Pancreatic Ductal Adenocarcinoma Cells Research Articles

Abstract C043: Hyperinsulinemia promotes pancreatic cancer progression by altering tumor metabolism

Abstract Metabolic diseases, such as type 2 diabetes (T2D), insulin resistance, and obesity, often accompany pancreatic ductal adenocarcinoma (PDAC), and they are associated with reduced survival. Hyperinsulinemia is a common hallmark symptom shared by those disorders and is independently associated with reduced survival of PDAC patients. While it has been established that endogenous hyperinsulinemia accelerates PDAC initiation by promoting the formation of KRAS-driven pre-cancerous lesions, its role in the progression of established tumors remains poorly understood. This represents a major knowledge gap in our understanding of whether insulin needs to be monitored and controlled during PDAC treatment, as it is not typically considered in current treatment paradigms. We hypothesized that hyperinsulinemia promotes the progression of PDAC tumors. Using patient-derived PDAC organoids (PDOs) and a mouse model of metabolic disorders, we found that insulin may promote PDOs’ growth by reprogramming tumor metabolism. A High-fat diet (HFD) treatment that induces hyperinsulinemia, but not hyperglycemia in mice accelerated the growth of PDO-derived orthotopic xenografts. Importantly, the fasting insulin level showed a significant positive correlation with the endpoint tumor volume, suggesting that endogenous insulin may positively contribute to PDOs’ growth in vivo. To assess the direct effect of insulin on the growth and molecular profile of PDOs, we treated an early passage PDO line with different concentrations of insulin and glucose at physiologically relevant levels. We observed a ∼1.2-fold increase in cell number after 7 days of culture in high insulin (10 nM) conditions with high (15 mM) or physiological (6 mM) levels of glucose, relative to the low insulin control. Phosphoproteomic analysis demonstrated significantly greater PI3K/AKT/mTOR, but not RAF/MEK/ERK, activity under high insulin with high or physiological levels of glucose compared to the control, directing the investigation toward the metabolic effects of insulin. Consistently, unbiased proteomics profiling revealed that metabolic proteins were significantly enriched in both high insulin conditions, and interestingly insulin enriched different sets of proteins in various metabolic pathways depending on glucose levels. Together, our preliminary results suggest that hyperinsulinemia may enhance the growth and progression of PDAC by altering tumor metabolism to support cell growth. Further experiments are needed to functionally link the insulin-mediated metabolic changes with increased PDO growth, as well as testing the direct effect of endogenous insulin on PDAC cells in vivo. Citation Format: Jeffrey Lin, James D Johnson, David Schaeffer, Vincent Richard, Christoph Borchers, Janel L Kopp. Hyperinsulinemia promotes pancreatic cancer progression by altering tumor metabolism [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 C043.

Abstract A079: SB-216 inhibits oncogenic beta-tubulin subtypes in PDAC

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest malignancies, with only 12% of patients living longer than five years after diagnosis. Recent studies have shown that inhibiting microtubule dynamics may be a promising therapeutic avenue for treating patients with this cancer. Microtubules are critical in cancer cell growth and division, as well as intracellular cytoskeletal support and internal trafficking. In PDAC, βIII- and βIVb-tubulin are highly upregulated in contrast to normal pancreas cells. Decreased expression of βIII- and βIVb-tubulin, through silencing or inhibition, correlates with reduced PDAC cell growth, tumorigenic potential, metastases in xenograft models, and chemoresistance. We hypothesized that a novel colchicine binding site beta-tubulin inhibitor, SB-216, will decrease cell growth while also decreasing the expression of oncogenic βIII- and βIVb-tubulin. To examine the anti-proliferative effects, PDCL110, a PDAC patient-derived cell line, cells were seeded (6,000 cells/well), and after 24 hours, treated with 0.1% DMSO (control), 2 nM, 5 nM, 10 nM, or 15 nM SB-216. Plates were analyzed on the IncuCyte Live-Cell Analysis System for six days, where phase contrast images of confluency were captured every four hours. To characterize the mRNA expression of βIII- and βIVb-tubulin, Panc-1 and PDCL110 cells were treated with 0.1% DMSO and 5 nM SB-216 for 48 hours. Next, total RNA was extracted using a TRIzol reagent. cDNAs were prepared using the SYBR Green RNA Reverse Transcription kit, and mRNA expression of beta-tubulin isotypes was quantified using the QuantStudio Real-Time PCR system. The expression of tubulin isotypes was normalized with the internal control ACTIN. Cell growth curves illustrated a significant (p<0.006) decrease in proliferation after 48 hours of treatment at all tested concentrations of SB-216. After 72 hours, cell growth inhibition became more significant (p<0.0001) in a dose-dependent manner. SB-216 treatment of a commercially available PDAC cell line (Panc-1) led to decreased mRNA expression of TUBB3 (class βIII, p<0.05) and TUBB4B (class βIVb, p<0.05) compared to DMSO control. Similarly, TUBB4B expression was significantly (p<0.05) decreased in the PDCL110 cells upon SB-216 treatment. These results enhance our confidence in utilizing SB-216 as a therapeutic agent against PDAC. Future studies are directed at elucidating the effects of SB-216 on metastases, chemoresistance, and mitochondrial function. Through an improved understanding of the mechanisms behind microtubule inhibition, we can meet the urgent clinical need of finding targeted treatments for this complex cancer. Citation Format: Lauren C Gattie, Chun Cai, Rui Wang, Wei Li, Evan Glazer. SB-216 inhibits oncogenic beta-tubulin subtypes in 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 A079.

Abstract C032: De novo pyrimidine biosynthesis inhibition synergizes with BCL-XL targeting in pancreatic cancer

Abstract Oncogenic KRAS, the genetic driver of 90% of PDAC, induces a metabolic rewiring characterized, in part, by dependency on de novo pyrimidine biosynthesis. Dihydroorotate dehydrogenase (DHODH) is the enzymatic ‘chokepoint’ of the de novo pyrimidine biosynthesis pathway and an in vivo metabolic liability in pancreatic ductal adenocarcinoma (PDAC). Targeting enzymes in the de novo pyrimidine synthesis pathway such as dihydroorotate dehydrogenase (DHODH) with clinically available inhibitor brequinar (BQ) starved the tumor of nucleotide synthesis and restrained growth of PDAC cells in vitro. However, therapeutic resistance limited BQ long-term effects in PDAC xenografts, which suggests compensatory pathways and that combinatorial strategies are required for enhanced efficacy. Here, we integrated a mass spectrometry (MS)-based quantitative temporal proteomics workflow, LC/MS- based metabolomics and in vitro and in vivo drug-anchored CRISPR/Cas9 genetic screens to identify compensatory pathways to DHODH inhibition (DHODHi) and targets for combination strategies. We demonstrate that DHODHi alters the apoptotic regulatory proteome thereby enhancing sensitivity to inhibitors of the anti-apoptotic BCL2L1 (BCL-XL) protein. Combinatorial regimens with DHODH and BCL-XL inhibition synergistically induce apoptosis in PDAC cell lines and patient-derived PDAC organoids. In vivo DHODH inhibition with Brequinar and BCL-XL degradation with DT2216, a proteolysis targeting chimera (PROTAC), significantly inhibits the growth of PDAC tumors. Our data defines mechanisms of adaptation to DHODH inhibition and identifies a combination therapy strategy in PDAC. Citation Format: huan zhang, Qijia yu, Naiara Santana-Codina, Clara Poupault, Claudia Campos, Xingping Qin, Aparna Padhye, Nicole Sindoni, Miljan Kuljanin, Junning Wang, Matthew J. Dorman, Andrew J. Aguirre, Stephanie K. Dougan, Kristopher A. Sarosiek, Joseph D. Mancias. De novo pyrimidine biosynthesis inhibition synergizes with BCL-XL targeting in 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 C032.

Abstract C025: A novel pan-RAS/β-catenin inhibitor, ADT-030, produces tumor regression in PDX models of pancreatic cancer

Abstract BACKGROUND: The absence of symptoms often leads to late diagnosis of pancreatic ductal adenocarcinoma (PDAC) limiting treatment options. Tumor cell proliferation and invasion/metastasis are usually driven by KRAS mutations and activation of Wnt/β-catenin pathway components. The recent approval of KRASG12C-specific inhibitors for lung cancer has validated RAS as a druggable target but has had limited application for PDAC given the rarity of this codon in PDAC. In addition, no Wnt/β-catenin inhibitors have been FDA approved. Thus, drugs which inhibit these important targets are urgently needed for PDAC and other cancers. Here, we describe treatment results with ADT-030, a novel dual pan-RAS/β-catenin inhibitor in murine models of PDAC and lung cancer. METHODS: In vitro assays of growth inhibition, colony formation, apoptosis, and cell cycle, along with western blotting for RAS signaling were performed to assess potency and selectivity of ADT-030. Murine 2838c3-Luc KRASG12D PDAC cells were implanted orthotopically into the pancreas of C57BL/6J mice and cells from two PDAC patient-derived xenograft (PDX) models harboring KRASG12D or KRASG12C mutations were implanted subcutaneously (SC) in NSG mice to evaluate the antitumor activity of ADT-030. ADT-030 was administered once daily by gavage 5x/week for 4 weeks. RESULTS: ADT-030 potently inhibited the growth of PDAC cells in vitro harboring the most prevalent KRASG12D mutation as well as those with KRASG12C. Annexin V assay revealed the capacity of ADT-030 to trigger apoptosis in KRAS mutant cells with minimal impact on BxPC3 RASWT cells. Cell cycle analysis using propidium iodide staining revealed that ADT-030 arrested cells in the G2/M phase. Other experiments showed that ADT-030 simultaneously suppressed both MAPK/AKT signaling and β-catenin transcriptional by inhibiting PDE10 and activating cGMP/PKG signaling. ADT-030 caused tumor regression in SC PDAC PDX mouse tumor models harboring either KRASG12D or KRASG12C mutations without discernable toxicity. Inhibition of orthotopic PDAC tumor growth by ADT-030 was dose-dependent, causing up to 80% reduction in tumor volume following a 4-week treatment period. In addition, ADT-030 significantly extended survival and yielded a high percentage of cures after stopping treatment in a murine model of KRAS- mutant lung cancer. CONCLUSION: ADT-030, novel dual pan-RAS/β-catenin inhibitor, showed strong and durable antitumor activity in clinically relevant and aggressive murine models of PDAC and lung cancer. ADT-030 is a promising development candidate for treatment of PDAC and other RAS-driven cancers, particularly given the complex KRAS mutation landscape of many cancers. Citation Format: Dhana Sekhar Reddy Bandi, Ganji P Nagaraju, Jeremy B Foote, Adam B Keeton, Xi Chen, Kristy L Berry, Yulia Y Maxuitenko, Upender Manne, Donald J Buchsbaum, Gary A Piazza, Bassel F El-Rayes. A novel pan-RAS/β-catenin inhibitor, ADT-030, produces tumor regression in PDX models of 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 C025.

Abstract C044: PI5P4Kα regulates cell fitness through iron homeostasis in pancreatic cancer

Abstract PI5P4Ks (Phosphatidylinositol 5-phosphate 4-kinases) are a family of lipid kinases that phosphorylate phosphatidylinositol 5-monophosphate (PI-5-P) at the 4-position to generate distinct pools of phosphatidylinositol 4,5-bisphosphate (PI-4,5-P2) largely on intracellular membranes. These lipid species are involved in cellular and metabolic stress responses, signaling and organelle communication. PI5P4Ks have been implicated in multiple cancers, with compelling evidence showing synthetic lethality when PI5P4Ks are inhibited in TP53-mutant cancer cells. Pancreatic ductal adenocarcinoma (PDAC) is characterized by extensive metabolic rewiring but despite the critical roles of PI5P4Ks in cellular metabolism, their role in PDAC remains completely unexplored. We have found that depletion of PI5P4Kα selectively induces apoptotic cell death in PDAC cells. To gain insights into the metabolic mechanisms underlying this apoptotic cell death, we quantified polar metabolites using gas chromatography- mass spectrometry (GC/MS) and found significant reductions in the ratio of monounsaturated to saturated fatty acids, lactate and TCA metabolites. Orthogonal rescue assays demonstrated that exogenous supplementation with fatty acids, pyruvate or lactate fail to rescue the observed apoptosis in PI5P4Kα-knockdown cells; however, TCA cycle metabolites did reverse the extent of apoptosis. Interestingly, we identify that these altered metabolic read-outs and the effects on cell fitness are dependent on iron. Consistent with these findings, intracellular iron levels are reduced upon PI5P4Kα inhibition, and this is directly linked to PI5P4Kα depletion disturbing the intracellular labile iron pools via iron import. Using a heterotopic xenograft mouse model, we demonstrate that PI5P4Kα knockdown leads to the abrogation of PDAC tumor growth due to increased intratumoral cell death. Validating the physiological relevance of PI5P4Kα in human PDAC, we find that PIP4K2A, the gene that encodes PI5P4Kα, is upregulated in human PDAC specimens relative to normal pancreas, and that PIP4K2A gene expression is strongly correlated with iron uptake related gene signatures. This study highlights the critical role of PI5P4Kα in PDAC and suggests that PI5P4Kα inhibition has therapeutic potential in pancreatic cancer. Citation Format: Gurpreet Kaur Arora, Ryan Loughran, Kyanh Ly, Cheska Marie Galapate, Alicia Llorente, Taylor R Anderson, Chantal Pauli, David A Scott, Yoav Altman, Cosimo Commisso, Brooke M Emerling. PI5P4Kα regulates cell fitness through iron homeostasis in 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 C044.

Abstract A017: Comprehensive evaluation of the therapeutic effects of whole-body hyperthermia for pancreatic ductal adenocarcinoma (PDAC) and the potential synergy with standard-of-care chemotherapeutics

Abstract Whole-body hyperthermia (WBHT) is a promising therapeutic approach that leverages elevated temperatures to enhance cancer treatment efficacy. By inducing controlled hyperthermia in the range of 39-42.5°C, cellular processes are disrupted and cancer cells can be sensitized to standard-of-care chemotherapies. However, the precise cellular and molecular mechanisms by which WBHT exerts its effects are not fully understood. Elucidating these mechanisms is particularly crucial for optimizing treatment protocols and improving outcomes in aggressive malignancies such as pancreatic ductal adenocarcinoma (PDAC). This study explores the impact of WBHT at 41.5°C on the cell cycle and associated molecular pathways in PDAC cells, providing insights into its therapeutic value and potential for combination regimens. PDAC cell lines, BxPC-3 and AsPC-1, were subjected to WBHT at 41.5°C for 1-6 hours and analyzed immediately, 24h and 48h post-WBHT Cell proliferation was assessed using the 5-ethynyl-2'-deoxyuridine (EdU) base-incorporation assay and proliferating cell nuclear antigen (PCNA) staining for immunofluorescence detection. Morphological changes post-WBHT were monitored using confocal fluorescence microscopy. Total RNA was extracted and sequenced using the Illumina platform. An in-house pipeline was used for QC, alignment and differential expression analysis. Enrichment analysis was performed to identify affected biological processes and pathways. In vivo experiments employed the chorioallantois membrane (CAM) assay per protocol published in STAR protocols. WBHT at 41.5°C significantly disrupted cell cycle progression in PDAC cell lines. The EdU incorporation assay revealed a dose-dependent decrease in DNA synthesis during and following WBHT, indicating cell cycle arrest in the S-phase. Decreases in Ki67, cyclin B1 and PCNA intensities further confirmed this reduction in cell proliferation. Moreover, cells showed notable signs of cellular and mitotic stress, such as the formation of multi-nucleated giant cells, aberrant metaphase morphology and micronuclei. Bulk RNA-seq confirmed alterations in gene expression profiles relating to the cell cycle immediately after HT exposure. Genes associated with the E2F targets and G2/M checkpoint pathways were negatively enriched, whereas genes involved in the Hallmark apoptosis pathway were positively enriched. Preliminary data from in ovo CAM experiments showed a significant inhibitory effect of WBHT monotherapy on growth of BxPC-3 tumors. Our data show that WBHT severely disrupts the cell cycle at the S- and M-phases, via multiple pathways involved in cell proliferation and apoptosis. These cancer hallmarks are targeted by conventional chemotherapeutics, paving the way for combination therapy as one-two punch cancer treatments. The synergy between WBHT with cisplatin and oxaliplatin, but also with gemcitabine and 5-fluorouracil (5-FU) could be optimized based on the improved understanding of the WBHT effects over time, in terms of WBHT duration, sequence of WBHT and chemo and the exact treatment schedule. Citation Format: Robin Colenbier, Tine Logghe, Gaëlle Boulet, Johannes Bogers, Jean-Pierre Timmermans. Comprehensive evaluation of the therapeutic effects of whole-body hyperthermia for pancreatic ductal adenocarcinoma (PDAC) and the potential synergy with standard-of-care chemotherapeutics [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 A017.

Abstract C016: Pancreatitis-induced resistance to KRAS targeted therapy: The role of cellular plasticity and underlying mechanisms

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease characterized by cell plasticity, particularly the epithelial-to-mesenchymal transition (EMT) phenotype, which significantly contributes to both pancreatic tumorigenesis and therapy resistance. Chronic pancreatitis, a key risk factor for PDAC, induces fibrosis, recruits macrophages, and elevates TGFβ, an EMT master regulator, in the tumor microenvironment (TME). Despite the observed negative correlation between dependence on oncogenic KRAS signaling and the quasi- mesenchymal (QM) subtype, the role of cellular plasticity and the underlying mechanisms of resistance remain unclear. To address this, we employed genetically engineered mouse models that mimic human pancreatitis and PDAC, alongside an in vitro 3-D tumor spheroid culture system. Our study demonstrates that chronic pancreatitis promotes resistance to KRAS- targeted therapy through a canonical TGFβ signaling pathway-dependent mechanism. Nuclear Factor of Activated T Cells 5 (NFAT5) forms a hetero-pentamer with canonical TGFβ factors SMAD3 and SMAD4, inducing EMT and promoting therapy resistance. Analysis of human samples indicates a positive correlation between NFAT5 expression and pancreatic tumorigenesis, with elevated NFAT5 expression correlating with poorer overall survival in the TCGA PDAC dataset. Functional studies reveal that genetic depletion or chemical inhibition of NFAT5 thwarts resistance to KRAS inhibition induced by TGFβ or pancreatitis, impairs the growth of QM-like escaper tumors, and synergizes with KRAS inhibitors to further suppress tumor growth in pre-clinical models. Mechanistically, we identified the chaperone protein and extracellular matrix regulator S100A4 as a key effector transcriptionally activated by the NFAT5- SMAD complex, mediating therapy resistance partially through the activation of key KRAS downstream MAPK and AKT signaling pathways. Furthermore, our investigation reveals that TGFβ stimulates PDAC cells to secrete the chemokine CCL2, which recruits circulating macrophages. In turn, these macrophages support PDAC cells in bypassing KRAS inhibition through paracrine TGFβ and S100A4. Overall, this study establishes a molecular foundation for KRAS-targeted therapy resistance associated with cellular plasticity in PDAC, highlighting pancreatitis as a potential risk factor, and proposes a novel strategy to overcome this resistance through NFAT5 inhibition or S100A4 blockade. Citation Format: Daiyong Deng, Pingping Hou. Pancreatitis-induced resistance to KRAS targeted therapy: The role of cellular plasticity and underlying mechanisms [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 C016.

Abstract A039: Repeat RNA mediated disruption of cellular plasticity in pancreatic cancer

Abstract Aberrant expression of repeat RNAs in pancreatic ductal adenocarcinoma (PDAC) mimics viral-like responses with implications for tumor cell state and the surrounding microenvironment. To better understand the relationship of repeat RNAs in human PDAC, we employed the NanoString CosMx™ spatial molecular imaging (SMI) platform, which utilizes a 1,000-plex RNA panel with custom repeat RNA probes targeting long interspersed nuclear element 1 (LINE-1) retrotransposon ORF1 and ORF2, HSATII satellite (SAT) repeat, and two human endogenous retroviruses (HERV-K and HERV-H) in 46 primary tumors. This analysis revealed correlations of high repeat RNA expression with alterations in the epithelial state of PDAC cells and the myofibroblast phenotype in cancer-associated fibroblasts (CAFs). The loss of cellular identity observed with dosing of extracellular vesicles (EVs) and individual repeat RNAs in PDAC and CAF cell culture models points to cell-cell intercommunication involving these viral-like elements. Differences in the PDAC and CAF response are driven by distinct innate immune signaling pathways through interferon regulatory transcription factor 3 (IRF3). Altogether, our data indicate that cell context-specific viral-like responses driven by tumor cells have broad impacts on single-cell heterogeneity within cancer cells and the pancreatic cancer microenvironment. Citation Format: Eunae You, Patrick Danaher, Chenyue Lu, Siyu Sun, Luli Zou, Ildiko Phillips, Alexandra Rojas, Natalie Ho, Yuhui Song, Michael Raabe, Katherine Xu, Peter Richieri, Hao Li, Natalie Aston, Rebecca Porter, Bidish Patel, Linda Nieman, Nathan Schurman, Briana Hudson, Khrystyna North, Sarah Church, Vikram Deshpande, Andrew Liss, Tae Kim, Yi Cui, Youngmi Kim, Benjamin Greenbaum, Martin Aryee, David Ting. Repeat RNA mediated disruption of cellular plasticity in 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 A039.

Abstract C070: Keratin 17 and GATA6 correlate with diffusely infiltrative versus gland-forming components of PDAC: Uncovering the transitional state in pancreatic ductal adenocarcinoma

Abstract Purpose: Optimization of pancreatic ductal adenocarcinoma (PDAC) patient survival will depend on advancing our understanding of the mechanisms governing tumor cell plasticity and effectively mitigating their invasive and metastatic capabilities. This study aims to investigate the correlation of cancer biomarkers crucial to these processes by integrating molecular signatures with histopathological characteristics using single-cell RNA sequencing (scRNA-seq) and multiplexed IHC/IF. Methods: Single-cell RNA sequencing (scRNA-seq) data from a comprehensive dataset of over 53,000 PDAC cells and validation of lead targets through 14-plex immunofluorescence, using a customized MICS platform (Miltenyi Biotec) and dual color IHC were employed to assess protein expression of defining biomarkers of basal and classical subtypes of PDAC including Keratin 17 (K17) and GATA6. Results: scRNAseq analysis revealed distinct patterns of K17 and GATA6 expression, highlighting intratumoral heterogeneity among PDAC tumors. Specifically, populations of cells expressing K17+/GATA6+ and K17+/GATA6- exhibited significant enrichment in pathways associated with invasion and cell migration, including PI3K/Akt/mTOR, TGF-β, and epithelial-mesenchymal transition (EMT) signaling. Conversely, GATA6-/K17- cells demonstrated enrichment in biosynthetic and metabolic pathways that govern cell cycle regulation. Additionally, mIF data assessment revealed correlations between histomorphological patterns and dynamic shifts in K17 and GATA6 expression within PDAC tumors. Diffusely infiltrative tumor cells (DITCs) uniformly expressed K17, contrasting with the gland-forming components (GFCs) of PDAC, which showed enrichment in GATA6 and sporadic expression of K17. E-cadherin, a key adhesion marker, exhibited the highest expression levels in the glandular and solid components of PDAC, underscoring their epithelial characteristics. Conversely, vimentin, a marker typically associated with epithelial-to-mesenchymal transition (EMT), was significantly expressed in DITCs, suggesting a mesenchymal-like phenotype in these infiltrative cells. Dual color IHC further validated these findings, reinforcing the correlation between morphologic plasticity and the expression patterns of K17 and GATA6, emphasizing their potential to distinguish distinct tumor cell populations within PDAC. Conclusions: These insights suggest that the interaction between these markers may indicate a distinct biological state characterized by altered cellular metabolism and potentially different therapeutic vulnerabilities. The robust association between K17 expression and diffusely infiltrative tumor cells underscores its potential as a marker for the most aggressive forms of this disease. Our results highlight the heterogeneous nature of PDAC and emphasize the critical importance of understanding these molecular distinctions in histopathological contexts to develop targeted therapies aimed at improving patient survival. Citation Format: Lyanne A Delgado- Coka, Brian Nelson, Michael Horowitz, Shayan Sarkar, Natalia Marchenko, Scott Powers, Luisa F Escobar-Hoyos, Kenneth Shroyer. Keratin 17 and GATA6 correlate with diffusely infiltrative versus gland-forming components of PDAC: Uncovering the transitional state in 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 C070.

Abstract C066: Identifying Transcriptional Drivers of Plasticity in Pancreatic Cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) remains a deadly disease with few effective treatments. Even with the advent of mutationally-directed therapies targeting the KRAS oncogene, PDAC transcriptional plasticity remains a major mechanism of therapeutic resistance. As a result, there is a desperate need to develop additional strategies to treat this deadly disease. In addition to the canonical genomic alterations in KRAS, TP53, CDKN2A, and SMAD4, PDAC tumors exhibit diverse transcriptional programs, or "cell states," with prognostic implications: PDAC tumors in the “classical state” are more responsive to upfront chemotherapy and have better outcomes, while those in the “basal state” are more aggressive with poorer prognoses. Currently, PDAC cell states are not used to direct therapy. Cell state is an integrative property shaped by both cell-intrinsic (e.g., mutations, epigenetics) and cell-extrinsic (e.g., tumor microenvironment) factors. In our prior work, single-cell analyses of clinical PDAC samples demonstrated notable cell state heterogeneity and plasticity. In addition, longitudinal monitoring of patient-derived organoids showed that cells can shift from classical to basal or coexpressor states as the environment changes, and that these cell states can drastically influence drug response. However, it remains unknown which signaling and transcriptional pathways induce basal or classical states, and whether these gene regulatory networks can be targeted therapeutically. Thus, there is a pressing need to devise scalable methods to determine essential drivers for prognostically relevant cancer cell states.Here, we aim to systematically and unbiasedly identify transcription factors (TFs) that drive basal and classical cell states in PDAC. We have successfully established a high-throughput screening pipeline to 1) over-express all human TF isoforms in a pooled format within a cohort of PDAC models; 2) sort cells using antibodies against state-specific cell surface markers; and 3) perform single-cell RNA sequencing on sorted cells to nominate individual TFs or combinations that induce specific cell state transitions. Preliminary results identify both previously reported (e.g., GATA4) and new TFs that can induce the classical state. By overexpressing TFs individually or in combination, we are able to engineer PDAC cell line models that more faithfully reflect the transcriptional phenotypes observed in patient tumors. Ultimately, we aim to use this approach to construct high-fidelity isogenic but state-variant PDAC models for use both in therapeutic screening and as a substrate for investigating mechanisms governing cancer cell state plasticity. Citation Format: Yuzhou Evelyn Tong, Aswanth Mahalingam, Walaa E Katan, Julia Joung, Alex K Shalek, Peter S Winter, Srivatsan Raghavan. Identifying Transcriptional Drivers of Plasticity in 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 C066.

Abstract C022: Elucidation and exploitation of RAS-inhibitor dependent macropinocytic regulation in pancreatic ductal adenocarcinoma

Abstract Alteration of essential metabolic pathways is a major mechanism by which oncogenic KRAS promotes tumor development and growth in pancreatic ductal adenocarcinoma (PDAC). KRAS- driven PDAC is dependent on nutrient scavenging pathways, including macropinocytosis and autophagy to fuel the metabolic demand of rapid proliferation. Thus, these metabolic processes are attractive targets for the development of treatments for PDAC. Acute KRAS loss results in downregulation of macropinocytosis in PDAC. Additionally, our lab demonstrated that KRAS loss or inhibition of ERK MAPK signaling decreased glucose consumption and glycolysis but increased autophagy, thereby enhancing dependency on autophagy for survival and growth. Accordingly, dual ERK MAPK and autophagy inhibition (via chloroquine) synergistically enhanced anti-tumor efficacy in PDAC. However, early clinical data has demonstrated that resistance to this treatment arises over time through unknown mechanisms. We observed that both autophagy induction and macropinocytosis downregulation are transient following RAS, MEK, or ERK inhibition—with autophagic and macropinocytic activity returning to or surpassing basal levels after within two weeks of treatment. Furthermore, we found that prolonged pharmacological inhibition of RAS, MEK, or ERK pathway consistently resulted in significant upregulation of macropinocytosis. We propose that prolonged MAPK inhibition upregulates macropinocytosis over time, consequently abrogating dependency on autophagy and therefore reducing sensitivity to autophagy inhibition. Furthermore, we found that chloroquine, which is commonly thought of as a lysosomotropic drug, does not prevent degradation of proteins engulfed via macropinocytosis. Together our data suggests that upregulation of macropinocytosis may mediate resistance to the combination of ERK MAPK inhibition and chloroquine. With the recent advance in development RAS inhibitors, the resistance mechanisms by which PDAC can overcome RAS inhibitor treatment is a major focus of the field. Given the dependence of PDAC on macropinocytic uptake for tumorigenic growth, it is imperative to understand how long-term treatment with RAS inhibitors regulate macropinocytosis. We demonstrated that PDAC cells with acquired resistance to RAS inhibition exhibit a 2- to 10-fold increase in macropinocytic uptake. We found that upregulated macropinocytosis following long-term RAS or MAPK inhibition is accompanied by increased albumin uptake and sensitivity to albumin-bound paclitaxel (nab-paclitaxel), a therapeutic that is included in the current standard of care for PDAC patients. These results suggest that RAS inhibitor pretreatment or concurrent treatment with nab-paclitaxel may represent a strategy to improve this current PDAC treatment. We conclude that a more complete understanding of the regulation of essential metabolic pathways following both acute and sustained RAS inhibition will better inform future RAS inhibitor combination therapies. Citation Format: Ryan Robb, Sarah Ackermann, Khalilah Taylor, Runying Yang. Elucidation and exploitation of RAS-inhibitor dependent macropinocytic regulation in 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 C022.

Abstract C045: Targeting metabolic heterogeneity in pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related death in the US. This poor prognosis is related to the complex tumor microenvironment and tumor heterogeneity that drives resistance to chemotherapy. Here the heterogeneity of the stromal, immune, and malignant epithelial cell populations enables signaling and metabolic crosstalk mechanisms that drive immune suppression and resistance to therapy. Recently, our group unveiled that multiple distinct metabolic subclasses of cancer cells coexist within individual PDAC tumors. Among these, two PDAC clonal populations can engage in metabolic symbiosis that supports mitochondrial metabolism. One subtype has a constitutively high integrated stress response (ISR), enabling resistance to mitochondrial inhibitors. Further, these cells release asparagine that supports the mitochondrial metabolism of the other subtype that are more sensitive to mitochondrial inhibition. Given these observations, we are developing approaches to disrupt this metabolic symbiosis. Clonal PDAC cells with constitutively active ISR exhibit increased sensitivity to inhibition of the redox activity of Redox factor 1/Apurinic-apyrimidinic endonuclease 1 (Ref-1). Importantly, this can be leveraged to disrupt the support of the mitochondrial metabolism of clonal symbiotic pairs. Moreover, polyclonal PDAC cell lines representative of the heterogeneity seen in PDAC patients show a highly synergistic response to the combination of Ref-1 redox inhibitors with mitochondrial inhibitors. These data suggest this treatment combination will be synergistic in polyclonal tumors that exist in most PDAC patients. Overall, these data have the potential to lead to new avenues to directly target heterogenous populations of cancer cells to treat pancreatic cancer- an urgent clinical need. Citation Format: Taryn Morningstar, Cecily Anaraki, Lorenzo Scipioni, Giulia Tedeschi, Michelle Digman, Aimee Edinger, Claus Jorgensen, Melissa Fishel, Christopher J Halbrook. Targeting metabolic heterogeneity in 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 C045.

Abstract A035: 3D High-Density Collagen I Improves the Modeling of Aggressive Pancreatic Cancer

Abstract Background: Fibrillar collagen type I constitutes the majority of the extracellular matrix in pancreatic ductal adenocarcinoma (PDAC), yet most in vivo murine models involve the use of basement membrane extracts, which instead contain collagen type IV, as matrices to embed PDAC cells. Type I collagen in the PDAC tumor microenvironment has been associated with worse prognosis. Our hypothesis is that PDAC cells grown in 3D high-density collagen I (HDC) gels behave more aggressively compared to the same cells grown using conventional basement membrane matrix (BMM). Methods: The KPC1199 2D cell line was previously generated from a tumor-bearing male KPC mouse. KPC1199 cells were cultured in vitro in HDC or BMM for one week. Orthotopic tumors were established by embedding KPC1199 cells in HDC (N=10) or BMM (N=10) in the pancreatic tail of syngeneic C57BL/6J mice via laparotomy. Half of the tumors were collected five weeks after tumor implantation, and the other half utilized for a survival study. Collagen was imaged by second harmonic generation (SHG) microscopy. Results: In vitro imaging of single KPC1199 cells grown in HDC revealed an aggressive phenotype with clonal network-like structures compared to a spheroidal, non-aggressive phenotype for cells grown in BMM over the course of seven days. SHG imaging confirmed consistent formation and organization of collagen in HDC tumors but minimal to faint collagen formation in BMM tumors. Gross examination of HDC mice showed 60% with visible liver metastasis compared with none in the BMM mice. In the survival study, the entire HDC group died before the BMM group (p-value < 0.01). Conclusions: Our pilot study is the first, to our knowledge, to successfully generate orthotopic PDAC tumors using 3D high-density collagen I in a mouse model. KPC1199 cells embedded in HDC showed an aggressive phenotype in vitro, and resultant tumors demonstrated more robust collagen organization compared to the same cells grown in the BMM substrate. This aggressive phenotype translated to visible liver metastasis within five weeks in the HDC mice and prior to death from primary tumor burden. This is not typical for most KPC-derived cell lines but is typical for human PDAC patients. Our preliminary findings support the use of HDC as an alternative substrate for better modeling of this aggressive cancer. Citation Format: Kim Nguyen-Ta, Sural Ranamukhaarachchi, Jeffrey Turner, Utsav Joshi, Himangshu Sonowal, Jorge de la Torre Medina, Herve Tiriac, Stephanie Fraley, Rebekah White. 3D High-Density Collagen I Improves the Modeling of Aggressive 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 A035.

Abstract C026: Concurrent inhibition of the RAS ERK-MAPK pathway and PIKfyve as a therapeutic strategy for pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized clinically by poor survival and mechanistically by KRAS- and autophagy-dependent growth. We and others previously demonstrated that inhibition of KRAS signaling via downstream inhibition of the RAF-MEK-ERK pathway enhanced autophagic flux and dependency of PDAC on autophagy. Furthermore, we demonstrated that concurrent treatment with the nonspecific autophagy inhibitor chloroquine (CQ) and ERK MAPK inhibitors synergistically blocked PDAC growth. These findings provided rationale for our initiation of Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT4132505) or ERKi (LY3214996; NCT04386057) with HCQ in PDAC. However, a limitation of this approach is that CQ/HCQ is limited in terms of specificity and potency. Thus, we aimed to identify alternative anti-autophagy strategies. We performed a CRISPR-Cas9 loss- of-function screen in PDAC cell lines that identified the lipid kinase PIKfyve as a growth- promoting gene. PIKfyve is a lipid kinase that is essential for the generation of PI(3,5)P2 and critical for the dynamic regulation of lysosomal recycling. Because PIKfyve has been implicated in regulation of autophagy in other cellular contexts, we evaluated the effects of PIKfyve inhibition on growth and autophagic flux in PDAC cell lines and tumors. We demonstrated that PIKfyve inhibition by the small molecule apilimod resulted in durable growth suppression, with much greater potency than CQ treatment. PIKfyve inhibition potently reduced autophagy as indicated by decreased autophagic flux and the robust accumulation of autophagy-related proteins. Additionally, PIKfyve inhibition resulted in the accumulation of LAMP1 positive vacuoles that exhibited impaired cargo degradation, likely due to a lack of required acidity. Next, we hypothesized that PIKfye inhibition would sensitize PDAC cells to RAS pathway inhibition. We demonstrated that PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and either the RAS ERK-MAPK pathway or RAS itself showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell cycle arrest and induction of apoptosis following loss of IAP proteins. These findings implicate PIKfyve as an effective anti-autophagy target when paired with RAS or ERK-MAPK pathway inhibition in pre-clinical models of PDAC. Citation Format: Jonathan M DeLiberty, Mallory K Roach, Clint A Stalnecker, Ryan Robb, Elyse G Schechter, Noah L Pieper, Runying Yang, Scott Bang, Khalilah E Taylor, Kristina Drizyte-Miller, John P Morris IV, Channing J Der, Adrienne D Cox, Kirsten L Bryant. Concurrent inhibition of the RAS ERK-MAPK pathway and PIKfyve as a therapeutic strategy for 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 C026.

Abstract B063: Elucidating treatment induced tumor antigen presentation in pancreatic cancer

Abstract Introduction Pancreatic ductal adenocarcinoma (PDAC), the most common form of pancreatic cancer, is projected to become the second leading cause of cancer-related deaths by 2030. The 5-year survival rate remains stubbornly low at just 13%. These discouraging statistics primarily stem from the lack of effective treatments till date. Despite the success of immunotherapy for several malignancies, immunotherapy for PDAC has yielded disappointing results alone or in combination with other therapies. Successful immunotherapy will require a deeper understanding of PDAC immunobiology and immune recognition of cancer during its progression, and treatment regimens. Immunosurveillance of cancer requires the presentation of peptide antigens on Major Histocompatibility complex class I (MHC-I). Understanding the repertoire of MHC-I associated peptides (pMHC-I), collectively termed as the “immunopeptidome”, is essential for anti-tumor immunity and successful immunotherapies. Using a collection of preclinical tools and human cell lines, we aim to uncover treatment induced antigens in PDAC that can be used for targeted immunotherapy approaches. Methods To understand the biogenesis of the immunopeptidome, our group has developed a novel genetically engineered mouse model (GEMM), which integrates inducible affinity tags on MHC-I (KbStrep) and ribosomes (RiboTag) into the Kras LSL-G12D/+ ; P53 fl/fl (KP) mouse model (KP/RiboMHC). This approach enabled us to precisely measure protein synthesis and antigen presentation in malignant PDAC cells in vivo with paired ribosome profiling and mass spectrometry based immunopeptidomics. In parallel we have employed a collection of human PDAC cell lines for immunopeptidomics analysis. About 93% of PDACs have KRAS mutation, with G12D mutation being the most common. In addition, PDAC is subjected to a deregulated metabolic milieu, with a strong dependence on glutamine utilization. Therefore, to examine treatment induced changes in PDAC antigen presentation, we examined the impact of KRAS inhibition (MRTX1133) and glutamine antagonism (6-diazo-5-oxo-L-norleucine, DON) on the biogenesis of the immunopeptidome in vitro and in vivo. Results Using our GEMM model, we have started to uncover MRTX1133 and DON induced alterations in protein synthesis and in pMHC-I repertoire in vivo using the KP/RiboMHC model and in vitro in mouse and human cell lines. We have identified unique antigen peptides that are induced by MRTX1133 and DON, including those derived from non-canonical translation events that were defined by ribosome profiling. Conclusions Our data demonstrates that KRAS inhibition with MRTX1133 or glutamine antagonism with DON reshapes the antigen landscape of PDAC. We are actively exploring which of the induced peptides harbor potential to mount an anti-tumor CD8+ T cell response. Collectively, these results could pave the way forward for rationally designed treatment regimens that pair targeted therapies with antigen specific immunotherapy in PDAC. Citation Format: Emma Adhikari, Andrew Weeden, Emily Brennan, Christopher Polera, Victoria lzumi, Bin Fang, John Koomen, Paul Stewart, Alex Jaeger. Elucidating treatment induced tumor antigen presentation in 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 B063.

Abstract IA-06: Elucidation of metabolic resistance mechanisms to RAS inhibition

Abstract Pancreatic ductal adenocarcinoma (PDAC) is characterized by KRAS- and autophagy-dependent growth. We previously determined that concurrent inhibition of autophagy, using the lysosomal inhibitor chloroquine (CQ), and of ERK, using a small molecule ERK inhibitor (ERKi), synergistically suppressed the growth of pancreatic ductal adenocarcinoma (PDAC) cell lines and patient xenograft-derived (PDX) organoids in vitro and PDX tumors in vivo. Our findings provided the rationale for our initiation of Phase I and Phase I/II clinical trials evaluating the combination of MEKi (binimetinib; NCT04132505) or ERKi (LY3214996; NCT04386057) with hydroxychloroquine (HCQ) in PDAC. We have extended these findings and determined that the combined inhibition of KRASG12C or KRASG12D and autophagy was effective in KRAS-mutant cancers. Our ongoing studies are centered on developing additional combinations for targeting metabolic resistance mechanisms to KRAS or ERK MAPK targeted therapies. First, we performed a CRISPR/Cas-9 mediated genetic loss-of-function screen in the presence of CQ to determine additional sensitizers to autophagy inhibition. Top sensitizers included multiple facilitators of the DNA damage response, mTOR pathway components, and genes involved in the upstream regulation of the autophagy pathway. Additionally, we identified PIKfyve, a lipid kinase critical for the recycling dynamics of lysosomes, as an essential autophagy-related gene in PDAC cells. PIKfyve inhibition with apilimod resulted in a potent reduction of autophagic flux and growth. Importantly, PIKfyve inhibition blocked the compensatory increases in autophagic flux associated both with MEK inhibition and with direct RAS inhibition. Accordingly, combined inhibition of PIKfyve and either the RAS ERK-MAPK pathway or RAS itself showed robust growth suppression across a panel of KRAS-mutant PDAC models. Growth suppression was due, in part, to potentiated cell cycle arrest and induction of apoptosis following loss of IAP proteins. We conclude that concurrent inhibition of RAS and PIKfyve is a synergistic, cytotoxic combination that may represent a novel therapeutic strategy for PDAC. In a parallel line of investigation, we determined that autophagic signaling is temporally regulated following RAS pathway inhibition, peaking at 48-72 hours, and returning to basal levels by 7 days. Interestingly, we found that as autophagic upregulation subsides, another nutrient scavenging pathway, macropinocytosis, increases. We hypothesize that further dissection of the dynamic regulation of autophagy and macropinocytosis will improve current anti-nutrient scavenging treatment strategies. We conclude that concurrent suppression of multiple metabolic processes, to block compensatory rebound activities, will be needed for effective PDAC treatment. Citation Format: Jonathan M. DeLiberty, Ryan Robb, Mallory K. Roach, Sarah E. Ackermann, Runying Yang, Noah L. Pieper, Khalilah E. Taylor, Scott Bang, Elisa Baldelli, Emanuel F. Petricoin III, John P. Morris IV, Clint A. Stalnecker, Kirsten L. Bryant. Elucidation of metabolic resistance mechanisms to RAS inhibition [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 IA-06.

Abstract A049: Collagen-driven kinome reprogramming reveals unique, actionable DDR1-signaling dependencies in pancreatic cancer cells

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) is the 3rd most common cause of cancer death in the United States, with a 5-year survival rate of only 13%, in part due to late detection and metastatic dissemination at early stages of progression. Most PDA tumors exhibit a dense fibro-inflammatory stroma consisting of fibroblasts, immune cells, and a dense collagen-rich extracellular matrix (ECM) that regulate disease progression. How distinct collagens (COLs) influence PDA progression remains unclear. COLs signal via specific cell surface receptors, of which the Discoidin Domain Receptors (DDRs) constitute a unique family of collagen-binding receptor tyrosine kinases (RTKs). DDR1 is expressed by PDA cancer cells, while DDR2 is expressed by mesenchymal cells. DDR1 is activated by both basement membrane COL (e.g. COL4) and fibrillar COLs (e.g. COLs 1, 2, 11). Compared to most RTKs, relatively little is understood about differential ligand activation and signaling downstream of DDRs. Our working hypothesis is that switches in ECM collagen composition over the course of PDA progression induce changes in DDR1-intracellular signaling cascades, which may reveal unexplored therapeutic susceptibilities to target and eliminate PDA cancer cells. Methods and Recent Advances: Using genetically engineered mouse models of PDA, we previously found that genetic ablation of Ddr1 impedes tumor progression, blocking the transition from well-differentiated to poorly-differentiated adenocarcinoma and, as a result, metastasis. Moreover, we showed that xenografts of DDR1-expressing human PDA cell lines display enhanced tumor growth exclusively when implanted within a COL1 scaffold. Leveraging an innovative high-throughput kinase-activity mapping (HT-KAM) platform and kinase network resource database (PhosphoAtlas), we started investigating mechanisms of differential activation of DDR1 by distinct COLs, including COL1 and COL11, which is an understudied fibrillar collagen that is prominent in the stroma of late-stage, metastatic PDA. New, Unpublished Findings: Using several PDA cell line models with or without DDR1 expression, our results indicate that DDR1 activates two distinct signaling networks: (1) a conserved, coordinated response that is modestly activated by COL1 but is hyperactivated by COL11 (e.g., ERBB, AKT, MEK/ERK, SRC), and (2) a unique set of phospho-signaling nodes that are only induced by COL11 –and not COL1 (e.g., specific RTKs and PKCs). Several of these kinases are associated with cancer cell proliferation and survival, as well as EMT induction, in part illuminating the malignancy-promoting effects of DDR1 in late-stage PDA in response to changes in COL composition. Conclusion: Differential activation of DDR1 represents a new paradigm on how distinct fibrillar COLs within the TME may drive PDA cell signaling. We are actively exploring how distinct COLs differentially regulate PDA tumor growth and metastatic behavior via DDR1, and how specific kinase-targeting interventions may prevent malignant phenotypes or induce tumor cell death. Citation Format: Anjum Sohail, Yeonjoo Hwang, Denise P Munoz, Yoshihiro Ishikawa, Rafael Fridman, Howard Crawford, Jean-Philippe Coppe. Collagen-driven kinome reprogramming reveals unique, actionable DDR1-signaling dependencies in pancreatic cancer cells [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 A049.

Abstract C052: Repurposing Statins to Target Gemcitabine Resistance in Pancreatic Cancer

Abstract Pancreatic ductal adenocarcinoma (PDA) is a lethal malignancy with a devastating prognosis. Gemcitabine, a pyrimidine anti-metabolite, is a cornerstone in PDA therapy, but resistance remains a major hurdle in clinical care. Metabolic reprogramming is an important driver of chemoresistance. Accordingly, targeting these altered metabolic pathways can improve response to treatment. To investigate metabolic vulnerabilities, we generated models of acquired PDA gemcitabine resistance. Screening common metabolic inhibitors we observed that gemcitabine-resistant cells were exquisitely susceptible to statins, inhibitors of the 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. This is due to a downregulation of the mevalonate biosynthesis pathway in gemcitabine-resistant cells. Importantly, the statin sensitivity correlates with the extent of the gemcitabine resistance, suggesting this metabolic vulnerability is gradually gained during acquisition of resistance. Rescue experiments showed that statins induce apoptosis by reduced protein geranylation of small GTPases. Finally, we find that pitavastatin inhibits growth of gemcitabine resistant tumors in immune competent models. Collectively, these data suggest that targeting the HMG-CoA reductase is a metabolic vulnerability that is gained in PDA cells upon the acquisition of gemcitabine resistance. The treatment combination of gemcitabine and pitavastatin, two widely used compounds in patients, has potential to translate into an immediate clinical benefit and improve survival in this deadly disease. Citation Format: Sabrina Calderon, Alica K. Beutel, Ethan Nghiem, Rima Singh, Natalie Yousefian, Cecily Anaraki, Kevin Gulay, Dennis Juarez, Alexander Kleger, Thomas Seufferlein, Alexander Muir, Cholsoon Jang, Herve Tiriac, David Fruman, Christopher Halbrook. Repurposing Statins to Target Gemcitabine Resistance in 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 C052.

Abstract C047: Autophagy-mediated proline-supply is important for cell survival in pancreatic cancers

Abstract Oncogenic KRAS mutations present in 90–95% of pancreatic cancer patients. KRAS mutation in pancreatic cancer increased the dependency on glutamine and consumption of proline. Proline is a non-essential amino acid but it is important for the synthesis of proteins such as collagen, and also for precursors of antioxidant molecules. Several enzymes such as P5CS(ALDH18A1),PYCR1 and PYCR2 are involving proline synthesis. The effects of proline synthesis enzymes on pancreatic cancer cells are relatively less studied than that of enzymes catalyzing proline. Here, we examined the impact of proline synthesis enzymes on pancreatic cancer cell survival and proliferation. We first examined that exogenous proline promotes cell survival under low glutamine conditions in various pancreatic cancer cells. Exogenous proline supplementation increased some pancreatic cell survival even when glutamine was not sufficiently available, whereas proline supplement is dispensible for certain cell types of pancreatic cancer. Next, to investigate the importance of proline synthesis enzymes in cell survival, we measured the cell proliferation of pancreatic cancer with knock-down the proline synthesis enzymes. The knockdown of ALDH18A1 and PYCR1,2, which are proline biosynthesis enzyme genes, reduced cell survival in pancreatic cancer cells, suggesting that in PDAC cells, the enzymes that synthesize proline from glutamine are crucial for cancer cell survival and growth. To test the effect of proline-sysnthesis enzymes on autophagy, we measured autophagy flux in the cell harboring GFP-RFP-LC3 or GFP-LC3 as a reporter. Knockdown of proline synthesis enzymes, particularly ALDH18A1 or PYCR1 led to an increase in autophagy levels, indicating that proline-independent cells can bypass proline synthesis by autophagy induction in the absence of proline synthesis genes. Furthermore gemcitabine-resistant cells showed reduced levels of proline synthesis enzymes. In pancreatic cancer cell lines, proline promotes cancer cell survival, generated by both biosynthesis from glutamine and starvation-induced autophagy, which also critically important for supporting cellular drug resistance. Taken together, these results indicate that proline synthesis is mechanistically associated with autophagy induced by glutamine-starvation, which is pivotal for supplying proline in pancreatic cancer cells. Citation Format: Ji hyeon Kim, Heesun Cheong. Autophagy-mediated proline-supply is important for cell survival in pancreatic cancers [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 C047.

Abstract A020: Exocytosed amino acids as a selective and side effect-free therapy for pancreatic cancer

Abstract Pancreatic adenocarcinoma (PDAC) remains one of the fatal cancers due to its poor prognosis and the lack of effective treatments, often diagnosed at advanced stages. There is an urgent need for innovative and selective therapies as current treatments show limited efficacy and significant side effects. Our proteomics analysis of extracellular vesicles from PDAC tumor cells revealed a distinctive imbalance in secreted amino acids. Specifically, we found that PDAC cells preferentially excrete proteins enriched with amino acids such as isoleucine and histidine, which are linked to disease progression. By targeting these exocytosed amino acids, we observed selective toxicity against PDAC cells in both in vitro and in vivo experiments. Additionally, we identified XRN1 as a potential target influenced by these amino acids. This approach demonstrated high selectivity, targeting tumor metabolism with minimal toxicity to normal tissues, and showed to be easy to administer with sustained tumor-killing effects. Our findings suggest that targeting exocytosed amino acids offers a promising therapeutic strategy for PDAC and potentially other cancers. Citation Format: Alfred Akinlalu, Zachariah Flaten, Komila Rasuleva, Ang Guo, Sijo Mathew, Dali Sun. Exocytosed amino acids as a selective and side effect-free therapy for 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 A020.