Murine PDAC Research Articles

Abstract PO-082: Delineating the molecular basis of early dissemination of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is often diagnosed at advanced stages rendering the already limited therapeutic options ineffective for many patients. Earlier diagnosis permits for “curative” surgery; however, 95% of these patients experience recurrence locally or distally and die within ten years of treatment. One hypothesis that can explain high recurrence is the early dissemination of PDAC progression. At the time of surgery, systemic subclinical dissemination of predominantly dormant cells found ubiquitously within patient livers has already occurred prior to the development of overt metastatic disease or bona fide recurrent PDAC. However, remarkably little is known about early PDAC cell dissemination despite these dismal outcomes, mainly due to the lack of human models to study disease progression. We previously demonstrated the cellular reprogramming “proof-of-principle” to model human PDAC progression (designated “10-22 cells”) and its use as a discovery tool for early biomarkers by unveiling a marker for stage 1 PDAC in human patients. We further analyzed transcriptomes of genetically tagged 10-22 cells progressing from pancreatic intraepithelial neoplasia (PanIN) to PDAC in mice and validated the results using the TCGA PDAC dataset, human clinical PanIN, PDAC tissues, and a well-established murine PDAC model. We found that extracellular vesicle transport and neuronal cell differentiation pathways were derepressed in the progression of PanINs to PDAC. HMG-Box Transcription Factor 1 (HBP1) emerged as a potential master factor regulating dynamically expressed genes in neuronal cell differentiation and dissemination during PDAC progression. HBP1 expression are aberrantly regulated in human and murine PanINs and PDAC tissue samples, and their mRNA expressions are inversely correlated with PDAC patients' prognosis. Ectopic overexpression of HBP1 increased proliferation and migration of normal and cancerous pancreatic cells. Conversely, knocking out HBP1 in PDAC cells decreased proliferation and migration. Altogether, our data indicate that HBP1 may confer the cell dissemination capacity in early PDAC progression. We are on the way to further dissect the mechanisms by which HBP1 regulates pancreatic cell migration and confers neuronal migratory phenotypes and how these signaling influence PDAC progression and prognosis. Citation Format: Taelor J. Ekstrom, Dmytro Grygoryev, Terry Morgan, Kenneth S. Zaret, Jungsun Kim. Delineating the molecular basis of early dissemination of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-082.

Abstract PO-064: ONC212 stimulates cytotoxic T-cell killing, increases tumor-immune cell interactions, and promotes tumor regression in combination with TLY012 in a PDAC murine model

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer with a 4.2% 5-year survival rate. There is an urgent need for innovative treatments for patients suffering from PDAC. Patients with PDAC often have immunosuppressed tumors that are apoptosis-resistant and have limited immune cell infiltration and/or activation. Tumor necrosis factor-Related Apoptosis-Inducing Ligand (TRAIL) selectively induces cancer cell apoptosis during innate immunity, while ONC212 is a potent fluorinated second-generation imipridone currently in IND-enabling studies that induces TRAIL signaling and the integrated stress response (ISR). TLY012 is a novel PEG’ylated trimeric TRAIL receptor agonist being clinically developed to overcome the short half-life of TRAIL. We hypothesized that combining a TRAIL pathway inducer and a TRAIL receptor agonist may be efficacious in PDAC and may activate immune cell killing based on prior work with imipridone ONC201. Immune cell co-culture experiments were conducted using PANC1 PDAC cells and TALL-104 CD8+ cytotoxic T-cells at a 1:1 effector-to-target cell ratio with or without ONC212. We observed a significant increase in T-cell killing of PDAC cells in the co-culture assay following treatment with ONC212 at several doses as compared to controls. We noted an increase in tumor-immune cell interactions as measured by the number of T-cells that were directly touching tumor cells at each timepoint. The BxPC-3 immunodeficient murine PDAC model revealed an increase in natural killer (NK) cell tumor infiltration by immunohistochemistry (IHC) staining for NK1.1 after 30 days of 50 mg/kg ONC212 treatment three times a week. The combination of ONC212 and TLY012 was evaluated using subcutaneously-implanted KPC-Luc PDAC cells in a syngeneic immunocompetent C57BL/6 mouse model. Mice treated with 25 mg/kg ONC212 alone twice a week, 10 mg/kg TLY012 alone twice a week, or combination therapy with ONC212 and TLY012 showed statistically significant decreases in tumor growth compared to controls after 5 weeks of treatment (n=6 mice per treatment condition) by tumor volume and in vivo bioluminescent imaging. We are analyzing murine plasma cytokine profiles, changes in tumor-infiltrating NK- and T-cells by flow cytometric and IHC analysis of murine spleen and KPC-Luc tumor samples. We report a novel immune stimulation of the TRAIL pathway and T-cell activation by ONC212 plus TLY012 leading to cytotoxic anti-PDAC responses in vivo. Citation Format: Kelsey E. Huntington, Anna Louie, Young Lee, Jared Mompoint, Isacco Ferrarini, Aakash Jhaveri, Varun V. Prabhu, Allen Melemed, Seulki Lee, Wafik S. El-Deiry. ONC212 stimulates cytotoxic T-cell killing, increases tumor-immune cell interactions, and promotes tumor regression in combination with TLY012 in a PDAC murine model [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-064.

Abstract PO-072: Inhibiting vasoactive intestinal peptide receptor signaling elicits T cell dependent anti-tumor response of pancreatic ductal adenocarcinoma to immune checkpoint therapy

Abstract Pancreatic ductal adenocarcinoma (PDAC) is unresponsive to immune checkpoint therapy largely due to a paucity of T cells within the tumor microenvironment (TME) and abundant immunosuppressive signaling pathways. In this study we show that human PDAC tumors over-express vasoactive intestinal peptide (VIP), an immunosuppressive neuropeptide that suppresses T cell effector properties and promotes the generation of regulatory T cells (Tregs). Therefore, we treated tumor-bearing mice with VIP receptor peptide antagonists and measured T cell homing, activation, and anti-tumor responses in preclinical murine models of PDAC. Pharmacological inhibition of VIP receptor (VIP-R) signaling using daily subcutaneous injections of peptide antagonists had no discernable toxicity in healthy and tumor-bearing mice. Treatment with VIP-R antagonists in combination with anti-PD-1 checkpoint blockade significantly decreased tumor burden, and improved survival in subcutaneous and orthotopic murine PDAC models. Combination therapy significantly enhanced T cell activation and proliferation and decreased frequencies of Tregs within the TME. Anti-tumor responses were T cell dependent, as the combination therapy failed to improve survival in CD4 or CD8 deficient mice using knock-out strains and antibody depletion. Furthermore, combination therapy significantly increased frequencies of tumor specific T cells (measured with a tetramer reagent) and provided protective immunity against tumor rechallenge. Combination therapy led to significant increases in the infiltration of adoptively transferred GFP+ T cells into PDAC tumors and decreased CXCR4 expression levels on T cells. Encouragingly, peptide-based VIP-R antagonists enhanced the in vitro activation of human T cells isolated from peripheral blood of PDAC patients. Human T cells cultured with VIP-R antagonists had increased proliferation, activation, and decreased proportions of T regs and exhausted T cells co-expressing PD-1, Tim-3 and Lag-3. Taken together, our findings show that VIP is a targetable mechanism of immune escape in PDAC. Inhibiting VIP receptor signaling improves T cell effector properties and synergistically improves anti-tumor responses to checkpoint inhibitors in mouse PDAC models. Additionally, as the VIP sequence is identical between human and mice, and since VIP-R antagonists have similar effects on human and murine T cells in culture, clinical translation is highly feasible. Citation Format: Sruthi Ravindranathan, Passang Tenzin, Jian Ming Li, Rohan Dhamsania, Michael Ware, Mohammad Zaidi, Shuhua Wang, Jingru Zhu, Maria Cardenas, Yuan Liu, Gaurav Joshi, Sanjeev Gumber, Brian Robinson, Anish Sen-Majumdar, Shanmuganathan Chandrakasan, Haydn Kissick, Alan Frey, Susan Thomas, Bassel El-Rayes, Gregory Lesinski, Edmund K. Waller. Inhibiting vasoactive intestinal peptide receptor signaling elicits T cell dependent anti-tumor response of pancreatic ductal adenocarcinoma to immune checkpoint therapy [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-072.

Abstract PO-125: The role of KDM6A in pancreatic cancer immune microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer death in the United States, with a 10% 5-year survival rate. Immunosuppressive myeloid cells, including tumor-associated neutrophils (TANs), contribute to tumor development and treatment resistance. Lysine (K)-specific demethylase 6A (KDM6A) is one of the most frequently mutated epigenetic genes and a tumor suppressor in PDAC. However, the molecular mechanisms by which KDM6A contributes to PDAC development and whether KDM6A impacts the tumor immune microenvironment are unknown. This study established a genetically engineered pancreas-specific Kdm6a-knockout Ptf1aCre;LSL-KrasG12D/+;LSL-p53R172H/+ (KPC) PDAC mouse model to investigate the influence of KDM6A loss on PDAC development and tumor immune microenvironment. We found that KDM6A loss accelerated PDAC progression and increased metastases. Pancreata of mice with Kdm6a deficiency developed aggressive undifferentiated PDACs. Additionally, KDM6A loss led to increased infiltration of TANs and neutrophil extracellular traps (NETs) formation. Mechanistically, we used Bru-seq technology to investigate the impact of KDM6A on nascent RNA transcription. We demonstrated that many chemotactic cytokines related to neutrophil recruitment, specifically CXCL1, were upregulated in KDM6A-knockout PDAC cells. We further confirmed increased CXCL1 mRNA and protein levels in KDM6A-deficient human and mouse PDAC cells. In addition, immunohistochemical staining also confirmed the upregulated CXCL1 expression in both human and murine PDAC cells with KDM6A loss. TANs were found to express CXCR2, the receptor of CXCL1, by immunofluorescent analysis. To further confirm that cancer cells with KDM6A loss can attract neutrophils in vitro, we performed chemotaxis assays using both human neutrophils derived from PLB-985 cells and mouse primary neutrophils isolated from bone marrow and conditioned media from KDM6A- knockout or knockdown PDAC cells. We found that PDAC cells with KDM6A loss promoted neutrophil recruitment in vitro compared to KDM6A-retained PDAC cells. Furthermore, the CXCL1 neutralizing antibody reversed the chemotactic property of KDM6A-deficient PDAC cells, confirming that CXCL1 is the primary chemokine mediating the neutrophil recruitment. In summary, these findings shed light on the mechanism by which KDM6A loss promotes PDAC development, regulates tumor immune microenvironment, and suggests that the CXCL1-CXCR2 axis may be a candidate target for the treatment of PDAC, especially those with KDM6A mutations. Citation Format: Lin Jin, Jing Yang, Zhujun Yi, Hong S. Kim, Feng Tian, Jiaqi Shi. The role of KDM6A in pancreatic cancer immune microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-125.

Abstract PO-014: VISTA: VIsual Semantic Tissue Analysis for pancreatic disease quantification in murine cohorts

Abstract Objective and quantifiable assessment of tissue pathology is necessary to study mechanistic disease progression; however, current quantification methods based on tissue staining have many drawbacks including cost, time, labor, batch effects, as well as uneven staining which can result in misinterpretation and investigator bias. Here we present VISTA, an automated deep learning tool for semantic segmentation and quantification of histologic features from hematoxylin and eosin (H&E) stained pancreatic tissue sections. VISTA is trained to identify four key tissue types in developing murine PDAC samples: normal acinar, acinar-to-ductal metaplasia (ADM), dysplasia, and other normal tissue. Predicted segmentations were quantitatively evaluated against pathologist annotation with Dice Coefficients, achieving scores of 0.79, 0.70, 0.79 for normal acinar, ADM, and dysplasia, respectively. Predictions were evaluated against biological ground truth using the mean structural similarity index to immunostainings amylase and pan-keratin (0.925 and 0.920, respectively). The total area of feature prediction was also correlated to the area of immunostaining in whole tissue sections using spearman correlation (0.86, 0.97, and 0.92 for DAPI, amylase, and cytokeratins, respectively). Importantly, our tool is not only able to predict staining information, but it is able to distinguish between ADM and dysplasia, which are not reliably distinguished with common immunostaining methods, showing VISTA’s potential to expand research beyond what is capable with current standards. As a use case example of VISTA, we quantified abundance of histologic features in murine cohorts with oncogenic Kras-driven disease. We observed stromal expansion, a reduction in normal acinar, and an increase in both ADM and dysplasia as the disease progresses, which matches known biology. Since VISTA is an automated algorithm, it can accelerate histological analysis and improve the consistency of quantification between laboratories and investigators. This work has been published in Nature Scientific Reports, and the code is available on github at https://github.com/GelatinFrogs/MicePan-Segmentation. Citation Format: Luke Ternes, Ge Huang, Christian Lanciault, Guillaume Thibault, Rachelle Riggers, Joe Gray, John Muschler, Young Hwan Chang. VISTA: VIsual Semantic Tissue Analysis for pancreatic disease quantification in murine cohorts [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-014.

Abstract 2475: Differential sensitivity of mouse PDAC KrasG12D cells to Ref-1/APE1 redox signalling inhibitors: Role of NFkB as a primary target of Ref-1/APE1 in Kras driven pancreatic ductal adenocarcinoma

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, with a five-year survival rate of less than ten percent. Kras is a common driver mutation of PDAC, contributing to progression and chemotherapy resistance. Additionally, inflammation is implicated in the development of PDAC and NFκB is recognized as a key mediator of inflammation and has been frequently observed to be upregulated in PDAC. Ref-1/APE1 (redox effector factor 1/apurinic endonuclease 1), a multifunctional protein involved in redox signaling of selected transcription factors (TF), is an upstream activator of NFκB and a therapeutic target. We have developed inhibitors to prevent Ref-1/APE1's redox signaling function, including activation of NFkB. We screened eight murine PDAC KC cell lines, which contain the KrasG12D mutation, to determine if there are subsets of these tumor cells that would respond differentially to Ref-1/APE1 redox signaling which recently completed phase I clinical trials for solid tumors. Additionally, we tested a series of second-generation inhibitors, including APX2009, APX2014 and others. The cell lines split into two distinct groups of sensitive and resistant to the APX compounds. In RNAseq analysis, the sensitive cell lines displayed enriched pathway genes and increased expression of TNFα-NFkB, epithelial to mesenchymal transition, inflammatory response and JAK/STAT3 signaling pathways as well as others, while the resistant lines were enriched for mTORC1, MYC and estrogen response pathways. Previous studies by our group have demonstrated the regulation of the JAK/STAT3 and NFkB pathways by Ref-1/APE1 and inhibition by APX compounds. To further tease out which of the downstream TFs and pathways were most dominant in the sensitivity response, we used KC3590 cells containing the KrasG12D mutation and expressing only a truncated, inactive form of NFκB. Rescue cells were created which have a full-length NFκB. When challenged with Ref-1/APE1 inhibitors APX3330, APX2009, and APX2014, cells containing the full-length, active NFκB were more sensitive to the APX compounds. To ascertain whether NFkB is the dominant TF reflecting APX inhibitor sensitivity, we studied whether knocking down RelA and STAT3, using siRNA, would alter the response of the cells to the APX compounds. STAT3 is known to be regulated by Ref-1/APE1 redox signaling. In these studies, the cellular response to APX inhibitors was unaffected following siRNA knock-down of RelA or STAT3. Overall, these experiments reveal suppression of NFκB as a primary determinant of Ref-1/APE1 cytotoxicity in KrasG12D cells. These findings allow us to begin to determine whether a specific gene signature within KRas driven PDAC tumors will inform which tumors would be more sensitive to Ref-1/APE1 redox signaling inhibitors. Citation Format: Rachel A. Caston, Randy Wireman, Lee Armstrong, Silpa Gampala, Olivia Babb, Nayela Chowdhury, Zonera Hassan, Christian Schneeweis, Gunter Schneider, Melissa L. Fishel, Mark R. Kelley. Differential sensitivity of mouse PDAC KrasG12D cells to Ref-1/APE1 redox signalling inhibitors: Role of NFkB as a primary target of Ref-1/APE1 in Kras driven pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2475.

Abstract 2467: Adiponectin receptor agonism induces a metabolic switch in pancreatic cancer cells from oxidative phosphorylation to glycolysis

Abstract Understanding the impact of risk factors on the progression of pancreatic cancer is critical for the development of novel therapeutics. Multiple studies have clearly shown that obesity positively correlates with increased progression of pancreatic adenocarcinoma (PDAC). We have demonstrated that individual adipose derived cytokines (aka adipokines) can elicit either pro-tumorigenic or anti-tumorigenic effects on PDAC progression. We previously demonstrated that adiponectin receptor agonism effectively suppresses pancreatic cancer growth in vitro as well as in vivo, through suppression of MAPK as well as STAT3 pathways. Yet, adiponectin is well known to stimulate the AMPK pathway and therefore we hypothesized that adiponectin agonism could also be affecting metabolic function in PDAC. In our current studies, we defined multiple metabolic alterations in pancreatic cancer cells in response to adiponectin receptor agonism using a seahorse-based assay. Human and murine PDAC cell lines were seeded into XFe96 microplates and subjected to a mitochondrial stress test as well as the glycolysis stress test. Our results demonstrate that adiponectin receptor activation impairs pancreatic cancer mitochondrial function leading to a metabolic switch from mitochondrial respiration to glycolysis. We further demonstrate that this switch is associated with AMPK phosphorylation-mediated inhibition of Acetyl Coenzyme A Carboxylase (ACC), the lipogenic enzyme whose inhibition induces fatty acid breakdown. Thus, our results support a role for adipose-secreted factors to influence the metabolic phenotype of pancreatic cancer. Since metabolic rewiring is often associated with development of chemotherapeutic resistance in cancer, we are currently testing whether dual therapeutic targeting of the mitochondrial respiration, via adiponectin receptor agonism, and glycolysis would provide an efficacious therapeutic. Citation Format: Sharon J. Manley, Jarrid Jack, Joseph Ambrose, Appolinaire A. Olou, Michael N. VanSaun. Adiponectin receptor agonism induces a metabolic switch in pancreatic cancer cells from oxidative phosphorylation to glycolysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2467.

Inhibition of ERAD synergizes with FTS to eradicate pancreatic cancer cells

BackgroundPancreatic ductal adenocarcinoma (PDAC), one of the most lethal cancers, is driven by oncogenic KRAS mutations. Farnesyl thiosalicylic acid (FTS), also known as salirasib, is a RAS inhibitor that selectively dislodges active RAS proteins from cell membrane, inhibiting downstream signaling. FTS has demonstrated limited therapeutic efficacy in PDAC patients despite being well tolerated.MethodsTo improve the efficacy of FTS in PDAC, we performed a genome-wide CRISPR synthetic lethality screen to identify genetic targets that synergize with FTS treatment. Among the top candidates, multiple genes in the endoplasmic reticulum-associated protein degradation (ERAD) pathway were identified. The role of ERAD inhibition in enhancing the therapeutic efficacy of FTS was further investigated in pancreatic cancer cells using pharmaceutical and genetic approaches.ResultsIn murine and human PDAC cells, FTS induced unfolded protein response (UPR), which was further augmented upon treatment with a chemical inhibitor of ERAD, Eeyarestatin I (EerI). Combined treatment with FTS and EerI significantly upregulated the expression of UPR marker genes and induced apoptosis in pancreatic cancer cells. Furthermore, CRISPR-based genetic ablation of the key ERAD components, HRD1 and SEL1L, sensitized PDAC cells to FTS treatment.ConclusionOur study reveals a critical role for ERAD in therapeutic response of FTS and points to the modulation of UPR as a novel approach to improve the efficacy of FTS in PDAC treatment.

Abstract LT009: Stromal BCAT1 drives branched-chain ketoacid dependency in stromal-rich PDAC tumours

Abstract Branched chain amino acids (BCAAs) in cancer serve as requisite precursors for protein synthesis, maintaining metabolite pools in the tricarboxylic acid (TCA) cycle, and sustaining production of nucleotides and lipids. However, the role of stromal cancer associated fibroblasts (CAFs) in support of BCAA metabolism in tumors is still poorly understood. Since most studies in pancreatic cancers have focused on systemic or cancer cell autonomous BCAA metabolism, understanding cancer-stromal ecosystem requires insight into the intersection of cancer-associated transformations in the stroma with reprogramming of their BCAA metabolism. Deciphering the precise role of various cellular components in BCAA metabolism of tumors is complicated by conflicting evidence from past studies and the challenging nature of the intricate tumor microenvironment (TME). Neither systemic in vivo BCAA metabolism nor cancer cells’ BCAA metabolism alone is sufficient to dissect the stromal role. The difficulty in understanding BCAA metabolism in the tumor milieu is exacerbated by nutrient-scarcity, exchange reactions, and metabolite sharing between cancer and stromal cells. Both, the fibrotic environment and nutrient scarcity are difficult to mimic in aggressive murine PDAC models. The metabolic fates of the BCAAs, leucine, valine, and isoleucine, are cell- and tissue-dependent. BCAA transaminases (BCAT1/2), first deaminate BCAAs to branched chain a-ketoacids (BCKAs). The second step in BCAA metabolism involves irreversible BCKA oxidation catalyzed by the mitochondrial BCKA dehydrogenase (BCKDH) complex. Further, oxidation of BCKAs results in succinyl-CoA and acetyl-CoA that act as anaplerotic or ketogenic sources for the TCA cycle. Our recent study revealed differential BCAA metabolism in cancer and stromal compartments of PDAC tumors. We identified a strikingly higher BCAA catabolic flux in CAFs but increased BCKA oxidative flux in cancer cells. Further, CAF-secreted BCKAs were used for maintaining protein synthesis, augmenting TCA cycle metabolite pools, and increasing oxidative phosphorylation in cancer cells. To corroborate the mechanistic underpinnings discovered in our human CAF and cancer cell-line model, we employed two patient-derived models: circulating tumor cells (CTCs) and tumor slice cultures. Collectively, we elucidated an undiscovered metabolic-signaling crosstalk between PDAC and stromal cells and demonstrated that targeting BCAA metabolism in PDAC tumors could mitigate PDAC aggression. Ziwen Zhu, Abhinav Achreja, Noah Meurs, Olamide Animasahun, Sarah Owen, Anjali Mittal, Pooja Parikh, TingWen Lo, Janusz Franco-Barraza, Jiaqi Shi, Mara Sherman, Edna Cuikerman, Andrew Pickering, Anirban Maitra, Vaibhav Sahai, Meredith Morgan, Sunitha Nagrath, Thedore Lawrence, Deepak Nagrath, "Tumor Reprogrammed Stromal BCAT1 Fuels Branched Chain Ketoacid Dependency in Stromal-Rich PDAC Tumors", Nature Metabolism, 2 (8), 2020. Citation Format: Deepak Nagrath. Stromal BCAT1 drives branched-chain ketoacid dependency in stromal-rich PDAC tumours [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT009.

Targeting eIF4A-Dependent Translation of KRAS Signaling Molecules.

Pancreatic adenocarcinoma (PDAC) epitomizes a deadly cancer driven by abnormal KRAS signaling. Here, we show that the eIF4A RNA helicase is required for translation of key KRAS signaling molecules and that pharmacological inhibition of eIF4A has single-agent activity against murine and human PDAC models at safe dose levels. EIF4A was uniquely required for the translation of mRNAs with long and highly structured 5' untranslated regions, including those with multiple G-quadruplex elements. Computational analyses identified these features in mRNAs encoding KRAS and key downstream molecules. Transcriptome-scale ribosome footprinting accurately identified eIF4A-dependent mRNAs in PDAC, including critical KRAS signaling molecules such as PI3K, RALA, RAC2, MET, MYC, and YAP1. These findings contrast with a recent study that relied on an older method, polysome fractionation, and implicated redox-related genes as eIF4A clients. Together, our findings highlight the power of ribosome footprinting in conjunction with deep RNA sequencing in accurately decoding translational control mechanisms and define the therapeutic mechanism of eIF4A inhibitors in PDAC. SIGNIFICANCE: These findings document the coordinate, eIF4A-dependent translation of RAS-related oncogenic signaling molecules and demonstrate therapeutic efficacy of eIF4A blockade in pancreatic adenocarcinoma.

Ion Channels Orchestrate Pancreatic Ductal Adenocarcinoma Progression and Therapy.

Pancreatic ductal adenocarcinoma is a devastating disease with a dismal prognosis. Therapeutic interventions are largely ineffective. A better understanding of the pathophysiology is required. Ion channels contribute substantially to the “hallmarks of cancer.” Their expression is dysregulated in cancer, and they are “misused” to drive cancer progression, but the underlying mechanisms are unclear. Ion channels are located in the cell membrane at the interface between the intracellular and extracellular space. They sense and modify the tumor microenvironment which in itself is a driver of PDAC aggressiveness. Ion channels detect, for example, locally altered proton and electrolyte concentrations or mechanical stimuli and transduce signals triggered by these microenvironmental cues through association with intracellular signaling cascades. While these concepts have been firmly established for other cancers, evidence has emerged only recently that ion channels are drivers of PDAC aggressiveness. Particularly, they appear to contribute to two of the characteristic PDAC features: the massive fibrosis of the tumor stroma (desmoplasia) and the efficient immune evasion. Our critical review of the literature clearly shows that there is still a remarkable lack of knowledge with respect to the contribution of ion channels to these two typical PDAC properties. Yet, we can draw parallels from ion channel research in other fibrotic and inflammatory diseases. Evidence is accumulating that pancreatic stellate cells express the same “profibrotic” ion channels. Similarly, it is at least in part known which major ion channels are expressed in those innate and adaptive immune cells that populate the PDAC microenvironment. We explore potential therapeutic avenues derived thereof. Since drugs targeting PDAC-relevant ion channels are already in clinical use, we propose to repurpose those in PDAC. The quest for ion channel targets is both motivated and complicated by the fact that some of the relevant channels, for example, KCa3.1, are functionally expressed in the cancer, stroma, and immune cells. Only in vivo studies will reveal which arm of the balance we should put our weights on when developing channel-targeting PDAC therapies. The time is up to explore the efficacy of ion channel targeting in (transgenic) murine PDAC models before launching clinical trials with repurposed drugs.

αvβ6 targeted CAR-T cells in immunocompetent orthotopic murine PDAC

αvβ6 targeted CAR-T cells in immunocompetent orthotopic murine PDAC

Abstract PO-026: The mitochondrial calcium uniporter contributes to PDAC development and invasion

Abstract Pancreatic ductal adenocarcinoma (PDAC) has an exceptionally poor prognosis, with about 50,000 new cases and 45,000 deaths each year. While novel chemotherapeutics targeted to driver pathways and immune responses have increased the overall survival rate of cancer patients in general, these improvements are rarely seen in PDAC. PDAC often metastasizes early, and metastases ultimately lead to death in many PDAC patients. A stronger understanding of disease mechanisms and new therapeutic modalities are desperately needed to improve outcomes for many patients. Previously, we showed that cancer cell lines of multiple types are “addicted” to constitutive Ca++ flow into mitochondria through the mitochondrial calcium uniporter (MCU) at endoplasmic reticulum-mitochondria contact sites. We hypothesized that mitochondrial Ca++ influx through MCU contributes to cancer cell development, proliferation, and metastasis in PDAC by promoting metabolic activity within mitochondria. We now show that mRNA expression of MCU is elevated in a subset of pancreatic cancer patients, and high MCU protein expression correlates with poor survival outcomes. In addition, MCU gene expression is associated with the expression of a number of genes which associate with metastasis and/or poor survival outcomes, including ADAM proteins, KRAS, and RAC1. To further examine the role of MCU in PDAC, we used the Pdx1cre; KrasLSL-G12D/+; p53fl/+, Rosa26LSL-YFP/LSL-YFP; Mcufl/fl (KPCY) murine model of PDAC. We generated KPCY-McuKO (knockout) animals and cell lines from their YFP-positive tissues for further analysis. Cell lines developed from KPCY-McuKO pancreatic tissues fail to take up Ca++ into mitochondria in a manner that is rescued by stable re-expression of MCU. This Ca++ uptake is associated with an increase in pyruvate dehydrogenase activation. In these cells, MCU expression is also associated with an increase in cell motility, self-renewal capacity, and cell proliferation. Re-expression of MCU in these cells is associated with a morphological change to a more fibroblastic morphology indicative of epithelial to mesenchymal transition (EMT), including decreased surface expression of e-cadherin. These findings suggest that MCU may contribute to growth and metastasis. Indeed, in an immunocompetent, syngeneic orthotopic model of murine PDAC using one of these cell lines, tumor growth and metastasis were greatly ablated. Such results suggest that MCU-mediated influx of mitochondrial Ca++ contributes to PDAC development and metastasis and may present a therapeutic target for cancer treatment. Citation Format: Jillian S. Weissenrieder, Jason R. Pitarresi, Emily Fernandez-Garcia, Ben Z. Stanger, Anil K. Rustgi, J. Kevin Foskett. The mitochondrial calcium uniporter contributes to PDAC development and invasion [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-026.

Abstract PO-023: Targeting a novel rewired pathway of nucleotide metabolism that drives chemoresistance in the most lethal molecular subtype of pancreatic cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) includes two molecular subtypes, of which the basal-like subtype is associated with the shortest survival and is highly resistant to chemotherapy. The basal-like subtype is defined by a 25-gene signature; however, the role of these genes in promoting tumor aggression remains unexplored. Here, we set out to uncover the mechanisms of chemoresistance and explore targeted therapies for this subtype. We focused on studying an oncofetal antigen, keratin 17 (K17), which is the most overexpressed hallmark gene of the basal-like PDAC. We manipulated the expression of K17 and found that in multiple in vitro and in vivo models of PDAC, spanning human and murine PDAC cells and orthotopic xenografts, K17 expression resulted in a greater than two-fold increase in resistance to Gemcitabine (Gem) and 5-fluorouracil, the major chemotherapeutic agents in standard-of-care treatments. To uncover the mechanisms associated with K17-induced chemoresistance, we performed unbiased metabolomic studies in isogenic PDAC cell lines and found that compared to control cells, K17 increases intracellular levels of deoxycytidine (dC) by four-fold that promote Gem (dC analogue) resistance. Based on previous findings that K17 enters nucleus to regulate gene expression, we explored whether K17 triggers metabolic reprogramming at the transcriptional level and found that enzymes involved in pyrimidine biosynthesis are positively correlated with K17 expression in PDAC cells. Given that it is still poorly understood how K17 regulates gene expression, we performed domain-prediction analyses. We discovered and validated a novel chromatin remodeling domain on K17 that is required for metabolic reprogramming. We are now performing ChIP-Seq and RNA-Seq to understand how this domain alters pyrimidine biosynthesis. To identify small molecules that could target K17-expressing PDACs potentially by disrupting metabolic reprograming, we performed an unbiased high-throughput drug screen and found that Podophyllotoxin (PPT), a microtubule inhibitor, significantly and selectively killed K17-positive compared to K17-negative PDAC cells. In the clinic, another microtubule inhibitor, Paclitaxel (PTX), is used in combination with Gem as a first line chemotherapy. Surprisingly, when combined with Gem, PPT but not PTX, was synergistic in inhibiting the viability of K17-expressing PDAC cells and enhanced survival of mice bearing K17-expressing PDACs. Currently, we are exploring the role of PPT in regulating pyrimidine biosynthesis. In summary, we identified a novel pathway of chemoresistance and a compound that could result in developing a biomarker-based personalized therapy. Citation Format: Chun-Hao Pan, Yuka Otsuka, BanuPriya Sridharan, Melissa Woo, Cindy V. Leiton, Sruthi Babu, Mariana Torrente Gonçalves, Ryan R. Kawalerski, Ji Dong K. Bai, Richard A. Moffitt, Jiang Zhao, David K. Chang, Andrew V. Biankin, Tim Duong, Pankaj K. Singh, Louis Scampavia, Timothy Spicer, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. Targeting a novel rewired pathway of nucleotide metabolism that drives chemoresistance in the most lethal molecular subtype of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-023.

Abstract PO-050: Solubilization of keratin 17 promotes the metastatic dissemination of the most lethal form of pancreatic cancer

Abstract CLINICAL NEED & OBJECTIVES: There is an unmet need for the discovery and development of novel therapeutic approaches for PDAC. Molecular subtyping, however, has recently uncovered potential biomarkers that, subject to further investigation, may be druggable to extend PDAC patient survival. Expression of Keratin 17 (K17), an oncofetal intermediate filament (IF) protein and hallmark of the most aggressive subtype, associates negatively with survival and drives chemotherapy resistance in PDAC. This suggests a functional role of K17 in PDAC, though the underlying mechanisms are unknown. Nuclear localization, however, has been suggested as a part of the overarching processes through which K17 promotes tumor growth. In related keratins, post-translational modifications (PTM) such as phosphorylation drive IF dissociation (solubilization) and can contribute to migration and metastatic dissemination. Thus, we hypothesized that K17 may harbor PTM- and/or solubilization-dependent oncogenic functions that could reveal new PDAC therapeutic options. METHODS & RESULTS: Using isogenic murine PDAC models, we found that K17 expression enhances tumor growth and metastatic potential. Furthermore, K17 solubility was associated with shorter survival, independent of total K17 expression or cell proliferation in both murine and human PDACs. To identify K17 PTMs potentially driving solubilization, we performed liquid chromatography mass spectrometry to sequence K17 isolated from patient PDACs and identified phosphorylation of a conserved N-terminal region (Ser 10-13) as the predominant PTMs. To determine if Ser 10-13 phosphorylation regulates K17 solubility, we generated mutants that mimic K17 Ser 10-13 phosphorylation and found that this was sufficient to solubilize K17 and promote nuclear localization. Building on these findings, we used kinase inhibitors to show that the PKC-MEK-RSK signaling pathway is involved in phosphorylation of K17 Ser 10-13. To assess the impact of K17 Ser 10-13 phosphorylation on tumors, nude mice were implanted orthotopically with isogenic human PDAC cell lines expressing either wild type (WT), loss of function (LOF) Ser 10-13 mutants, or Ser 10-13 phosphorylation gain of function (GOF) mutants. Compared to tumors expressing WT K17, GOF tumors resulted in increased metastases, and LOF mutants abrogated these characteristics. CONCLUSION: These results suggest that enhanced K17 solubility by phosphorylation at Ser 10-13 is sufficient to impact PDAC metastasis and survival and reveal novel druggable sites on K17. These results motivate future studies to uncover K17 phosphorylation targeting approaches for PDAC. If successful, they will be coupled with the identification of PDAC patients by tumor K17 expression, providing the basis of a potential precision medicine strategy. Citation Format: Ryan R. Kawalerski, Mariana T. Gonçalves, Lucia Roa-Peña, Cindy V. Leiton, Chun-Hao Pan, Luke A. Torre-Healy, Taryn Boyle, Natasha T. Snider, Kenneth R. Shroyer, Luisa F. Escobar-Hoyos. Solubilization of keratin 17 promotes the metastatic dissemination of the most lethal form of pancreatic cancer [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-050.

Fibrotic Response to Neoadjuvant Therapy Predicts Survival in Pancreatic Cancer and Is Measurable with Collagen-Targeted Molecular MRI.

To evaluate the prognostic value of posttreatment fibrosis in human PDAC patients, and to compare a type I collagen targeted MRI probe, CM-101, to the standard contrast agent, Gd-DOTA, for their abilities to identify FOLFIRINOX-induced fibrosis in a murine model of PDAC. Ninety-three chemoradiation-treated human PDAC samples were stained for fibrosis and outcomes evaluated. For imaging, C57BL/6 and FVB mice were orthotopically implanted with PDAC cells and FOLFIRINOX was administered. Mice were imaged with Gd-DOTA and CM-101. In humans, post-chemoradiation PDAC tumor fibrosis was associated with longer overall survival (OS) and disease-free survival (DFS) on multivariable analysis (OS P = 0.028, DFS P = 0.047). CPA increased the prognostic accuracy of a multivariable logistic regression model comprised of previously established PDAC risk factors [AUC CPA (-) = 0.76, AUC CPA (+) = 0.82]. In multiple murine orthotopic PDAC models, FOLFIRINOX therapy reduced tumor weight (P < 0.05) and increased tumor fibrosis by collagen staining (P < 0.05). CM-101 MR signal was significantly increased in fibrotic tumor regions. CM-101 signal retention was also increased in the more fibrotic FOLFIRINOX-treated tumors compared with untreated controls (P = 0.027), consistent with selective probe binding to collagen. No treatment-related differences were observed with Gd-DOTA imaging. In humans, post-chemoradiation tumor fibrosis is associated with OS and DFS. In mice, our MR findings indicate that translation of collagen molecular MRI with CM-101 to humans might provide a novel imaging technique to monitor fibrotic response to therapy to assist with prognostication and disease management.

Abstract 955: Long-term gemcitabine treatment reshapes the pancreatic tumor microenvironment and sensitizes murine carcinoma to combination immunotherapy

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death with a median survival time of 6-12 months. As most patients present with disseminated disease, the majority are offered palliative chemotherapy. With no approved treatment modalities for patients who progress on chemotherapy, we explored the effects of long-term Gemcitabine on the tumor microenvironment in order to identify potential therapeutic options in chemo-refractory PDAC. Using a combination of mouse models, patient-derived xenografts, primary and established cell lines, we first evaluated chemotherapy-induced alterations in the tumor secretome and immune surface proteins by high throughput proteomic arrays. In addition to enhancing antigen presentation and immune checkpoint expression, Gemcitabine consistently increased the synthesis of CCL/CXCL chemokines and TGFβ-associated signals. These secreted factors altered the composition of the tumor stroma, conferring Gemcitabine-resistance to cancer-associated fibroblasts in vitroand further enhancingTGFβ1 biosynthesis. We therefore combined Gemcitabine with anti-PD-1 in transgenic models of murine PDAC, which failed to alter disease course unless mice also underwent genetic or pharmacologic ablation of TGFβ signaling. In the setting of TGFβ signal deficiency, Gemcitabine and anti-PD-1 led to a robust CD8+ T-cell response and decrease in tumor burden, markedly enhancing overall survival. These results suggest that Gemcitabine may prime PDAC tumors for immune checkpoint inhibition by enhancing antigen presentation, but requires the inhibition of an immunosuppressive cytokine barrier. Given the current lack of third line treatment options, this approach warrants consideration in the clinical management of Gemcitabine-refractory PDAC. Citation Format: Daniel R. Principe, Matthew Narbutis, Sandeep Kumar, Alex Park, Navin Viswakarma, Matthew J. Dorman, Suneel D. Kamath, Paul Grippo, Melissa L. Fishel, Rosa F. Hwang, Dinesh Thummuri, Patrick W. Underwood, Hidayatullah G. Munshi, Jose G. Trevino, Ajay Rana. Long-term gemcitabine treatment reshapes the pancreatic tumor microenvironment and sensitizes murine carcinoma to combination immunotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 955.

Long-Term Gemcitabine Treatment Reshapes the Pancreatic Tumor Microenvironment and Sensitizes Murine Carcinoma to Combination Immunotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death with a median survival time of 6-12 months. Most patients present with disseminated disease and the majority are offered palliative chemotherapy. With no approved treatment modalities for patients who progress on chemotherapy, we explored the effects of long-term gemcitabine administration on the tumor microenvironment to identify potential therapeutic options for chemorefractory PDAC. Using a combination of mouse models, primary cell line-derived xenografts, and established tumor cell lines, we first evaluated chemotherapy-induced alterations in the tumor secretome and immune surface proteins by high throughput proteomic arrays. In addition to enhancing antigen presentation and immune checkpoint expression, gemcitabine consistently increased the synthesis of CCL/CXCL chemokines and TGFβ-associated signals. These secreted factors altered the composition of the tumor stroma, conferring gemcitabine resistance to cancer-associated fibroblasts in vitro and further enhancing TGFβ1 biosynthesis. Combined gemcitabine and anti-PD-1 treatment in transgenic models of murine PDAC failed to alter disease course unless mice also underwent genetic or pharmacologic ablation of TGFβ signaling. In the setting of TGFβ signaling deficiency, gemcitabine and anti-PD-1 led to a robust CD8+ T-cell response and decrease in tumor burden, markedly enhancing overall survival. These results suggest that gemcitabine successfully primes PDAC tumors for immune checkpoint inhibition by enhancing antigen presentation only following disruption of the immunosuppressive cytokine barrier. Given the current lack of third-line treatment options, this approach warrants consideration in the clinical management of gemcitabine-refractory PDAC. SIGNIFICANCE: These data suggest that long-term treatment with gemcitabine leads to extensive reprogramming of the pancreatic tumor microenvironment and that patients who progress on gemcitabine-based regimens may benefit from multidrug immunotherapy.See related commentary by Carpenter et al., p. 3070 GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/15/3101/F1.large.jpg.

INOS Regulates the Therapeutic Response of Pancreatic Cancer Cells to Radiotherapy.

Pancreatic ductal adenocarcinoma (PDAC) is highly resistant to radiotherapy, chemotherapy, or a combination of these modalities, and surgery remains the only curative intervention for localized disease. Although cancer-associated fibroblasts (CAF) are abundant in PDAC tumors, the effects of radiotherapy on CAFs and the response of PDAC cells to radiotherapy are unknown. Using patient samples and orthotopic PDAC biological models, we showed that radiotherapy increased inducible nitric oxide synthase (iNOS) in the tumor tissues. Mechanistic in vitro studies showed that, although undetectable in radiotherapy-activated tumor cells, iNOS expression and nitric oxide (NO) secretion were significantly increased in CAFs secretome following radiotherapy. Culture of PDAC cells with conditioned media from radiotherapy-activated CAFs increased iNOS/NO signaling in tumor cells through NF-κB, which, in turn, elevated the release of inflammatory cytokines by the tumor cells. Increased NO after radiotherapy in PDAC contributed to an acidic microenvironment that was detectable using the radiolabeled pH (low) insertion peptide (pHLIP). In murine orthotopic PDAC models, pancreatic tumor growth was delayed when iNOS inhibition was combined with radiotherapy. These data show the important role that iNOS/NO signaling plays in the effectiveness of radiotherapy to treat PDAC tumors. SIGNIFICANCE: A radiolabeled pH-targeted peptide can be used as a PET imaging tool to assess therapy response within PDAC and blocking iNOS/NO signaling may improve radiotherapy outcomes.

Macropinocytosis Renders a Subset of Pancreatic Tumor Cells Resistant to mTOR Inhibition

SummaryPancreatic ductal adenocarcinoma (PDAC) features a near-universal mutation in KRAS. Additionally, the tumor suppressor PTEN is lost in ∼10% of patients, and in mouse models, this dramatically accelerates tumor progression. While oncogenic KRAS and phosphatidylinositol 3-kinase (PI3K) cause divergent metabolic phenotypes individually, how they synergize to promote tumor metabolic alterations and dependencies remains unknown. We show that in KRAS-driven murine PDAC cells, loss of Pten strongly enhances both mTOR signaling and macropinocytosis. Protein scavenging alleviates sensitivity to mTOR inhibition by rescuing AKT phosphorylation at serine 473 and consequently cell proliferation. Combined inhibition of mTOR and lysosomal processing of internalized protein eliminates the macropinocytosis-mediated resistance. Our results indicate that mTORC2, rather than mTORC1, is an important regulator of protein scavenging and that protein-mediated resistance could explain the lack of effectiveness of mTOR inhibitors in certain genetic backgrounds. Concurrent inhibition of mTOR and protein scavenging might be a valuable therapeutic approach.