnic mesenchyme, a layer of mesenchyme adjacent to the fetal pancreatic epithelium, gives rise to the majority of the total CAF population. Here, I aim to test the hypothesis that both iCAFs and myCAFs originate from the splanchnic mesenchyme. myCAFs have been previously observed to be proximal to tumor cells and express αSMA at a higher level, while iCAFs have been observed to be distal to tumor cells and express αSMA at a lower level. Here, we used a genetically engineered mouse PDAC model carrying KrasG12D/+;p53Frt/+;Pdx1FlpO/+;Isl1cre/+;R26Tomato/+ alleles. In this model, epithelial specific FlpO expression leads to Kras activation and p53 deletion, causing tumorigenesis. Simultaneously, splanchnic specific Cre expression leads to permanent tomato expression in the splanchnic descendants. The pancreata of these mice were harvested, fixed, and mounted onto slides. Co-immunostaining with αSMA and tomato was then done. The stained slides were scanned, and the Inform software was used to segment and phenotype the cells. More than 95% of αSMA high cells were tomato positive, and more than 95% of αSMA low cells were also tomato positive. We thus conclude that both myCAFs and iCAFs originate from the splanchnic mesenchyme. Consistent with previous in vitro studies, this lineage tracing study suggests that different CAF subtypes are likely due to patterning influenced by different factors within the tumor microenvironment. Citation Format: Thomas S. Walter, Lu Han, Michael Ostrowski. myCAFs and iCAFs have similar lineage [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1344.

Increased GPR35 expression in human colorectal and pancreatic cancer samples: A preliminary clinical validation of a new biomarker.

G protein-coupled receptor 35 (GPR35) is involved in carcinogenesis; however, limited experimental data are available on its actual expression in patients with colorectal cancer (CRC) and pancreatic adenocarcinoma (PDAC). We aimed to measure the relative expression of GPR35 in samples from patients with CRC or PDAC. Using real-time polymerase chain reaction (RT-PCR), we have examined GPR35 expression in surgery samples from 40 CRC and 17 PDAC patients, and performed analysis of the results. The analysis of GPR35 expression in patients with CRC revealed correlations between relative GPR35 mRNA expression and several tumor characteristics, with statistical significance for higher American Joint Committee on Cancer (AJCC) stages, T stages and histological grades. GPR35 expression was significantly higher in tumor samples compared to the paired healthy samples collected from the same patient. Similar, although not statistically significant trends were found in PDAC tumor samples for sex (lower expression in women) and for samples with no nodal involvement (lower expression). Samples with higher tumor T stages and higher histological grades or considered inoperable had higher GPR35 expression. We have identified correlations which confirm our expectation of high GPR35 expression in CRC and PDAC. Our findings suggest the prognostic value of GPR35 testing in patients with an increased risk of CRC or PDAC development, and warrant further clinical confirmation.

Abstract A051: Understanding the Arf-p53 axis in PDAC suppression

Abstract Understanding the gene regulatory networks that contribute to PDAC initiation and progression is critical for improving early detection and more effectively treating this disease. Activating mutations in KRAS are often accompanied by mutations in the TP53, CDKN2A, and SMAD4 tumor suppressor genes in ~72%, 30%, and 32% of PDAC cases (Raphael et al. 2017). Understanding the consequences of deletion of individual tumor suppressor genes can delineate specific genotype-phenotype correlations that can provide new clinical insights. Using a genetically engineered mouse PDAC model based on KrasG12D expression and tumor suppressor gene inactivation through Ptf1aCreER expression in adult acinar cells, we showed previously that p53 deletion or point mutation dramatically accelerates PDAC relative to mice with intact p53. To understand tumor evolution in mice with different p53 statuses, we analyzed the transcriptomic profiles of PDACs developing with intact or mutant p53 by spatial transcriptomics and both bulk and single cell RNA-sequencing. These experiments have provided insight into the pathways that become dysregulated as PDAC develops in the context of different p53 alleles. In addition, as oncogenic signals trigger p53 stabilization via the p19ARF (ARF) protein in some contexts, we sought to determine whether ARF acts upstream of p53 in PDAC suppression. Indeed, the frequent deletion of the CDKN2A locus, which encodes both the ARF and p16INK4A (p16) tumor suppressor genes, suggests the importance of both these genes in suppressing PDAC, but the specific role of ARF in PDAC suppression has not been tested. Using mouse models to definitively interrogate the importance of ARF in suppressing PDAC, we found that Arf inactivation promotes KrasG12D-driven PDAC, and with a latency similar to p53 inactivation. Interestingly, concurrent inactivation of both p53 and Arf accelerated oncogenic KRAS-driven PDAC development relative to p53 inactivation alone, suggesting a p53-independent role for ARF in PDAC suppression. Transcriptomic profiling of tumors from these mice revealed both p53-dependent and p53-independent programs of ARF-mediated tumor suppression, which provides insight into how tumors develop in the absence of ARF. Characterization of the immune and fibroblast components of the tumor microenvironment in tumors with intact tumor suppressors, p53 deficiency, Arf deficiency and combined p53;Arf deficiency reveals genotype-dependent differences, including different CAF profiles observed in tumors of different genotypes. Ongoing analysis of human PDAC will help to further parse the roles of ARF and p16 in human pancreatic cancer development. Together, these studies will help to illuminate how p53 and Arf inactivation contribute to cancer cell evolution and crosstalk with the tumor microenvironment during PDAC development, understanding with potential for improving clinical interventions in PDAC. Citation Format: Laura D. Attardi, Brittany M. Flowers, Kathryn Hanson, Abigail S. Mulligan, Sofia Ferreira, Sohinee Bhattacharyya, Hannes Vogel, Laura D. Wood, Mara Sherman. Understanding the Arf-p53 axis in PDAC suppression [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A051.