Development Of Pancreatic Ductal Adenocarcinoma Research Articles

Genetic assessment of IPMN for predicting concomitant pancreatic ductal adenocarcinoma.

Krüppel-like transcription factor 4(KLF4) mutations are more frequently observed in low-grade lesions than in high-grade lesions of intraductal papillary mucinous neoplasms (IPMN) of the pancreas. However, the role of KLF4 mutations in IPMNs with concomitant pancreatic ductal adenocarcinoma (PDAC) remains unclear. This study clarified the rate and effect of KLF4 mutations in IPMN with concomitant PDAC. DNA was extracted from 65 formalin-fixed and paraffin-embedded samples from 52 patients including 13 IPMN with concomitant PDAC and 39 IPMN alone. A comprehensive screening was performed using next-generation sequencing (NGS) for the 5 IPMNs with concomitant PDAC and 5 IPMNs alone, followed by targeted sequencing for KLF4, GNAS, and KRAS mutations. In NGS screening, KRAS mutations were observed in all samples except for one, GNAS mutation in two IPMNs with concomitant PDAC, and a KLF4 mutation in one IPMN with concomitant PDAC. Targeted sequence detected KLF4 mutations in 11 of the 52 IPMNs. Concomitant PDAC developed only in the non-intestinal, non-invasive, and branch duct IPMN cases, and KLF4 mutations were more frequent in this IPMN type than in the other type (36% vs. 10%, p = 0.04). For this IPMN type with KLF4 mutation, PDAC-prediction sensitivity, specificity, and accuracy were 63%, 82%, and 79%, respectively. For selected IPMNs with non-intestinal, non-invasive, and branch duct, genetic assessment might be a helpful tool for predicting the possible development of concomitant PDAC, although a prospective validation study using a larger study population is needed.

MKLN1-AS promotes pancreatic cancer progression as a crucial downstream mediator of HIF-1α through miR-185-5p/TEAD1 pathway

In pancreatic ductal adenocarcinomas (PDAC), profound hypoxia plays key roles in regulating cancer cell behavior, including proliferation, migration, and resistance to therapies. The initial part of this research highlights the important role played by long noncoding RNA (lncRNA) MKLN1-AS, which is controlled by hypoxia-inducible factor-1 alpha (HIF-1α), in the progression of PDAC. Human samples of PDAC showed a notable increase in MKLN1-AS expression, which was linked to a worse outcome. Forced expression of MKLN1-AS greatly reduced the inhibitory impact on the growth and spread of PDAC cells caused by HIF-1α depletion. Experiments on mechanisms showed that HIF-1α influences the expression of MKLN1-AS by directly attaching to a hypoxia response element in the promoter region of MKLN1-AS.MKLN1-AS acts as a competitive endogenous RNA (ceRNA) by binding to miR-185-5p, resulting in the regulation of TEAD1 expression and promoting cell proliferation, migration, and tumor growth. TEAD1 subsequently enhances the development of PDAC. Our study results suggest that MKLN1-AS could serve as a promising target for treatment and a valuable indicator for predicting outcomes in PDAC. PDAC is associated with low oxygen levels, and the long non-coding RNA MKLN1-AS interacts with TEAD1 in this context.Graphical abstract

Resolution of acinar dedifferentiation regulates tissue remodeling in pancreatic injury and cancer initiation.

Background and AimsAcinar to ductal metaplasia (ADM) is crucial in the development of pancreatic ductal adenocarcinoma (PDAC). However, our understanding of the induction and resolution of ADM remains limited. We conducted comparative transcriptome analyses to identify conserved mechanisms of ADM in mouse and human. MethodsWe identified Sox4 among the top upregulated genes. We validated the analysis by RNA in situ hybridization (ISH). We performed experiments in mice with acinar-specific deletion of Sox4 (Ptf1a: CreER; Rosa26-LSL-YFPLSL-YFP; Sox4fl/fl ) with and without an activating mutation in Kras (KrasLSL-G12D/+). Mice were given caerulein to induce pancreatitis. We performed phenotypic analysis by immunohistochemistry, tissue decellularization and single cell RNA sequencing. ResultsWe demonstrated that Sox4 is reactivated in ADM and PanINs. Contrary to findings in other tissues, Sox4 actually counteracts cellular dedifferentiation and helps maintain tissue homeostasis. Moreover, our investigations unveiled the indispensable role of Sox4 in the specification of mucin-producing cells and tuft-like cells from acinar cells. We identified Sox4-dependent non-cell-autonomous mechanisms regulating the stromal reaction during disease progression. Notably, Sox4-inferred targets are activated upon KRAS inactivation and tumor regression. ConclusionsOur results indicate that our transcriptome analysis can be used to investigate conserved mechanisms of tissue injury. We demonstrate that Sox4 restrains acinar dedifferentiation and is necessary for the specification of acinar-derived metaplastic cells in pancreatic injury and cancer initiation and is activated upon Kras ablation and tumor regression in mice. By uncovering novel potential strategies to promote tissue homeostasis, our findings offer new avenues for preventing the development of PDAC.

The scRNA-sequencing landscape of pancreatic ductal adenocarcinoma revealed distinct cell populations associated with tumor initiation and progression

Pancreatic ductal adenocarcinoma (PDAC) stands as a formidable malignancy characterized by its profound lethality. The comprehensive analysis of the transcriptional landscape holds immense significance in understanding PDAC development and exploring novel treatment strategies. However, due to the firm consistency of pancreatic cancer samples, the dissociation of single cells and subsequent sequencing can be challenging. Here, we performed single-cell RNA sequencing (scRNA-seq) on 8 PDAC patients with different lymph node metastasis status. We first identified the crucial role of MMP1 in the transition from normal pancreatic cells to cancer cells. The knockdown of MMP1 in pancreatic cancer cell lines decreased the expression of ductal markers such as SOX9 while the overexpression of MMP1 in hTERT-HPNE increased the expression of ductal markers, suggesting its function of maintaining ductal identity. Secondly, we found a S100A2+ tumor subset which fueled lymph node metastasis in PDAC. The knockdown of S100A2 significantly reduced the motility of pancreatic cancer cell lines in both wound healing and transwell migration assays. While overexpression of S100A2 led to increased migratory capability. Moreover, overexpression of S100A2 in KPC1199, a mouse pancreatic cancer cell line, caused a larger tumor burden in a hemi-spleen injection model of liver metastasis. In addition, epithelial-mesenchymal transition-related genes were decreased by S100A2 knockdown revealed by bulk RNA sequencing. We also identified several pivotal contributors to the pro-tumor microenvironment, notably OMD+ fibroblast and CCL2+ macrophage. As a result, our study provides valuable insights for early detection of PDAC and promising therapeutic targets for combatting lymph node metastasis.

Human P2X7 receptor variants Gly150Arg and Arg276His polymorphisms have differential effects on risk association and cellular functions in pancreatic cancer

BackgroundThe purinergic P2X7 receptor (P2X7R) plays an important role in the crosstalk between pancreatic stellate cells (PSCs) and cancer cells, thus promoting progression of pancreatic ductal adenocarcinoma (PDAC). Single nucleotide polymorphisms (SNPs) in the P2X7R have been reported for several cancers, but have not been explored in PDAC.Materials and methodsBlood samples from PDAC patients and controls were genotyped for 11 non-synonymous SNPs in P2X7R and a risk analysis was performed. Relevant P2X7R-SNP GFP variants were expressed in PSCs and cancer cells and their function was assayed in the following tests. Responses in Ca2+ were studied with Fura-2 and dye uptake with YO-PRO-1. Cell migration was monitored by fluorescence microscopy. Released cytokines were measured with MSD assay.ResultsRisk analysis showed that two SNPs 474G>A and 853G>A (rs28360447, rs7958316), that lead to the Gly150Arg and Arg276His variants, had a significant but opposite risk association with PDAC development, protecting against and predisposing to the disease, respectively. In vitro experiments performed on cancer cells and PSCs expressing the Gly150Arg variant showed reduced intracellular Ca2+ response, fluorescent dye uptake, and cell migration, while the Arg276His variant reduced dye uptake but displayed WT-like Ca2+ responses. As predicted, P2X7R was involved in cytokine release (IL-6, IL-1β, IL-8, TNF-α), but the P2X7R inhibitors displayed varied effects.ConclusionIn conclusion, we provide evidence for the P2X7R SNPs association with PDAC and propose that they could be considered as potential biomarkers.

Abstract CT022: Mutant KRAS peptide-based vaccine in patients at high risk of developing pancreatic cancer: Preliminary analysis from a phase I study

Abstract Background: The development of pancreatic ductal adenocarcinoma (PDAC) from the first driver event takes >10 years, providing a unique window of opportunity for early interception. As a frequent alteration identified in both PDAC and pancreatic premalignancy, mutant KRAS (mKRAS) has emerged as a potential target for immune-based interception strategies in individuals at high risk of developing pancreatic cancer. Methods: This is a single-arm, open-label, second-in-human phase I study of a pooled synthetic long peptide (SLP) vaccine with poly-ICLC adjuvant targeting up to six mKRAS subtypes (G12D, G12R, G12V, G12A, G12C, G13D) in patients with hereditary predisposition to developing PDAC (NCT05013216). Key inclusion: radiographic evidence of a pancreatic abnormality and eligibility under at least one of three high-risk groups based on family history of PDAC and/or germline mutation status. Vaccination schema: mKRAS vaccine given on weeks 1, 3, 5, and 13. Peripheral blood collected at baseline and weeks 5, 13, and 17 for analysis of immunogenicity by IFN-gamma ELISpot. Co-primary endpoints: safety and mKRAS-specific T cell response. Results: At the time of data cutoff (November 15, 2023), 15 out of 20 planned patients were vaccinated on study and evaluable for immunogenicity. All 15 patients achieved a mKRAS-specific T cell response, defined by the maximal fold change in the pooled average IFN-gamma-producing mKRAS-specific T cell density within 17 weeks post-vaccination (median: 12.3, range: 3.1-167). Differential immunogenicity elicited by individual mKRAS epitopes was noted in this cohort. Specifically, the mKRAS G12V peptide induced the highest fold change increase in antigen-specific T cell expansion (median: 32.6, range: 2.9-243) while the mKRAS G12D peptide was the least immunogenic (median: 7.6, range: 0.4-104). Regarding safety, all adverse events were grade 1 or grade 2 in severity per NCI CTCAE v5.0. The most frequent vaccine-related adverse events were injection site reactions (93.8%), fatigue (62.5%), chills (43.8%), and headache (37.5%). No safety signals were observed. Conclusions: For the first time, we demonstrate mKRAS-specific T cell responses induced by vaccination in a cohort of high-risk individuals with inherited predisposition to PDAC. These findings directly serve as proof-of-concept to advance vaccine strategies focused on intercepting the development of pancreatic cancer in high-risk populations. Support: Stand Up To Cancer, Lustgarten Foundation, and NIH/NCI. Citation Format: Saurav D. Haldar, Amanda Huff, Elizabeth Abou Diwan, Anna Ferguson, Carol Judkins, Jiayun Lu, Hao Wang, Hassan Sinan, Christopher Thoburn, Katherine M. Bever, Mark Yarchoan, Amy M. Thomas, Julie M. Nauroth, Daniel A. Laheru, Michael G. Goggins, Elizabeth M. Jaffee, Nilofer S. Azad, Neeha Zaidi. Mutant KRAS peptide-based vaccine in patients at high risk of developing pancreatic cancer: Preliminary analysis from a phase I study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT022.

Immunohistochemical characteristics of epithelial-mesenchymal transition in pancreatic ductal adenocarcinoma

The epithelial-mesenchymal transition (EMT) plays a crucial role in the development of pancreatic ductal adenocarcinoma (PDAC). There are insufficient and contradictory data in the scientific literature on the peculiarities of epithelial and mesenchymal marker expression in PDAC ducts and EMT zone, which requires further research on the role of EMT in the development and progression of PDAC. Aim: to conduct a comprehensive assessment of epithelial and mesenchymal markers of EMT in the PDAC ducts and EMT zone at different degrees of differentiation. Materials and methods. A comprehensive pathomorphological and immunohistochemical examination including 49 cases of surgical material from patients with PDAC, divided into groups of moderate (G2) and low degree of tumor differentiation (G3). Results. PDAC was characterized by low levels of E-cadherin expression in both ducts and EMT – Me = 22.58 % [12.81; 36.23] and Me = 25.17 % [19.04; 35.37], respectively (p > 0.05). Only membrane expression of the marker was detected in the ducts and membrane-cytoplasmic one – in the EMT zone without significant differences in the groups. There was a significant decrease in the ductal β-catenin expression (p < 0.05) in G2 with membrane-cytoplasmic staining (Me = 15.58 % [10.42; 26.24]), in G3 – with membrane (Me = 4.42 % [2.35; 5.93]); in the EMT zone, only membrane-cytoplasmic expression was observed in both groups without significant difference (p > 0.05). Membrane expression of CK7 was positive in 100 % of PDAC, significantly lower at G2 (Me = 19.51 % [10.70; 27.24]; Me = 26.19 % [20.93; 30.05], p < 0.05), and CK18 (Me = 21.34 % [9.68; 29.96] – in G2, in G3 – Me = 22.50 % [8.24; 40.08], p > 0.05). Expression of α-SMA and Vim was seen in spindle-shaped stromal cells, around tubular and trabecular structures with membrane-cytoplasmic staining. There was no significant difference between α-SMA in G2 and G3, the level of vimentin was significantly lower in G3 (p < 0.05). Conclusions. The features of PDAC E-cadherin, β-catenin, CK7 and CK18 marker expression indicate a greater EMT process at the periphery of the tumor and its severity increases with tumor progression. The optimal markers to determine the mesenchymal phenotype of PDAC cells are α-SMA and vimentin.

STIM1/SOX2 proteins are co-expressed in the tumor and microenvironmental stromal cells of pancreatic ductal adenocarcinoma and ampullary carcinoma

Pancreatic ductal adenocarcinoma (PDAC) and ampullary carcinoma (AAC) are lethal malignancies with modest benefits from surgery. SOX2 and STIM1 have been linked to anticancer activity in several human malignancies. This study included 94 tumor cases: 48 primary PDAC, 25 metastatic PDAC, and 21 primary AAC with corresponding non-tumor tissue. All cases were immunohistochemically stained for STIM1 and SOX2 and results were correlated with clinicopathologic data, patient survival, and BCL2 immunostaining results. Results revealed that STIM1 and SOX2 epithelial/stromal expressions were significantly higher in PDAC and AAC in comparison to the control groups. STIM1 and SOX2 expressions were positively correlated in the primary and metastatic PDAC (P = 0.016 and, P = 0.001, respectively). However, their expressions were not significantly associated with BCL2 expression. SOX2 epithelial/stromal expressions were positively correlated with the large tumor size in the primary AAC group (P = 0.052, P = 0.044, respectively). STIM1 stromal and SOX2 epithelial over-expressions had a bad prognostic impact on the overall survival of AAC (P = 0.002 and P = 0.001, respectively). Therefore, STIM1 and SOX2 co-expression in tumor cells and intra-tumoral stroma could contribute to the development of PDAC and AAC. STIM1/SOX2 expression is linked to a bad prognosis in AAC.

Abstract 4323: Deletion of the lipid kinase PIKfyve in Kras-driven pancreatic genetically engineered mouse model

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) accounts for 95% of all pancreatic cancers and, as one of the most lethal cancers, has a five-year survival rate of approximately 6%. Autophagic processes have been reportedly elevated in PDAC; thus, the inhibition of autophagy leads to decreased tumor growth. Autophagic processes are catabolic mechanisms that recycle cellular components. Cells in stressful conditions, such as starvation, have elevated autophagy to redistribute energy to sustain cell survival; however, this can also lead to cell death when attempts to maintain cell viability fail. In addition, autophagy is known to promote immune evasion by tumors and cancer progression and appears to be required for the optimal development of PDAC. The lipid kinase PIKfyve converts phosphatidylinositol-3-phosphate to phosphatidylinositol 3,5-bisphosphate and is required in the late stage of the autophagic pathway. Blocking PIKfyve disrupts autophagic processes. However, the role of PIKfyve in PDAC development has not been fully understood. We investigated the role of PIKfyve in the progression of PDAC using a genetically engineered mouse model. Given the poor clinical outcomes of pharmacological inhibition of autophagy by chloroquine, we have evaluated the PIKfyve inhibitor ESK981 in the context of PDAC progression. Methods: The pancreatic-specific deletion of PIKfyve was established on a Kras G12D, Tp53 R172H, and p48 Cre (KPC) or Kras G12D and p48 Cre (KC) background. The ratio of pancreatic mass to body weight was monitored at various time points. Histopathology evaluations were conducted based on hematoxylin and eosin staining and immunohistochemistry of cytokeratin 19 staining. Pharmacological inhibition of PIKfyve was performed on KPC mice using ESK981 monotherapy. Four weeks after vehicle or ESK981 treatment, pancreatic tissues were collected intact and weighed. Histopathology evaluations were performed to evaluate the lesion. Conclusion: We demonstrated that the deletion of PIKfyve in pancreatic tissue significantly reduces the pancreatic mass weight compared to the PIKfyve wildtype counterpart and is coupled with less pancreatic intraepithelial neoplasia (PanIN) and PDAC development in KPC and KC mice. Compared to the vehicle treatment, pharmacological inhibition of PIKfyve by ESK981 on KPC mice resulted in a significant increase in the percentage of normal pancreatic tissue and reduced pancreatic lesion development. Citation Format: Sarah N. Yee, Xia Jiang, Caleb Cheng, Ahmet Korkaya, Rahul Mannan, Rohit Mehra, Yuanyuan Qiao, Arul M. Chinnaiyan. Deletion of the lipid kinase PIKfyve in Kras-driven pancreatic genetically engineered mouse model [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 4323.

Abstract 6631: Aging influences the pancreatic ductal adenocarcinoma tumor immune microenvironment to promote features of immunosuppression

Abstract Advancing age is the strongest independent risk factor for the development of pancreatic ductal adenocarcinoma (PDAC) and a negative prognostic factor. PDACs are typically immunologically “cold” tumors and do not often respond to currently approved immunotherapy treatment approaches. How aging may alter non-malignant cells, such as fibroblasts and immune cell populations, in the PDAC tumor microenvironment (TME) to decrease anti-tumor immune responses is not understood. We identified that aged microenvironments significantly alter the growth and progression of PDAC tumors. Orthotopic KPC and Panc02 mouse PDAC cell line injections into the pancreata of aged (>64-week-old) compared to young (6-8-week-old) C57BL/6 mice result in more rapid tumor growth and increased frequency of liver metastases in vivo. Tumors from aged mice have high expression of the TGFβ family member GDF-15 and hyperactivation of AKT signaling driven by the release of secreted factors from aged pancreatic fibroblasts. We found that in aged microenvironments there is a marked reduction in the number of CD8+ T cells and an increase in FoxP3+ regulatory T cells in primary orthotopic pancreatic tumors using the KPC and Panc02 models. In addition, we performed portal vein injections to establish liver metastases to study the differences in immune cell infiltration in metastases from young vs. aged microenvironments. Aged mice form more and larger liver metastases compared to young mice, and metastatic lesions in aged mice have decreased CD8+ T cell infiltration when compared to young mice. Since tumors in aged microenvironments have increased AKT activation compared to young microenvironments, we next tested if AKT signaling was sufficient to alter immune cell infiltration in PDAC tumors. In young mice, treatment with recombinant GDF-15, which activates AKT signaling, reduced CD8+ T cell infiltration into tumors. Conversely, in KPC tumors grown in aged mice treated with the AKT inhibitor MK-2206, there was a trend toward increased CD8+ T cell infiltration and a significant reduction in immunosuppressive FoxP3+ Tregs. Together, these data highlight age-dependent differences in the immune infiltration in PDAC tumors. They suggest that activation of AKT signaling can drive features of immunosuppression in PDAC, providing the rationale for targeting AKT signaling to improve antitumor immune responses in PDAC. Citation Format: Emma Kartalia, James M. Leatherman, Jae W. Lee, Elizabeth M. Jaffee, Ashani T. Weeraratna, Daniel J. Zabransky. Aging influences the pancreatic ductal adenocarcinoma tumor immune microenvironment to promote features of immunosuppression [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 6631.

Abstract 3939: Ror2, a novel key regulator driving cell fate decisions throughout pancreatic tumor progression

Abstract Introduction: Reprogramming of pancreas cell fate drives development of pancreatic ductal adenocarcinoma (PDAC). Acinar cells, the most probable origin of pancreatic cancer, undergo a rapid cell identity switch towards a duct-like phenotype upon KrasG12D expression and when combined with pancreatitis or the loss of acinar differentiation factors. Metaplastic and dysplastic duct-like cells are heterogeneous with a proportion acquiring features reminiscent of gastric lineages. While some gastric signatures are maintained in the classical PDAC subtype, they are eroded in the more aggressive, basal-like PDAC. Since subtype identity has a major impact on prognosis and therapeutic targetability, druggable targets that regulate cellular reprogramming in pancreatic cancer can be exploited to increase sensitivity to therapy. Methods: To elucidate mechanisms responsible for early reprogramming, pancreatic tissue of mice with conditional KrasG12D expression and loss of Pdx1 was analyzed by single-nucleus ATAC-seq. Expression of identified target genes was studied in precancerous lesions and PDAC by using multiplex RNAscope and IHC staining. Computational analyses of publicly available sequencing data were used to establish correlation to PDAC subtype identity. Genes were functionally studied in PDAC cell lines. Results: By performing snATAC-seq and RNA-seq of early transformed pancreatic tissue, we discovered that acinar cells with the combined expression of KrasG12D and loss of Pdx1 activate expression of a gastric metaplastic gene signature accompanied by elevated levels of the receptor kinase Ror2. Ror2 is also highly expressed in distinct subpopulations of metaplastic and dysplastic cells, associated with a gastric neck cell phenotype and enhanced proliferative capacity. In contrast, Ror2Low lesions are characterized by a gastric pit cell-like and a senescent phenotype. Genetic ablation of Ror2 resulted in a shift in lesion identity, enriching those with a pit cell and senescent identity. In PDAC, we found that Ror2 anti-correlates with a similar gastric pit cell phenotype that is maintained in the classical subtype PDAC but is strongly associated with the more aggressive basal-like subtype. Overexpression of ROR2 in human PDAC cell lines with a classical differentiation induced loss of the classical gene signature as well as epithelial-to-mesenchymal transition. Moreover, ROR2 enforces a strong dependency on AKT signaling, causing increased vulnerability of ROR2-expressing cells to AKT inhibition, but increased resistance to the KRAS inhibitor MRTX1133. Conclusions: We discovered Ror2 as a critical determinant of precancerous lesion as well as PDAC subtype identity. Its role in driving an aggressive PDAC phenotype that is inherently resistant to Kras inhibition suggests that inhibiting this receptor tyrosine kinase will enhance sensitivity to the new generation of targeted therapies. Citation Format: Simone Benitz, Alexander Steep, Malak Nasser, Jonathan Preall, Ujjwal Mahajan, Ian Loveless, Holly McQuithey, Erick Davis, Hui-Ju Wen, Daniel Long, Thomas Metzler, Samuel Zwernik, Daniel Salas-Escabillas, Ling Huang, Nina Steele, Ivonne Regel, Filip Bednar, Howard Crawford. Ror2, a novel key regulator driving cell fate decisions throughout pancreatic tumor progression [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 3939.

Abstract 1206: Three-dimensional immune atlas of pancreatic cancer precursor lesions reveals large inter- and intra-lesion heterogeneity

Abstract Immunotherapies are generally ineffective in pancreatic ductal adenocarcinoma (PDAC). Paradoxically, inflammation is believed to play a key role in PDAC development and invasion: patients suffering from chronic pancreatitis have a 13-fold increase in risk of developing PDAC. PDAC cells are surrounded by a dense network of fibrotic tissue containing immunosuppressive cells such as regulatory T cells, tumor-associated macrophages, and cancer-associated fibroblasts. Better understanding of the role of inflammation in PDAC could lead to the design of effective immunotherapies. Pancreatic intraepithelial neoplasia (PanIN) is a precursor to PDAC. While most of us will develop PanINs, few of these lesions will progress to invasive cancer. Efforts to determine which PanINs are likely to progress have shown that the size, incidence, and genetic variation of these lesions is high. Here, we add to these efforts through in-depth quantitative analysis of the pancreatic immune microenvironment. CODA is a novel 3D imaging technique used extensively to study the structure, transcriptomic signatures, and genetic heterogeneity of PanINs. In serial histological samples, CODA integrates image registration, cell detection, and segmentation of nine pancreatic structures: PanIN, normal ducts, islets, acini, nerves, vasculature, fat, stroma, and immune aggregates. Through integration with CD45, CD3, and FoxP3 labelled images, we explored immune infiltration in 48 large (cm3) samples of human pancreas tissues containing >1,000 PanIN lesions to describe spatial correlations between local immune hotspots, 3D tumor morphology, and the surrounding pancreas structures. We quantified inter- and intra-sample heterogeneity in immune response to pancreatic cancer precursor lesions. We determined that this cohort varied greatly in both the number of PanIN (range 3 - 92 per cm3) and overall immune cell density (range 3,000 - 40,000 immune cells/mm3). We determined that, while PanIN size is not related to local immune cell density, overall PanIN burden is highly correlated to global immune cell density. This suggests that PanIN content does not have a short-range relationship to inflammation, but does have a long-range relationship with pancreatic structures and immune infiltration. As chronic inflammation in the pancreas shows correlation with development of pancreatic cancer, increased knowledge of the heterogeneous, local immune cell environment in precancerous samples will help us determine the role of inflammation in reacting to and controlling malignant progression. Citation Format: Ashley L. Kiemen, Cristina Almagro-Pérez, Valentina Matos-Romero, Alicia M. Braxton, Jaanvi Mahesh-Babu, Elizabeth D. Thompson, Toby C. Cornish, PeiHsun Wu, Laura D. Wood, Arrate Muñoz-Barrutia, Ralph H. Hruban, Denis Wirtz. Three-dimensional immune atlas of pancreatic cancer precursor lesions reveals large inter- and intra-lesion heterogeneity [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 1206.

Abstract 4774: Inhibition of pancreatic acinar ductal metaplasia by novel YAP/TAZ inhibitor, CV-4-26

Abstract Current challenges for pancreatic ductal adenocarcinoma (PDAC) treatment include overcoming the disease’s fibrous tumor microenvironment with limited cytotoxicity- in which tissue stiffening is believed to occur even pre-disease onset. Acinar to ductal metaplasia (ADM), the transdifferentiation of acinar cells into duct-like cells, is a predisposing event to PDAC development that is also linked to Hippo signaling, a major pathway involved in fibrosis and inflammation. Hippo activation is initiated by the yes-associated protein (YAP) in conjunction with the PDAZ binding motif (TAZ) and TEA domain (TEAD) transcription factor. Thus, the synthesis of the novel covalent TEAD autopalmitoylation inhibitor, CV-4-26, presented an opportunity to therapeutically target ADM. CV-4-26 binds to the TEAD lipid binding pocket, which, in turns, hinders TEAD palmitoylation, thereby leading to the inhibition of the Hippo pathway. Utilizing our 3D organoid p48Cre/+LSL-KRASG12D/+ (KC) mouse model, we demonstrated pronounced phenotypic and molecular inhibition of ADM by CV-4-26, resulting in a conserved acinar phenotype, as well as downregulation of downstream markers of the YAP/TAZ complex. The compound did not reverse ADM to the acinar state once ducts were visibly formed. Yet, CV-4-26 demonstrated a lack of cytotoxicity (up to 25 µM) as determined by Calcein AM staining. Biomechanical measurements of extracellular matrix stiffness acquired through calculations of fluorescent nanoparticle displacement further displayed decreased moduli as a response to inhibition treatment. Moreover, a 56-fold downregulation of collagen (COL1A1, COL1A2) mRNA, markers for PDAC development, was observed. However, less reduction in the collagens and extracellular matrix stiffness was observed in the ADM reversal treatments, corroborating our morphology and gene expression results. Our data suggest that combining CV-4-26 by ADM inhibition with standard of care therapies for PDAC may enhance the penetration of small molecules through the tumor’s extracellular matrix. Citation Format: Corey M. Perkins, Chen Zhou, Martha Campbell-Thompson, Yating Mao, Kalina R. Atanasova, Jinmai Jiang, Ranjala Ratnayake, Hendrik Luesch, Jamel Ali, Chenglong Li, Thomas D. Schmittgen. Inhibition of pancreatic acinar ductal metaplasia by novel YAP/TAZ inhibitor, CV-4-26 [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 4774.

Abstract 3427: Serum metabolites and pancreatic ductal adenocarcinoma in two prospective cohorts

Abstract Objective: Metabolomics may provide insight to understand the etiology and potential mechanisms for pancreatic ductal adenocarcinoma (PDAC) development beyond established risk factors of smoking, diabetes, and adiposity. We extended our previous metabolomic study by including additional participants from the Prostate, Lung, Colorectal, and Ovarian Cancer Screening Trial (PLCO) with the aim to examine prospective associations between serum metabolites and incident PDAC. Method: We measured 1483 known metabolites in pre-diagnostic serum (up to 24 years) in two PDAC nested case-control studies within the PLCO (360 cases-control pairs) of mostly non-smoker men and women and Alpha-Tocopherol, Beta-Carotene Cancer Prevention Study (ATBC, 372 cases-control pairs) male smokers using liquid chromatography, high resolution/accurate mass spectrometry. Controls were incidence-density sampled and matched to cases by age, blood draw date, sex, race/ethnicity, and cohort. We used conditional logistic regression to calculate odds ratios and 95% confidence intervals for PDAC per standard deviation increase in log10-metabolite levels in each cohort and combined using fixed-effect meta-analyses and stratified by follow-up time. We also performed ElasticNet regression in each cohort to identify metabolites. We assessed associations of 72 predefined metabolic pathways with PDAC by comparing Fisher’s statistic of metabolite p-values per pathway with a null distribution based on 500,000 permutations. Results: Sixty-six metabolites were associated with PDAC at a false discovery rate (FDR)<0.05 with 26 below the Bonferroni threshold (P<3.4 × 10−5). Among the 66 metabolites, 14 and 15 were selected in ElasticNet regressions in ATBC and PLCO, respectively, with two in both cohorts. Notable findings include: fibrinopeptide B(1-9); seven modified, di- or poly-peptides; eight xenobiotics related to tobacco smoke; amino acids, carbohydrates, and TCA cycle metabolites known to be regulated by mutant KRAS (aspartate, glutamate, lactate, pyruvate, and α-ketoglutarate); four secondary bile acids and three aromatic amino acids (tryptophan, phenylalanine, and tyrosine) that were positively, while four fibrinogen cleavage peptides were inversely associated with PDAC. Among the top identified metabolites, aspartate, α-glutamyltyrosine, pyroglutamylglutamine, and Glu-Gly-Asn-Val showed time-varying associations overall (P≤0.05) with greater magnitudes within the first 5 years after blood draw. Thirteen metabolic pathways were associated with PDAC (Bonferroni P≤0.05). Conclusion: Our study suggests pre-diagnostic circulating metabolites, particularly those potentially related to subclinical disease, microbiome, and tobacco metabolism are associated with PDAC. The identified metabolites along with other known risk factors might be useful in algorithms to model disease risk and progression. Citation Format: Ting Zhang, Steven C. Moore, Sheng Fu, Kevin Wang, Demetrius Albanes, Stephanie J. Weinstein, Kai Yu, Rachael Z. Stolzenberg-Solomon. Serum metabolites and pancreatic ductal adenocarcinoma in two prospective cohorts [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 3427.

Abstract 2087: Targeting lipid metabolism in pancreatic cancer

Abstract Background PIKfyve is a lipid kinase that serves as the sole source of cellular PI(3,5)P2 and PI5P, critical phosphatidylinositols for autophagy and lysosome function. Pancreatic ductal adenocarcinoma (PDAC) is known to upregulate and depend on lysosomal functions such as autophagy to exist in its harsh, nutrient-disrupted microenvironment. Thus, we aimed to establish PIKfyve as a therapeutic target in PDAC. Using a PIKfyve-knockout genetically engineered mouse model of pancreatic cancer (KC and KPC), we determined that PIKfyve was necessary for PDAC development. Given these results, we hypothesized that PIKfyve was critical for maintaining metabolic homeostasis in PDAC and that inhibiting PIKfyve would be an efficacious therapeutic strategy for PDAC. Methods and results We first validated the known role of PIKfyve in regulating autophagy using CRISPRi mediated knockdown, PIKfyve inhibitors Apilimod and ESK981, and a PIKfyve degrader, which all demonstrated that PIKfyve perturbation disrupted autophagic flux and suppressed PDAC cell growth. Little is known about the metabolic impacts of PIKfyve inhibition in PDAC beyond disruption of autophagy. Thus, we paired RNA-seq and a metabolic CRISPR screen to discover that PIKfyve inhibition obligated PDAC cells to upregulate de novo lipid synthesis. Specifically, PDAC cells upregulated fatty acid synthesis genes ACACA and FASN upon PIKfyve inhibition. Additionally, these genes were synthetically critical in the context of PIKfyve inhibition. We further performed metabolomics and lipidomics which revealed accumulation of sphingolipids upon PIKfyve inhibition. Given this, we concluded that PIKfyve regulates lipid homeostasis in PDAC. To further establish PIKfyve as a target in PDAC, we investigated the role of PIKfyve upon inhibition of KRAS, which drives PDAC through MAPK pathway signaling. First, KRAS/MAPK inhibition diminished expression of FASN and ACACA, genes synthetically essential upon PIKfyve inhibition. Additionally, KRAS/MAPK inhibition upregulated autophagy which was attenuated upon concurrent PIKfyve inhibition. Given this metabolic conflict between KRAS/MAPK and PIKfyve, we assessed the effects of dual inhibiting KRAS/MAPK and PIKfyve in a syngeneic orthotopic model. Encouragingly, combination therapy of trametinib (MEK inhibitor) and ESK981 eliminated tumor burden in most of mice, while single treatments only had modest effects. Further, we established a patient-derived xenograft model and showed that while the treatment with individual compounds trametinib, MRTX-1133 (KRASG12D inhibitor), and ESK981 all suppressed tumor growth, the combination of ESK981 and trametinib or ESK981 and MRTX-1133 caused substantial tumor regression in nearly all mice. Taken together, targeting lipid metabolism through inhibition of PIKfyve is a promising therapeutic strategy for PDAC, particularly in combination with the numerous KRAS/MAPK inhibitors in clinical development. Citation Format: Caleb Cheng, Jasmine P. Wisniewski, Ahmet Korkaya, Bailey Jackson, Rahul Mannan, Somnath Mahapatra, Nicholas Rossiter, Rupam Bhattacharyya, Pietro Morlacchi, Sydney Peters, Yuanyuan Qiao, Costas A. Lyssiotis, Arul M. Chinnaiyan. Targeting lipid metabolism 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 2087.

Abstract 6627: Boosting macrophage-specific BCAA oxidation enhances immune activation within the tumor microenvironment and diminishes tumor growth in pancreatic cancer

Abstract Treating pancreatic ductal adenocarcinoma (PDAC) poses a significant challenge, mainly due to therapy resistance linked to the immunosuppressive tumor microenvironment (TME) within PDAC tumors. This TME is characterized by a fibrotic stroma and an abundance of immunosuppressive cells, notably tumor-associated macrophages (TAMs). TAMs crucially hinder effector T cell activity and contribute to T cell exhaustion. Redirecting TAMs toward an immunostimulatory state has emerged as a promising approach in PDAC treatment. Recent investigations, including our own studies, reveal a profound influence of metabolic pathways on the immune phenotype of TAMs. Specifically, dysregulation in branched-chain amino acid (BCAA) metabolism has been observed in PDAC patients, manifesting as elevated circulating BCAA levels correlating with a doubled risk of PDAC development. However, the extent to which BCAA metabolism influences TAM phenotype and anti-tumor immune responses remains a largely unexplored territory, presenting a challenge for PDAC treatment. Our focus revolves around understanding how heightened BCAA levels in PDAC affect TAM phenotype, the immunosuppressive tumor environment, and tumor progression. Employing an orthotopic PDAC model and genetically modified mice, specifically BCKDKfl/fl BCKDK LysMcre/+ (mimicking increased BCAA oxidation), alongside a BCKDK-targeting pharmacological inhibitor (BT-2), multicolor flow cytometry, and in vitro models, our research has revealed crucial insights. We found a diminished expression of BCAA oxidation genes in TAMs compared to normal pancreatic macrophages. Interestingly, M1 and M2 macrophages seemed to utilize BCAAs differently, reflected in varying levels of TCA cycle intermediates derived from BCAA oxidation. Notably, boosting BCAA oxidation specifically in TAMs through genetic modifications resulted in reduced PDAC growth associated with an enhanced immunostimulatory TAM phenotype and activation of CD8+ T cells in the TME. Conversely, administering BCAAs induced an immunosuppressive TAM phenotype, fostering PDAC growth, suggesting a direct influence of BCAAs on TAM phenotype. Pharmacologically enhancing BCAA oxidation via BT-2 bolstered immune activation within the tumor environment and reduced PDAC growth. Intriguingly, our analysis of publicly available data unveiled a significant correlation between high BCAA oxidation and favorable responses to anti-PD1 therapy in cancer patients. Our findings emphasize the pivotal role of BCAA metabolism in shaping the immune landscape of PDAC, offering potential avenues for therapeutic interventions targeting TAMs and mitigating TME immunosuppression. Citation Format: Gauri Mirji, Sajad Ahmad Bhat, Zachary Schug, Ben Z. Stanger, Zoltan Arany, Rahul S. Shinde. Boosting macrophage-specific BCAA oxidation enhances immune activation within the tumor microenvironment and diminishes tumor growth 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 6627.

A 15-Inflammation-Related Gene Signature Predicts the Prognosis of Patients With Pancreatic Ductal Adenocarcinoma

Chronic inflammation promotes the development of pancreatic ductal adenocarcinoma (PDAC) and PDAC-related inflammatory tumor microenvironment facilitates tumor growth and metastasis. Thus, we aimed to study the association between inflammatory response and prognosis in patients with PDAC. We conducted the whole transcriptomic sequencing using tissue samples collected from patients diagnosed with PDAC (n = 106) recruited from Shandong Cancer Hospital. We first constructed a prognostic signature using 15 inflammation-related genes in The Cancer Genome Atlas (TCGA) cohort (n = 177) and further validated it in an independent International Cancer Genome Consortium (ICGC) cohort (n = 90) and our in-house cohort. PDAC patients with a higher risk score had poorer overall survival (OS) (P < 0.001; HR, 3.02; 95% CI, 1.94-4.70). The association between the prognostic signature and OS remained significant in the multivariable Cox regression adjusting for age, sex, alcohol exposure, diabetes, and stage (P < 0.001; HR, 2.91; 95% CI, 1.73-4.89). This gene signature also robustly predicted prognosis in the ICGC cohort (P = 0.01; HR, 1.94; 95% CI, 1.14-3.30) and our cohort (P < 0.001; HR, 2.40; 95% CI, 1.45-3.97). Immune subtype C3 (inflammatory) was enriched and CD8+ T cells were higher in patients with a lower risk score (P < 0.05). Furthermore, PDAC patients with higher risk scores were more sensitive to chemotherapy, immunotherapy, and PARP inhibitors (P < 0.05). In sum, we identified a novel gene signature that was associated with inflammatory response for risk stratification, prognosis prediction, and therapy guidance in PDAC patients. Future studies are warranted to validate the clinical utility of the signature.

Prevención y detección precoz del cáncer de páncreas

In western countries, pancreatic ductal adenocarcinoma (PDAC) has shown a rising incidence and is currently one of the leading causes of cancer related mortality. Due to its aggressive biology, most patients diagnosed with PDAC present with advanced disease not suitable for curative treatment. An early detection strategy could potentially improve results by detecting more PDAC patients within the initial stages of the disease.Currently, diagnosis of PDAC is mostly based in cross sectional imaging, including computed tomography and magnetic resonance imaging (MRI) as well as biomarkers such as CA 19-9. Selected cases might be further studied using endoscopic ultrasound (EUS) with the option of EUS guided biopsy if histology is needed to confirm diagnosis.Due to its low incidence, screening general population for PDAC has been shown to be an ineffective and expensive approach. For this reason, efforts have shifted towards surveillance of specific groups of patients known to be at risk for development of PDAC. In these groups, screening has shown to have a higher yield for early cases and improved results in terms of resection rates and survival. These groups include carriers of certain oncogenic germline mutations, patients with strong family history for PDAC, and patients diagnosed with mucinous cystic neoplasms of the pancreas. Patients over 50 years old diagnosed with new-onset diabetes and patients living with chronic pancreatitis are also considered to be at risk for PDAC development. Patients selected for surveillance should be included in screening protocols including periodic imaging with MRI or EUS.Development of biomarkers is an area of active research in the field of pancreatic cancer. New genetic or molecular biomarkers, to be measured in an isolated or combined manner, are an interesting approach looking to improve current diagnostic capabilities and to provide new tools for effective screening in wider groups of patients.The aim of this article is to describe strategies currently available for early detection of PDAC, including promising research areas for the near future.

Integrin-αvβ6 targeted peptide-toxin therapy in a novel αvβ6-expressing immunocompetent model of pancreatic cancer

Previously we reported that a novel αvβ6-specific peptide-drug conjugate (SG3299) could eliminate established human pancreatic ductal adenocarcinoma (PDAC) xenografts. However the development of effective therapies for PDAC, which is an essential need, must show efficacy in relevant immunocompetent animals. Previously we reported that the KPC mouse transgenic PDAC model that closely recapitulates most stages of development of human PDAC, unlike in humans, failed to express αvβ6 on their tumours or metastases. In this study we have taken the KPC-derived PDAC line TB32043 and engineered a variant line (TB32043mb6S2) that expresses mouse integrin αvβ6. We report that orthotopic implantation of the αvβ6 over-expressing TB32043mb6S2 cells promotes shorter overall survival and increase in metastases. Moreover, systemic treatment of mice with established TB32043mb6S2 tumours in the pancreas with SG2399 lived significantly longer (p < 0.001; mean OS 48d) compared with PBS or control SG3511 (mean OS 25.5d and 26d, respectively). Thus SG3299 is confirmed as a promising candidate therapeutic for the therapy of PDAC.

Peptidase inhibitor 16 promotes proliferation of pancreatic ductal adenocarcinoma cells through OASL signaling.

Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive cancer with a poor prognosis and a 5-year survival rate of less than 11%. As a member of the CAP superfamily of proteins, the role of peptidase inhibitor 16 (Pi16) in tumor progression is still unclear. Immunohistochemistry and quantitative RT-PCR methods were used to detect the expression levels of Pi16 protein and mRNA in PDAC patients. CRISPR/Cas9 technology was used to knock out the expression of Pi16 in PDAC cell lines. In vivo and in vitro experiments were used to verify the effect of Pi16 on PDAC proliferation ability. By RNA sequencing, we found that oligoadenylate synthetase L(OASL) can serve as a potential downstream target of Pi16. The expression of Pi16 was higher in PDAC tissues than in matched adjacent tissues. High expression of Pi16 was associated with PDAC progression and poor prognosis. Overexpression of Pi16 could promote the proliferation of PDAC cells in vitro and in vivo. Bioinformatics analysis and coimmunoprecipitation assays showed that Pi16 could bind to OASL. Moreover, the functional recovery test confirmed that Pi16 could promote the proliferation of PDAC via OASL. Our present study demonstrates that Pi16 might participate in the occurrence and development of PDAC by regulating cell proliferation by binding to OASL, indicating that Pi16 might be a promising novel therapeutic target for PDAC.