Aggressive Pancreatic Ductal Adenocarcinoma Research Articles

Correlating tissue and plasma‑specific piRNA changes to predict their possible role in pancreatic malignancy and chronic inflammation.

The aggressiveness of pancreatic ductal adenocarcinoma is primarily due to lack of effective early detection biomarkers. Circulating non-coding RNAs serve as diagnostic or prognostic biomarkers in multiple types of cancer. Comparison of their expression between diseased tissue and relevant body fluids such as saliva, urine, bile, pancreatic juice, blood etc. may reveal mechanistic involvement of common non-coding RNAs. piwi-interacting RNAs (piRNAs) are a class of non-coding RNAs. The aim of the present study was to investigate plasma and tumour tissue piRNA changes in patients with pancreatic cancer (PC) and explore the possible role in tumorigenesis and pancreatic inflammation. Sequencing of circulating plasma small RNAs from patients with PC and chronic pancreatitis (CP) was performed and differentially expressed piRNAs were compared with those in tissues. Subsequent search for target genes for those piRNAs was performed followed by pathway and cluster analysis. A total of 36 piRNAs were shown to be deregulated in pancreatic tumour tissue and alteration of 11 piRNAs was detected in plasma of patients with PC. piRNAs hsa-piR-23246, hsa-piR-32858 and hsa-piR-9137 may serve a key role in PC development as their expression was correlated in both plasma and tumour tissue. Key piRNA-target interactions interfering with key biological pathways were also characterized. A total of 19 deregulated piRNAs in plasma samples of patients with CP was identified; these targeted genes responsible for chronic inflammation. Therefore, the present study provides a comprehensive description of piRNA alteration in pancreatic malignancy and inflammation; these may be explored for biomarker potential in future.

Abstract C063: Inhibition of PRMT5 reduces aggressiveness in basal-like pancreatic ductal adenocarcinoma

Abstract The basal-like pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive type of mesenchymal PDAC that resists treatment and has a poor prognosis. Understanding the mechanisms driving basal-like PDAC biology is crucial for unraveling the intricacies of PDAC plasticity and developing effective treatment strategies for this aggressive tumor. Recent studies from our laboratory have shown that protein arginine methyl transferase 5 (PRMT5) significantly contributes to PDAC plasticity and progression towards the basal-like subtype. Specifically, our data show that patients with high levels of PRMT5 expression have a worse outcome, thus arguing that PRMT5 plays a role in PDAC aggressiveness and outcome. Moreover, our experiments indicate that inhibiting PRMT5, either genetically or chemically, in basal-like PDAC cells induces the expression of epithelial markers like GATA6 and E-CADHERIN, suggesting that PRMT5 promotes mesenchymal features in PDAC. Using wound healing and transwell assays, we have demonstrated that pharmacological inhibition of PRMT5 significantly suppresses cell migration and invasion. Additionally, colony formation and cell proliferation are markedly reduced upon PRMT5 inhibition in basal-like PDAC. Collectively, these findings suggest that inhibiting PRMT5 can effectively constrain the aggressive cellular features characteristic of basal-like PDAC, such as cell proliferation, clonogenicity, and migration. Recent research indicates that PRMT5 preferentially methylates splicing factors in various cell types, implying that RNA splicing regulation is crucial to PRMT5’s role in enhancing tumor plasticity and aggressiveness. We are currently investigating whether PRMT5-mediated RNA splicing alterations contribute to the maintenance of the basal-like PDAC phenotype. These studies provide key evidence that PRMT5 is essential for maintaining the aggressive characteristics of basal-like PDAC, highlighting its potential as a therapeutic target for this challenging cancer subtype. Citation Format: Christina Wang, Zamira Guerra Soares, Dakarai Esgdaille, Stephano Spano Mello. Inhibition of PRMT5 reduces aggressiveness in basal-like pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C063.

Abstract B079: Disassembly of embryonic keratin filaments promotes pancreatic cancer metastases

Abstract Keratin 17 (K17), an intermediate filament protein typically expressed during embryonic development, is a hallmark of one of the most aggressive pancreatic ductal adenocarcinoma (PDAC) subtypes. Its expression is also associated with poor patient survival and resistance to first-line therapies. However, the mechanistic roles of K17 in malignancy remain largely unexplored. Here, we demonstrate that K17 expression and disassembly enhance tumor growth, increase metastatic potential, and shorten overall survival. Using the Clinical Proteomic Tumor Analysis Consortium (CPTAC) stratified by K17 phosphorylation status, we found that increased K17 phosphorylation correlated with worse survival. Mass spectrometry of untreated PDAC patient samples identified a phosphorylation hotspot at serines 10-13, which is conserved across species and type I keratin proteins. We found that K17 phosphorylation, mediated by the PKC/MEK/RSK pathway, led to increased disassembly and nuclear translocation in pancreatic cancer cells. Mice with phosphomimetic mutations at the serine hotspot showed increased metastases and decreased survival compared to wild-type and phosphorylation-resistant K17. Lastly, detergent-soluble nuclear K17 promoted metastasis- related genes in both patient and murine tumors. These findings suggest that disassembly of K17 mediated by phosphorylation at serines 10-13 is sufficient to promote metastases and decrease overall survival in PDAC. This opens the door for further investigation into K17 phosphorylation as a novel therapeutic target to enhance PDAC patient survival, especially in those with more aggressive, chemoresistant subtypes. Citation Format: Deanne E Yugawa, Ryan Kawalerski, Mariana Torrente Gonçalves, Chun-Hao Pan, Robert Tseng, Lucia Roa-Pena, Cindy V Leiton, Luke A Torre-Healy, Taryn Boyle, Sumedha Chowdhury, Natasha T Snider, Kenneth R Shroyer, Luisa F Escobar-Hoyos. Disassembly of embryonic keratin filaments promotes pancreatic cancer metastases [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B079.

Abstract B090: Evaluating the molecular determinants of PDAC racial health disparities

Abstract Pancreatic cancer is the 3rd leading cause of cancer-related deaths, with a 5-year survival rate of just 13%. Pancreatic ductal adenocarcinoma (PDAC), accounting for over 90% of pancreatic malignancies, has the highest incidence rate, mortality rate, and shortest survival times in non- Hispanic Black (defined here as African and/or African American ancestry) patients compared to other races. To identify molecular features that may contribute to racial health disparities in PDAC, we used quantitative mass spectrometry to determine the proteomic signatures unique to tumor racial origin in a cohort of 30 Caucasian and 12 African American patients. This analysis of the bulk tumor proteome revealed 183 proteins were differentially expressed between these groups. The protein with the highest expression in tumors from Black patients relative to Whites was Ring Finger Protein 2 (RNF2) that acts as an epigenetic transcriptional repressor in developmental processes pertaining to cellular differentiation. To determine the molecular pathways in PDAC regulated by RNF2, chromatin immunoprecipitation and sequencing (ChIP-seq) was performed on MIA-PaCa2 and a primary PDAC cell line derived from a Black patient to identify binding sites for RNF2 in PDAC cells. From the 1,142 replicated peaks identified, RNF2 peaks were localized over the transcription start site of 644 unique transcripts, including GATA6, which is an important determinant in PDAC subtype delineation with higher GATA6 expression associated with the less aggressive and more chemosensitive Classical subtype. Modulating RNF2 levels/activity by exogenous expression, short hairpin RNA (shRNA) knockdown, and small molecule inhibition confirms that GATA6 is a target of RNF2 repression in PDAC. Multi-omic (transcriptomic, proteomic, and phosphoproteomic) characterization was performed on MIA-PaCa2 cells with CRISPR-Cas9 knockout (KO) of RNF2. Differential gene expression (DE) analysis revealed changes in platinum drug resistance, EMT, and innate immune response, which are characteristic of the aggressive Inflammatory PDAC subtype we previously reported using proteomics. Total and phospho-proteomics corroborated these results identifying changes in DNA repair, inflammation response, TNFα mediated signaling, and apoptotic signaling. These results reflect the enrichment of Inflammatory subtype markers in tumors originating in Black patients. In agreement with these results, RNF2 KO shows a significant reduction in migration, as well as an increase in sensitivity to oxaliplatin. Our work identifies differences in the underlying proteome of PDAC tumors associated with tumor racial origin and describes how elevated expression of RNF2 in PDACs from Black patients contribute to inflammation and subtype delineation. This study delineates the associations between tumor racial origin, subtype specification, and response to therapy that could mitigate PDAC health disparities and be broadly applied across all PDAC patients. Citation Format: Anthony E Johansen-Sallee, Alexa M Barber, Henry C.-H. Law, Vignesh Vudatha, Jose Trevino, Nicholas T Woods. Evaluating the molecular determinants of PDAC racial health disparities [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B090.

Abstract C061: Loss of CaMK2B accelerates tumor formation and enhances metastatic competency in genetically engineered mouse models of PDAC

Abstract Background Pancreatic ductal adenocarcinoma (PDAC) is the fourth leading cause of cancer- related deaths in the United States and is often not diagnosed until it has already metastasized. Our prior work established that calcium signaling pathways facilitate a pro-metastatic phenotype through the induction of an epithelial-to-mesenchymal transition (EMT) program in PDAC tumor cells, but the downstream signaling molecules mediating this phenotype have remained elusive. MethodsUsing a novel genetically engineered model of pancreatic cancer, we demonstrate that loss of Calcium/calmodulin-dependent kinase-II B (CaMK2B), a downstream effector kinase activated by calcium, enhances cellular plasticity, and increases metastasis. To this end, we generated a genetically engineered mouse model where Camk2b was knocked out in tumor cells in a lineage-labeled Kras- and p53-driven PDAC mouse model (herein KPCY-Camk2bcKO). Results Genetic deletion of Camk2b in the KPCY genetically engineered model accelerated tumor formation and dramatically reduced the overall median survival of KPCY- Camk2b cKO mice (78 days) relative to KPCY controls (136 days). Pathological and immunofluorescence analysis of KPCY-Camk2b cKO animals showed an increased number of poorly differentiated tumor regions and a higher metastatic burden, with >85% of KPCY- Camk2b cKO animals presenting with multi-focal metastases. Further analysis of tumor cells in KPCY-Camk2b cKO mice showed enhanced expression of markers of the highly aggressive squamous PDAC subtypes. Strikingly, Camk2b deletion combined with oncogenic Kras G12D (in the absence of p53 alterations), was sufficient to form lethal metastatic PDAC tumors, while age-matched control Kras G12D tumors with intact Camk2b only progressed to the acinar-to-ductal metaplasia (ADM) or Pancreatic Intraepithelial Neoplasia (PanIN) stage. Collectively, these results establish Camk2b loss as a new oncogenic and pro-metastatic mechanism in PDAC Conclusion This work demonstrates that Camk2b loss enhances tumor cell evolution and plasticity within PDAC to promote metastasis. In this context, we speculate that loss of Camk2b rewires calcium signaling to drive an aggressive subtype of PDAC with increased metastasis. Citation Format: Jessica Peura, Nikita Bhalerao, Calvin Johnson, Yamini Ogoti, Faith Keller, Emma Watson, Zhen Zhao, Jason Pitarresi. Loss of CaMK2B accelerates tumor formation and enhances metastatic competency in genetically engineered mouse models of PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C061.

Abstract C075: Ror2, a Novel Key Regulator Driving Cell Fate Decisions throughout Pancreatic Carcinogenesis

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 neoplastic 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 neoplastic 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, Alec Steep, Malak Nasser, Jonathan Preall, Ujjwal Mukund Mahajan, Holly McQuithey, Ian Loveless, Erick T. Davis, Hui-Ju Wen, Daniel W. Long, Thomas Metzler, Samuel Zwernik, Michaela Louw, Donald Rempinski, Daniel Salas-Escabillas, Sydney Brender, Linghao Song, Ling Huang, Brian K. Theisen, Zhenyu Zhang, Nina G. Steele, Ivonne Regel, Filip Bednar, Howard C. Crawford. Ror2, a Novel Key Regulator Driving Cell Fate Decisions throughout Pancreatic Carcinogenesis [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C075.

Abstract A055: Multiomics profiling reveals druggable tumor-stroma interactions in ATM-deficient pancreatic cancer

Abstract The complex biology of pancreatic ductal adenocarcinoma (PDAC) remains a significant challenge and limits overcoming its dismal prognosis. The tumor microenvironment (TME), shaped by dynamic interactions between cancer-associated fibroblasts (CAFs), immune and tumor cells, plays a crucial role in PDAC pathogenesis. Uncharted field lies in the impact of specific gene mutations within the tumor epithelium on orchestrating distinct oncogenic TME patterns. Our investigation into homologous recombination deficiency (HRD), caused by mutations in genes like ATM, reveals its role in promoting PDAC aggressiveness and triggering a pronounced desmoplastic reaction, suggesting a unique TME programing. Through single-cell-resolved and multiomics analyses of murine ATM and/or P53-depleted PDACs, and validation in human cases, we unveil transcriptional, translational, and secretory regulatory events, shed light on the intricate interplay between tumor genetics and PDAC microenvironment and pave the way for the elaboration of novel personalized therapies. Murine tumor analysis using single-nucleus multiomics RNA and ATAC sequencing, alongside histological validation in human tissues, revealed specific enrichment of myCAFs in ATM-deficient tumors and reduced populations of T cells and macrophages. Molecular signatures and cell-cell communication inference uncovered ATM-specific interactions within the TME, highlighting TGF-β signaling as pivotal in mediating dialog between CAFs and an aggressive EMT-like tumor cell subset prevalent in ATM-depleted tumors. Additionally, distinct communication patterns were observed between CAFs and immune cells, associated with specific epigenetic and molecular profiles. Mechanistically, transcriptomics, secretomics and proteomics investigations confirmed increased TGFβ release by ATM-deficient PDAC cells and identified a reactive oxygen species (ROS)-mediated mechanism affecting tumor cell behavior and interactions with CAFs. Consistently, a combinatorial therapy targeting TGF-β-mediated tumor-stroma dialog reversed cancer-promoting TME remodeling and exacerbated FOLFIRINOX cytotoxic effects, in vivo, exclusively in ATM-deficient HRD PDACs. Dissecting the biology of ATM-deficient HRD malignant cells, our study unveils their pivotal role in orchestrating oncogenic communication networks with CAFs and immune cells, along with specific tumor promoting stromal reprogramming. Our findings paint a comprehensive picture of how distinct tumor-stroma interactions reshape the TME, steering it towards a cancer-promoting outcome and shed light on the potential to exploit genotype-specific vulnerabilities via tailored tumor-stroma interference strategies. Citation Format: Elodie Roger, Hannah M. Mummey, Eleni Zimmer, Julia Ragg, Yuna Lee, Kyle J. Gaulton, Alica K. Beutel, Christopher J. Halbrook, Lukas Perkhofer, Johann Gout, Alexander Kleger. Multiomics profiling reveals druggable tumor-stroma interactions in ATM-deficient pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr A055.

Identification of Ppy-lineage cells as a novel origin of pancreatic ductal adenocarcinoma.

The Ppy gene encodes pancreatic polypeptide (PP) secreted by PP- or γ-cells, which are a subtype of endocrine cells localised mainly in the islet periphery. For a detailed characterisation of PP cells, we aimed to establish PP cell lines. To this end, we generated a mouse model harbouring the SV40 large T antigen (TAg) in the Rosa26 locus, which is expressed upon Ppy-promoter-mediated Cre-loxP recombination. Whereas Insulin1-CreERT-mediated TAg expression in beta cells resulted in insulinoma, surprisingly, Ppy-Cre-mediated TAg expression resulted in the malignant transformation of Ppy-lineage cells. These mice showed distorted islet structural integrity at 5 days of age compared with normal islets. CK19+ duct-like lesions contiguous with the islets were observed at 2 weeks of age, and mice developed aggressive pancreatic ductal adenocarcinoma (PDAC) at 4 weeks of age, suggesting that PDAC can originate from the islet/endocrine pancreas. This was unexpected as PDAC is believed to originate from the exocrine pancreas. RNA-sequencing analysis of Ppy-lineage islet cells from 7-day-old TAg+ mice showed a downregulation and an upregulation of endocrine and exocrine genes, respectively, in addition to the upregulation of genes and pathways associated with PDAC. These results suggest that the expression of an oncogene in Ppy-lineage cells induces a switch from endocrine cell fate to PDAC. Our findings demonstrate that Ppy-lineage cells may be an origin of PDAC and may provide novel insights into the pathogenesis of pancreatic cancer, as well as possible therapeutic strategies. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

ZKSCAN5 activates LAPTM5 expression by recruiting SETD7 to promote metastasis in pancreatic ductal adenocarcinoma.

Lysosomal-associated transmembrane protein 5 (LAPTM5) has been associated with poor prognosis in cancer patients. Its role in regulating metastasis in pancreatic ductal adenocarcinoma (PDAC), however, remains vague. The study here aimed to expound the metastasis-promoting properties of LAPTM5 in PDAC and the detailed mechanism. LAPTM5 was overexpressed in metastatic PDAC cells and was related to the dismal prognosis of patients in GEO datasets. By using lentiviral vectors harboring short hairpin RNA, we found that LAPTM5 downregulation reduced PDAC cell viability, proliferation, and aggressiveness in vitro and liver metastasis in vivo. Zinc finger with KRAB and SCAN domains 5 (ZKSCAN5) was predicted and verified to mediate LAPTM5 transcription in PDAC cells. Both ZKSCAN5 and SET domains, containing lysine methyltransferase 7 (SETD7) bound to the LAPTM5 promoter, and ZKSCAN5 recruited SETD7 to form a complex promoting LAPTM5 transcription. LAPTM5 knockdown reversed the promoting effect of ZKSCAN5 on the metastasis of PDAC cells. Thus, our findings on the ZKSCAN5/SETD7/LAPTM5 axis provide insights into the underlying mechanism of liver metastasis dissemination in PDAC.

Positive feedback regulation between glycolysis and histone lactylation drives oncogenesis in pancreatic ductal adenocarcinoma

BackgroundMetabolic reprogramming and epigenetic alterations contribute to the aggressiveness of pancreatic ductal adenocarcinoma (PDAC). Lactate-dependent histone modification is a new type of histone mark, which links glycolysis metabolite to the epigenetic process of lactylation. However, the role of histone lactylation in PDAC remains unclear.MethodsThe level of histone lactylation in PDAC was identified by western blot and immunohistochemistry, and its relationship with the overall survival was evaluated using a Kaplan-Meier survival plot. The participation of histone lactylation in the growth and progression of PDAC was confirmed through inhibition of histone lactylation by glycolysis inhibitors or lactate dehydrogenase A (LDHA) knockdown both in vitro and in vivo. The potential writers and erasers of histone lactylation in PDAC were identified by western blot and functional experiments. The potential target genes of H3K18 lactylation (H3K18la) were screened by CUT&Tag and RNA-seq analyses. The candidate target genes TTK protein kinase (TTK) and BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) were validated through ChIP-qPCR, RT-qPCR and western blot analyses. Next, the effects of these two genes in PDAC were confirmed by knockdown or overexpression. The interaction between TTK and LDHA was identified by Co-IP assay.ResultsHistone lactylation, especially H3K18la level was elevated in PDAC, and the high level of H3K18la was associated with poor prognosis. The suppression of glycolytic activity by different kinds of inhibitors or LDHA knockdown contributed to the anti-tumor effects of PDAC in vitro and in vivo. E1A binding protein p300 (P300) and histone deacetylase 2 were the potential writer and eraser of histone lactylation in PDAC cells, respectively. H3K18la was enriched at the promoters and activated the transcription of mitotic checkpoint regulators TTK and BUB1B. Interestingly, TTK and BUB1B could elevate the expression of P300 which in turn increased glycolysis. Moreover, TTK phosphorylated LDHA at tyrosine 239 (Y239) and activated LDHA, and subsequently upregulated lactate and H3K18la levels.ConclusionsThe glycolysis-H3K18la-TTK/BUB1B positive feedback loop exacerbates dysfunction in PDAC. These findings delivered a new exploration and significant inter-relationship between lactate metabolic reprogramming and epigenetic regulation, which might pave the way toward novel lactylation treatment strategies in PDAC therapy.

Abstract 1458: Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness

Abstract Pancreatic ductal adenocarcinoma (PDAC) represents a very aggressive type of pancreatic cancer (9% survival rate at 5 years). PDX1 is an important transcription factor for embryonic development of the pancreas, endocrine lineage differentiation and the maintenance of mature beta cells. Notably, in PDAC patients, the expression of PDX1 is downregulated in tumor cells compared to adjacent non-tumoral tissue. Interestingly, increasing tumor PDX1 expression has been shown to enhance the overall survival rate in PDAC patients. Therefore, our aim was to analyze the role of PDX1 on tumor aggressiveness of PDAC cells. To induce PDX1 expression, PANC1 cells were treated with BRD7552 (a PDX1 inducer) for 9 days. Overexpression of PDX1 was confirmed by Western Blot analysis and no cytotoxic effects were observed by MTT or Trypan Blue exclusion assays on treated cells compared to control. Wound healing and transwell migration assays showed a significant reduction in the migration rate compared to the control group. A higher proportion of cells in G1 phase was observed in treated cells compared to control by propidium iodide staining followed by flow cytometry assay. Furthermore, the cell confluence assay showed a significant reduction in proliferation rate in treated cells compared to control, but no differences were observed on the proportion of KI67+ and PH3+ cells by immunostaining. To assess the in ovo effects of BRD7552, treated PANC1 cells were implanted onto the chorioallantoic membrane of chick embryos and tumor growth was measured at different stages observing a significant reduction in tumor growth in PANC1 cells compared to control between days 3 and 8 post implantation. No significant differences in morphology or color were observed between treated and control implanted cells. Hematoxylin-eosin staining showed reduced invasion of treated cells into the chorioallantoic membrane compared to control. In conclusion, the overexpression of PDX1 affects cell cycle, reduces proliferation rate and inhibits migratory potential in vitro. Moreover, in an in ovo model this overexpression diminished tumor growth and invasion in PANC1 cells. In conclusion, the overexpression of PDX1 induced by BRD7552 drives pancreatic ductal adenocarcinoma to a less aggressive tumoral phenotype. Citation Format: Jimena Mosna, Federico Garde, Marcelo Stinson, Candela Pastore, Pablo Sanchis, Abel Carcagno. Anti tumoral role of PDX1 on pancreatic ductal adenocarcinoma aggressiveness [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 1458.

Abstract 6927: A refined view of pancreatic cancer genomic evolution through single-nucleus DNA sequencing

Abstract Numerous next-generation sequencing studies have provided an overview pancreatic ductal adenocarcinoma (PDAC)’s genomic evolution. But how its genome evolves through treatment and metastasis has not been extensively studied due to lack of samples and technical limitation of bulk sequencing on low-tumor purity samples. Herein we applied targeted single-nucleus DNA sequencing (snDNA-seq) to a cohort of 18 patients, each with >= 2 multiregional/longitudinal samples, totaling 65 samples. These included 5 early-stage resections, 11 stage IV autopsies and 2 biopsy-derived organoids taken pre- and post-treatment. We also developed a set of computational methods for this new datatype to delineate genomic evolution at high resolution. Despite higher sensitivity than bulk, snDNA-seq did not uncover significant subclonal single-nucleotide variations (SNVs) on our panel targeting 253 select genes for PDAC. But it detected a higher frequency of CDKN2A (72% against 30%) and SMAD4 (56% against 32%) alterations, mostly attributed to chromosomal deletions. Many were subclonal or only affected a few hundred base-pairs of the gene, making them elusive to bulk sequencing. Except for one case driven by BRCA2 mutations rather than the canonical KRAS oncogene, other patients had mostly linear phylogenies with more SNVs occurring before the most recent common ancestor (MRCA) of clones, for early/late-stage and longitudinal cases alike. This early fixation of drivers and rapid clonal sweep afterwards likely correlates with the particular aggressiveness of PDAC. Metastatic clones almost always fell towards the end of phylogenies and were shared among spatially separated sites, indicating late metastatic dissemination in molecular evolution time. Metastases to the liver and diaphragm appeared to be more genetically evolved than other sites, likely due to harsher selective pressure cast by the metastatic routes/distal microenvironments. 16 of 18 PDACs were observed to converge towards tumor cell-intrinsic TGF-β unresponsiveness by mutating various components of the pathway. Such convergent evolution usually happened at the primary site, indicating a strong selective advantage of the phenotype in the desmoplastic, nutrient-poor pancreas microenvironment. Continuous evolution was seen through treatment and metastasis, driven by seemingly random genome-scale copy number variations (CNVs) and focal amplification/deletions to genes such as KRAS, CDKN2A, SMAD4, MYC. These insights on PDAC’s genomic evolution inform more precision medicine efforts to come. Early fixation of driver SNVs sculpts a largely homogeneous disease that could be uniformly targeted. But special care should be taken to obviate resistance mechanisms conferred by continuously evolving CNV events- compared to SNVs, these mechanisms provide faster remodeling of the genome, and thus faster generation of new phenotypes and adaptation. Citation Format: Haochen Zhang, Palash Sashittal, Elias-Ramzey Karnoub, Akhil Jakatdar, Shigeaki Umeda, Nicolas Lecomte, Jungeui Hong, Katelyn Mullen, Akimasa Hayashi, Caitlin A. McIntyre, Benjamin J. Raphael, Christine A. Iacobuzio-Donahue. A refined view of pancreatic cancer genomic evolution through single-nucleus DNA sequencing [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 6927.

Abstract 6167: Spatial transcriptomic analysis identifies pancreatic cancer cell surface markers for imaging and therapy

Abstract Pancreatic cysts and precancer lesions have been reported in up to 50% of adults; however, few of the precancer lesions develop into aggressive pancreatic ductal adenocarcinoma (PDAC). Strategies for the differentiation of precancers and PDAC are needed. Recently proposed molecular imaging targets to accomplish this goal include integrin αvβ6 and fibroblast activation protein (FAP). To discover and quantitatively evaluate potential imaging and therapeutic targets, we performed Visium spatial transcriptomic (ST) sequencing on human surgical sections of normal tissue adjacent to precancers, intraductal papillary mucinous neoplasms and PDAC. All human tissues were collected in accordance with the Stanford Institutional Review Board. The resulting data were correlated with immunohistochemistry (IHC) and combined with analysis with publicly available spatial and single cell PDAC datasets to create a quantitative PDAC cancer surfaceome score. Pseudotime and receiver operating characteristic (ROC) analyses were also performed. We found that clusters with high cancer surfacesome scores were spatially correlated with a cellular morphology consistent with cancer. In the cancer cluster, the expression of surface markers CLDN4, GPRC5A, TSPAN8, and CEACAM5 was enhanced more than 13-fold compared with the normal pancreas, while FAP and ITGB6 were differentially expressed by less than 4-fold. Within cancer clusters, the fraction of spots overexpressing CLDN4, S100P, ITGB6, MUC5AC, TFF1, CEACAM5, and FAP was 88%, 87%, 78%, 66%, 59%, 47%, and 38%, respectively. The Pearson’s correlation across space and expression intensity between CLDN4, S100P, GPRC5A, TSPAN8, MUC1 and TFF1 was greater than 0.6 for all combinations. The correlation of mRNA encoding ITGB6 and FAP with cancer surfaceome markers was smaller, e.g. 0.37 and 0.01 versus S100P, respectively. The area under the curve (AUC) for the differentiation of PDAC from normal or precancerous tissue for CLDN4, S100P, MUC5AC, TFF1, CEACAM5, and TSPAN8 was 0.9, 0.5, 0.3, 0.5, 0.8, and 0.4, respectively. From pseudotime trajectory analysis, we found that MUC5AC and TFF1 increased at a precancerous pseudotime whereas CLDN4 increased in time and space in spots corresponding to a PDAC-specific surfaceome score. This work demonstrates the feasibility of using spatial transcriptomics and IHC in human pancreatic cancer samples to discover and compare the expression of cell surface markers that can be employed in imaging and therapeutic protocols. Citation Format: James Wang, Aris J. Kare, Martn K. Schneider, Jai Woong Seo, Andrei Iagaru, Gregory W. Charville, Walter G. Park, Katherine W. Ferrara. Spatial transcriptomic analysis identifies pancreatic cancer cell surface markers for imaging and therapy [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 6167.

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 6818: Obesity-mediated perineural invasion promotes PDAC tumorigenesis

Abstract Obesity is a modifiable host factor that has been reported to increase the risk of pancreatic ductal adenocarcinoma (PDAC) by ~60% and the mortality by two-fold, however, the exact mechanisms by which obesity enhances PDAC aggressiveness is incompletely understood. Given that nearly 74% of U.S. adults are overweight or obese (US Center for Disease Control), resolving the complex association between obesity and PDAC may unveil new targets for prevention and/or intervention to decrease incidence and improve outcomes. The prevailing notion is that obesity-induced tumor in situ factors (i.e., oxidative, hypoxic, nutrient, and biochemical) play crucial roles in enhancing cancer cell fitness and survival. However, these concepts have yet to lead to the identification of any actionable target in patients, with obesity-related cancer in general and PDAC in particular. Prior studies and our preliminary data show the presence of a distinctive tumor immune profile in PDAC models under conditions of obesity, which encompasses immune suppressive myeloid and type 2 immune cells, namely M2-like tumor-associated macrophages (TAMs), and myeloid-derived suppressor cells (MDSCs), that inhibit effector CD8+ T cell expansion and function enabling PDAC progression. However, the genesis of this ‘type 2 immune cell’ dominant tumor-immune microenvironment (TIME) of obesity-driven PDAC remains to be fully understood. This led us to take a holistic approach utilizing multiple genetically engineered mouse models (GEMMs), pharmacological inhibitors, transcriptomic analysis (RNA-seq, spatial transcriptomic, and scRNA-seq), fresh PDAC patient samples, and imaging technologies (confocal microscopy) to understand the molecular mechanisms. Our novel mechanistic study using some of the above-mentioned tools shows that obesity-driven PDAC has significantly higher perineural invasion (PNI) that enhances PDAC progression. We found that sympathetic nerve terminals (of the PNI) via neurotransmitter release control the expression of integral cytokines that cause a dramatic remodeling of the TIME. Specifically, the PNI-mediated neuro-adrenergic signaling activates β2-adrenergic receptor (β2-AR) on PDAC cells, leading to increased tumor cell production/proliferation and secretion of the ‘alarmins’ IL-33 and IL-1β. Importantly, we showed that mitigation of adrenergic signaling using two separate approaches: (i) pharmacological antagonists of β2-ARs (β-blockers), and (ii) the use of a thermoneutral model leads to substantial abrogation of obesity-mediated tumor progression. These, and other key data raise the provocative question that reducing adrenergic signaling-dependent IL-33 expression and release favor a pro-inflammatory TIME and significantly increase patient survival in obesity-driven PDAC. Citation Format: Shyamananda Singh Mayengbam, Aftab Alam, Sharon Senchanthisai, Irmak Bektas, Prasenjit Dey. Obesity-mediated perineural invasion promotes PDAC tumorigenesis [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 6818.

Connectivity mapping-based identification of pharmacological inhibitor targeting HDAC6 in aggressive pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) remains highly lethal due to limited therapeutic options and expensive/burdensome drug discovery processes. Utilizing genomic-data-driven Connectivity Mapping (CMAP) to identify a drug closer to real-world PC targeting may improve pancreatic cancer (PC) patient outcomes. Initially, we mapped CMAP data to gene expression from 106 PC patients, identifying nine negatively connected drugs. These drugs were further narrowed down using a similar analysis for PC cell lines, human tumoroids, and patient-derived xenografts datasets, where ISOX emerged as the most potent agent to target PC. We used human and mouse syngeneic PC cells, human and mouse tumoroids, and in vivo mice to assess the ability of ISOX alone and in combination with 5FU to inhibit tumor growth. Global transcriptomic and pathway analysis of the ISOX-LINCS signature identified HDAC 6/cMyc as the target axis for ISOX. Specifically, we discovered that genetic and pharmacological targeting of HDAC 6 affected non-histone protein cMyc acetylation, leading to cMyc instability, thereby disrupting PC growth and metastasis by affecting cancer stemness. Finally, KrasG12D harboring tumoroids and mice responded effectively against ISOX and 5FU treatment by enhancing survival and controlling metastasis incidence. Overall, our data validate ISOX as a new drug to treat advanced PC patients without toxicity to normal cells. Our study supports the clinical utility of ISOX along with 5FU in future PC clinical trials.

Limited Role of the Apparent Diffusion Coefficient (ADC) for Tumor Grade and Overall Survival in Resectable Pancreatic Ductal Adenocarcinoma.

This study evaluated the relationship between apparent diffusion coefficient (ADC) values in pancreatic ductal adenocarcinoma (PDAC) and tumor grades based on WHO, Adsay, and Kalimuthu classifications, using whole-mount pancreatectomy specimens. If glandular formation plays a key role in the degree of diffusion restriction, diffusion-weighted imaging could facilitate non-invasive grading of PDAC. A freehand region of interest (ROI) was drawn along tumor borders on the preoperative ADC map in each tumor-containing slice. Resection specimens were retrospectively graded according to WHO, Adsay, and Kalimuthu classifications and correlated with overall survival and the 10th percentile of whole-volume ADC values. Findings from 40 patients (23 male, median age 67) showed no correlation between ADC p10 values and WHO differentiation (p = 0.050), Adsay grade (p = 0.955), or Kalimuthu patterns (p = 0.117). There was no association between ADC p10 and overall survival (p = 0.082) and other clinicopathological variables. Survival was significantly lower for poor tumor differentiation (p = 0.046) and non-glandular Kalimuthu patterns (p = 0.016) and there was a trend towards inferior survival for Adsay G3 (p = 0.090) after correction for age, tumor location, and stage. Preoperative ADC measurements for determining PDAC aggressiveness had limited clinical utility, as there was no correlation with histological parameters or overall survival in resectable PDAC.

Nutrient scavenging-fueled growth in pancreatic cancer depends on caveolae-mediated endocytosis under nutrient-deprived conditions.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by its nutrient-scavenging ability, crucial for tumor progression. Here, we investigated the roles of caveolae-mediated endocytosis (CME) in PDAC progression. Analysis of patient data across diverse datasets revealed a strong association of high caveolin-1 (Cav-1) expression with higher histologic grade, the most aggressive PDAC molecular subtypes, and worse clinical outcomes. Cav-1 loss markedly promoted longer overall and tumor-free survival in a genetically engineered mouse model. Cav-1-deficient tumor cell lines exhibited significantly reduced proliferation, particularly under low nutrient conditions. Supplementing cells with albumin rescued the growth of Cav-1-proficient PDAC cells, but not in Cav-1-deficient PDAC cells under low glutamine conditions. In addition, Cav-1 depletion led to significant metabolic defects, including decreased glycolytic and mitochondrial metabolism, and downstream protein translation signaling pathways. These findings highlight the crucial role of Cav-1 and CME in fueling pancreatic tumorigenesis, sustaining tumor growth, and promoting survival through nutrient scavenging.

Nutrient restriction-activated Fra-2 promotes tumor progression via IGF1R in miR-15a downmodulated pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a lethal disease, characterized by an intense desmoplastic reaction that compresses blood vessels and limits nutrient supplies. PDAC aggressiveness largely relies on its extraordinary capability to thrive and progress in a challenging tumor microenvironment. Dysregulation of the onco-suppressor miR-15a has been extensively documented in PDAC. Here, we identified the transcription factor Fos-related antigen-2 (Fra-2) as a miR-15a target mediating the adaptive mechanism of PDAC to nutrient deprivation. We report that the IGF1 signaling pathway was enhanced in nutrient deprived PDAC cells and that Fra-2 and IGF1R were significantly overexpressed in miR-15a downmodulated PDAC patients. Mechanistically, we discovered that miR-15a repressed IGF1R expression via Fra-2 targeting. In miR-15a-low context, IGF1R hyperactivated mTOR, modulated the autophagic flux and sustained PDAC growth in nutrient deprivation. In a genetic mouse model, Mir15aKO PDAC showed Fra-2 and Igf1r upregulation and mTOR activation in response to diet restriction. Consistently, nutrient restriction improved the efficacy of IGF1R inhibition in a Fra-2 dependent manner. Overall, our results point to a crucial role of Fra-2 in the cellular stress response due to nutrient restriction typical of pancreatic cancer and support IGF1R as a promising and vulnerable target in miR-15a downmodulated PDAC.

Innate and adaptive immune-directed tumour microenvironment in pancreatic ductal adenocarcinoma.

One of the most deadly and aggressive cancers in the world, pancreatic ductal adenocarcinoma (PDAC), typically manifests at an advanced stage. PDAC is becoming more common, and by the year 2030, it is expected to overtake lung cancer as the second greatest cause of cancer-related death. The poor prognosis can be attributed to a number of factors, including difficulties in early identification, a poor probability of curative radical resection, limited response to chemotherapy and radiotherapy, and its immunotherapy resistance. Furthermore, an extensive desmoplastic stroma that surrounds PDAC forms a mechanical barrier that prevents vascularization and promotes poor immune cell penetration. Phenotypic heterogeneity, drug resistance, and immunosuppressive tumor microenvironment are the main causes of PDAC aggressiveness. There is a complex and dynamic interaction between tumor cells in PDAC with stromal cells within the tumour immune microenvironment. The immune suppressive microenvironment that promotes PDAC aggressiveness is contributed by a range of cellular and humoral factors, which itself are modulated by the cancer. In this review, we describe the role of innate and adaptive immune cells, complex tumor microenvironment in PDAC, humoral factors, innate immune-mediated therapeutic advances, and recent clinical trials in PDAC.