KPC Cells Research Articles

Abstract PO-110: Targeting cathepsin B in the pancreatic stellate cells stimulates CD8+ T cell dependent anti-tumor immune response

Abstract Background: Cathepsin B (CTSB) is a lysosomal cysteine protease which has been described to have a role in pancreatic cancer development. However, its significance in the tumor microenvironment has never been investigated. Pancreatic stellate cells (PSCs) are one of the main sources of Cancer Associated Fibroblasts (CAFs) in the pancreatic cancer microenvironment. Herein, we describe the effects of targeting CTSB in the pancreatic stellate cells (PSCs) in pancreatic cancer (PDAC). Methods: To specifically study the role of CTSB in PSCs, PSCs were isolated from C57BL/6J (WT) or CTSB -/- mice and were orthotopically injected with KPC cancer cells into the tail of pancreas of 6-week old female WT mice. Tumors were analyzed at endpoint using flow cytometry. To further study the dependence of effects of CTSB in PSCs on adaptive immune cells, WT or CTSB -/- PSCs were co-injected with KPC cells into Rag 1-/- mice or CD8+ T cell depleted mice (anti-CD8 mAb, 250ug/dose every 3 days). In vitro, WT or CTSB-/- PSCs were co-cultured with KPC across a transwell. CD8+ T cells, isolated from KPC tumor bearing mice, were exposed to conditioned media from this co-culture ex-vivo and their cytotoxicity was measured using calcein release assay. In another experiment, WT mice injected with KPC cancer cells were treated with vehicle control or the CTSB inhibitor, CA-074 methyl ester (10 mg/kg), through daily intraperitoneal injections. Results: Co-injections of WT PSC with KPC cells formed larger tumors as compared to KPC cancer cells alone, however, co-injection with CTSB-/- PSCs abrogated this tumor promoting effect. Flow cytometric analysis revealed increased infiltration of Ifn-γ secreting CD8+ T cells in the KPC+CB-/- PSC group as compared to KPC+WT PSC group. Tumor decreasing effect of CTSB-/- PSCs was lost when co-injections were performed in Rag 1-/- mice or CD8+ T cell depleted mice, indicating that this effect relies on a functional adaptive immune response, specifically CD8+ T cells. In vitro, co-culture of CTSB-/- PSCs with KPC led to reduced expression of inflammatory CAF (iCAF) markers like IL-6 and IL-11 when compared to co-culture of WT PSCs with KPC. CD8+ T cells exposed to conditioned media from CTSB-/- PSC co-culture showed increased cytotoxicity against KPC cells ex-vivo, as compared to those exposed to conditioned media from WT PSC co-culture. Finally, treatment with CA-074 was able to decrease orthotopic PDAC tumor growth with increased CD8+ T cell infiltration evident on flow cytometry. Conclusion: Targeting CTSB in the PSCs stimulates anti-tumor adaptive immune response against pancreatic cancer by inhibiting conversion of PSCs into the tumor-promoting iCAF phenotype. CTSB might emerge as a novel target for CAF-directed therapy in pancreatic cancer. Citation Format: Bharti Garg, Tejeshwar Jain, Utpreksha Vaish, Vikas Dudeja. Targeting cathepsin B in the pancreatic stellate cells stimulates CD8+ T cell dependent anti-tumor immune response [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-110.

Abstract PO-122: Combined CDK and BET inhibition reprograms the tumor and stromal compartments to enhance anti-tumor immunity in immunologically-cold CDKN2A-deficient pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has a highly immunosuppressive tumor microenvironment (TME). The immune landscape of these tumors is further influenced by cross-talk with fibroblasts present in stroma. Importantly, genomic features also influence immune and stromal composition within PDAC and may provide opportunity for personalized immunotherapy. Given the central role of CKDN2A in PDAC (being lost in up to half of patients, and the second most commonly alteration across all metastatic disease), we hypothesize that the CDKN2A-deficient PDAC TME can be adapted for therapeutic targeting to sensitize PDAC to immunotherapy. Methods: Bioinformatic analysis of PDAC samples from the TCGA database evaluated how CDKN2A loss alters expression of T cell-inflamed gene signatures. Preclinical in vitro and in vivo studies were conducted with CDKN2A-targeted CRISPR-Cas9-edited luciferase-expressing KPC cells (KPC-luc) to generate isogenic KPC-lucΔCKDN2A cells. The BET inhibitor JQ-1 and CDK inhibitor palbociclib were used to evaluate impact on apoptosis and immune-related signaling using isogenic KPC-luc cells and patient-derived PDAC organoids. Spheroids were used to evaluate impact of CDK/BET inhibition on cross-talk with pancreatic cancer-associated fibroblast (CAF) spheroids. Finally, in vivo studies tested the ability of CDK/BET inhibition to enhance T cell migration, and delay tumor growth when combined with PD-L1 blockade. Results: CDKN2A loss was enriched in tumors lacking a T cell-inflamed gene signature. Similarly, CDKN2A loss decreased T cell migration in vitro and into the KPC-lucΔCDKN2A TME in vivo. Loss of CDKN2A rendered KPC-luc cells susceptible to direct apoptosis from dual CDK/BET inhibition. Combined CDK/BET inhibition at sublethal doses also modulated immune phenotype of tumors, inducing DNA damage and cGAS/STING to increase type I interferon and T cell migration in vitro. Additionally, treatment with BETi alone or combined with CDKi also modulated checkpoint ligands on KPC-luc cells and patient-derived organoids. In stroma, treatment of PDAC CAFs with JQ-1 polarized CAFs towards a myofibroblast CAF (myCAF) phenotype. This could also be mediated via cross-talk between tumor and CAF cells, as JQ-1-treated KPC-lucΔCDKN2A cells or supernatants cultured with CAF spheroids also induced myCAFs. This cross-talk was bidirectional, as supernatants from BETi-treated CAFs modulated expression of checkpoint ligands on KPC-luc cells. In vivo, combined BET/CDK inhibition increased T cell infiltration into PDAC tumors, and when combined with PD-L1 blockade diminished tumor growth. Conclusion: While CDKN2A loss promotes an immunosuppressive PDAC TME, it also renders tumors susceptible to BET/CDK inhibition. This approach polarizes CAF cells towards a myCAF phenotype, directing cross-talk between tumors and stroma to promote a more immunogenic phenotype. This suggests combined CDK/BET inhibition may be a personalized therapeutic approach for patients with CKDN2A-deficient PDAC, and a means to sensitize PDAC to immunotherapy. Citation Format: Brian M. Olson, Alison J. Thomas, Michael B. Ware, Gregory B. Lesinski. Combined CDK and BET inhibition reprograms the tumor and stromal compartments to enhance anti-tumor immunity in immunologically-cold CDKN2A-deficient 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-122.

Abstract PO-127: A uPA/uPAR axis in both the tumor cell and stromal compartment drives PDAC disease progression

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal solid tumor malignancy with a 5-year survival rate of 9%. In both patients and animal models of disease, PDAC is associated with robust coagulation system activity. Intriguingly, in addition to being a rich source of procoagulant factors, PDAC tumors highly express fibrinolytic system components. Supporting this concept, urokinase plasminogen activator (uPA) and uPA receptor (uPAR) expression positively correlates with reduced overall patient survival. Here, we tested the hypothesis that the expression and activity of plasminogen activation (PA) system components are functionally linked to PDAC tumor growth and disease progression. We generated C57Bl/6-derived KPC (i.e., KRasG12D, TRP53R172H) PDAC cell lines in which uPA and uPAR were knocked out using CRISPR-Cas9. We then analyzed orthotopic tumor growth and experimental metastasis in mice carrying null or functional mutations in uPA, uPAR, or plasminogen to evaluate the interplay of PA components derived from tumor cells and/or stromal cells in mediating PDAC progression. Although both KPC cell CRISPR variants retained procoagulant function, elimination of tumor cell uPA or uPAR yielded significantly smaller tumors when compared to Cas9 control tumor cells in wildtype mice. Similarly, the growth of WT KPC tumor cells in C57Bl/6 background uPA-KO or uPAR-KO mice also resulted in reduced tumor growth. To our surprise, the metastasis potential of WT KPC tumor cells in uPA-KO or uPAR-KO mice did not change when compared to wildtype mice. Regarding to the uPA/uPAR axis downstream effector plasminogen, the growth of WT KPC tumors in plasminogen-KO mice was also significantly reduced, but not to the same extent as when eliminating uPA or uPAR. In addition, eliminating plasminogen drastically reduced WT KPC tumor cells metastasis potential. In conclusion, our data suggest a mechanism whereby uPA functions through uPAR in both the tumor cell and stromal cell compartments to promote PDAC progression through plasminogen-dependent and -independent mechanisms. Citation Format: Yi Yang, Sara R. Abrahams, Aditi Kothari, Harshi Matada, Keely Davey, Alisa S. Wolberg, Matthew J. Flick. A uPA/uPAR axis in both the tumor cell and stromal compartment drives PDAC disease progression [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-127.

Abstract PO-113: The prolyl isomerase PIN1 plays a critical role in fibroblast differentiation states to support pancreatic cancer

Abstract PIN1 is a phosphorylation-directed prolyl isomerase that alters the conformation and, therefore, the function of many proteins. PIN1 overexpression in cancer contributes to cancer cell-intrinsic phenotypes including cellular proliferation and migration. While its pro-tumor functions have generated interest in therapeutic targeting of PIN1 for cancer treatment, the effects of PIN1 inhibition on tumor-associated stromal phenotypes have not yet been studied. We assessed pancreatic cancer xenografts and genetically engineered p48-Cre; LSL-KrasG12D; p53R172H (KPC) mice that were treated with small molecule PIN1 inhibitors or crossed into a full body PIN1 knockout (Pin1−/−), and found that PIN1 inhibition or loss decreased tumor growth and extended overall survival. To interrogate a direct role for PIN1 in the stroma, we orthotopically injected a KPC cell line into syngeneic Pin1+/+ or Pin1−/− hosts and found dramatic reduction of tumor cell growth in Pin1−/− hosts. Further analysis of the Pin1−/− tumor microenvironment revealed decreased expression of alpha-SMA, a marker of myofibroblastic cancer associated fibroblasts (myCAFs), as well as decreased ECM deposition and/or organization. Pancreatic stellate cells (PSCs) activated in the tumor microenvironment play a major role in the deposition of ECM and secrete growth factors to support tumor cell proliferation and survival. We, therefore, interrogated the role of PIN1 in PSCs. We found that loss of PIN1 in PSCs inhibits TGF-beta-induced stellate cell activation into a myofibroblast phenotype. Single cell ATAC-seq analysis demonstrated that a subset of TGF-beta responsive changes to chromatin accessibility are impaired in the absence of PIN1, and suggests that specific transcription factor families may play a role in the PIN1-dependent response to TGF-beta. Further analysis of PSCs or CAFs with PIN1 loss indicated that, at baseline, these cells express gene programs consistent with the recently described antigen presenting CAFs (apCAFs). Finally, in addition to changes in cellular state and plasticity, we found that loss of PIN1 alters PSC secretion of paracrine factors that support oncogenic phenotypes. For example, PSCs with loss of PIN1 have reduced expression of HGF and increased expression of VEGF, resulting in altered cancer cell and vascular phenotypes. This work establishes a role for PIN1 in regulating fibroblast function and suggests that targeting PIN1 in cancer will have a broad anti-tumor effect. Our ongoing work continues to use 2D co-cultures, heterotypic 3D bioprinted tissues, and in vivo mouse models to interrogate the precise mechanisms by which PIN1 controls fibroblast phenotypes and impact of these changes on tumor phenotypes and outcomes. Citation Format: Ellen M. Langer, Isabel A. English, Vidhi Shah, Kevin MacPherson, Kayleigh M. Kresse, Brittany L. Allen-Petersen, Colin J. Daniel, Mara H. Sherman, Andrew Adey, Rosalie C. Sears. The prolyl isomerase PIN1 plays a critical role in fibroblast differentiation states to support 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-113.

Comparative Untargeted Metabolomic Profiling of Induced Mitochondrial Fusion in Pancreatic Cancer.

Mitochondria are dynamic organelles that constantly alter their shape through the recruitment of specialized proteins, like mitofusin-2 (Mfn2) and dynamin-related protein 1 (Drp1). Mfn2 induces the fusion of nearby mitochondria, while Drp1 mediates mitochondrial fission. We previously found that the genetic or pharmacological activation of mitochondrial fusion was tumor suppressive against pancreatic ductal adenocarcinoma (PDAC) in several model systems. The mechanisms of how these different inducers of mitochondrial fusion reduce pancreatic cancer growth are still unknown. Here, we characterized and compared the metabolic reprogramming of these three independent methods of inducing mitochondrial fusion in KPC cells: overexpression of Mfn2, genetic editing of Drp1, or treatment with leflunomide. We identified significantly altered metabolites via robust, orthogonal statistical analyses and found that mitochondrial fusion consistently produces alterations in the metabolism of amino acids. Our unbiased methodology revealed that metabolic perturbations were similar across all these methods of inducing mitochondrial fusion, proposing a common pathway for metabolic targeting with other drugs.

Abstract 1757: IFN-γ signaling in myeloid and fibroblastic cells regulates pancreatic cancer growth and metastasis

Abstract Pancreatic ductal adenocarcinoma (PDAC) represents a significant healthcare problem which is on the rise, primarily because of the late stage diagnosis, early metastasis, and rapidly emerging resistance to therapy. As cancer cells in a typical pancreatic tumor are outnumbered by stromal and immune cells, the role of tumor microenvironment in cancer growth and progression is essential. One of the key regulators of tumor microenvironment in numerous cancers is IFN-γ signaling pathway, which is implicated into regulation of multiple cell types. Using a genetic murine model with conditional IFN-γR2 deficiency in myeloid cells, we performed an orthotopic injection of cell line derived from Pdx1Cre-Kras-P53 model (“KPC” cells). The absence of IFN-γ signaling in myeloid cells led to an increase of tumor burden and size of tumors in these mice. Further analysis demonstrated that IFN-γR2 deletion in myeloid cells changes the differentiation of cancer cells in primary tumors, resulting in a loss of characteristic histological pattern and shifting it towards more aggressive phenotype. Subsequent intrasplenic injection/liver spread model revealed that loss of IFN-γR2 in myeloid cells led to increased metastatic outgrowth of PDAC KPC cells. This effect was partially dependent on the gut microbiota. Deficiency of IFN-γR2 in myeloid cells increases recruitment and infiltration of monocytes in normal and tumor liver tissue. Meanwhile, deletion of IFN-γR2 in several subsets of cancer-associated fibroblasts demonstrated that IFN-γ signaling in different subsets of fibroblast/myofibroblasts may play opposing role in PDAC tumor growth. Our data identifies cell type specific IFN-γ signaling pathway as an important regulator of pancreatic cancer progression and metastasis, that can be potentially exploited as a novel immunooncology target or a biomarker of the disease progression. Citation Format: Elena Ivleva, Ashley Crisostomo, Natalia Andreeva, Sergei Grivennikov. IFN-γ signaling in myeloid and fibroblastic cells regulates pancreatic cancer growth and metastasis [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 1757.

Deuterium MRSI characterizations of glucose metabolism in orthotopic pancreatic cancer mouse models.

Detecting and mapping metabolism in tissues represents a major step in detecting, characterizing, treating and understanding cancers. Recently introduced deuterium metabolic imaging techniques could offer a noninvasive route for the metabolic imaging of animals and humans, based on using 2 H magnetic resonance spectroscopic imaging (MRSI) to detect the uptake of deuterated glucose and the fate of its metabolic products. In this study, 2 H6,6' -glucose was administered to mice cohorts that had been orthotopically implanted with two different models of pancreatic ductal adenocarcinoma (PDAC), involving PAN-02 and KPC cell lines. As the tumors grew, 2 H6,6' -glucose was administered as bolii into the animals' tail veins, and 2 H MRSI images were recorded at 15.2T. 2D phase-encoded chemical shift imaging experiments could detect a signal from this deuterated glucose immediately after the bolus injection for both the PDAC models, reaching a maximum in the animals' tumors ~ 20 min following administration, and nearly total decay after ~ 40 min. The main metabolic reporter of the cancers was the 2 H3,3' -lactate signal, which MRSI could detect and localize on the tumors when these were 5mm or more in diameter. Lactate production time traces varied slightly with the animal and tumor model, but in general lactate peaked at times of 60 min or longer following injection, reaching concentrations that were ~ 10-fold lower than those of the initial glucose injection. This 2 H3,3' -lactate signal was only visible inside the tumors. 2 H-water could also be detected as deuterated glucose's metabolic product, increasing throughout the entire time course of the experiment from its ≈10 mM natural abundance background. This water resonance could be imaged throughout the entire abdomen of the animals, including an enhanced presence in the tumor, but also in other organs like the kidney and bladder. These results suggest that deuterium MRSI may serve as a robust, minimally invasive tool for the monitoring of metabolic activity in pancreatic tumors, capable of undergoing clinical translation and supporting decisions concerning treatment strategies. Comparisons with in vivo metabolic MRI experiments that have been carried out in other animal models are presented and their differences/similarities are discussed.

TP53 missense mutations in PDAC are associated with enhanced fibrosis and an immunosuppressive microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal cancer, which is refractory to all currently available treatments and bears dismal prognosis. About 70% of all PDAC cases harbor mutations in the TP53 tumor suppressor gene. Many of those are missense mutations, resulting in abundant production of mutant p53 (mutp53) protein in the cancer cells. Analysis of human PDAC patient data from The Cancer Genome Atlas (TCGA) revealed a negative association between the presence of missense mutp53 and infiltration of CD8+ T cells into the tumor. Moreover, CD8+ T cell infiltration was negatively correlated with the expression of fibrosis-associated genes. Importantly, silencing of endogenous mutp53 in KPC cells, derived from mouse PDAC tumors driven by mutant Kras and mutp53, down-regulated fibrosis and elevated CD8+ T cell infiltration in the tumors arising upon orthotopic injection of these cells into the pancreas of syngeneic mice. Moreover, the tumors generated by mutp53-silenced KPC cells were markedly smaller than those elicited by mutp53-proficient control KPC cells. Altogether, our findings suggest that missense p53 mutations may contribute to worse PDAC prognosis by promoting a more vigorous fibrotic tumor microenvironment and impeding the ability of the immune system to eliminate the cancer cells.

Abstract LT012: The prolyl isomerase PIN1 plays a critical role in fibroblast plasticity to impact pancreatic cancer

Abstract PIN1 is a phosphorylation-directed prolyl isomerase that alters the conformation and, therefore, the function of many proteins. Due to its role in activation and stabilization of many oncogenes, we hypothesized that targeting PIN1 in pancreatic ductal adenocarcinoma (PDA) would slow tumor growth. We tested this hypothesis in vitro and in vivo with PIN1 inhibitors and/or genetic model systems. Pancreatic cancer cell lines knocked down for PIN1 or treated with PIN1 inhibitors showed decreased proliferation, invasion, and anchorage independent growth compared to control lines. Consistent with these in vitro results, treatment of pancreatic cancer xenografts or genetically engineered p48-Cre; LSL-KrasG12D; p53R172H (KPC) mice with PIN1 inhibitors decreased tumor growth and extended overall survival. Similar results were seen in KPC mice that were crossed into a full body PIN1 knockout (PIN1−/−). Further analysis of KPC PIN1−/− tumors revealed not only reduced size of pancreatic tumors, but also decreased alpha-SMA expression and decreased ECM deposition in the stroma surrounding the tumors. PDA is characterized by a dense, desmoplastic tumor stroma that contributes to tumor growth, metastasis, and therapeutic resistance. Pancreatic stellate cells (PSCs) that are activated in the tumor microenvironment play a major role in the deposition of ECM and secrete growth factors to support tumor cell proliferation and survival. To interrogate a direct role for PIN1 in the stroma, we first orthotopically injected a KPC cell line into syngeneic PIN1+/+ or PIN1−/− mice and found dramatic reduction of tumor cell growth in PIN1−/− hosts. Next, we analyzed PSCs in vitro and found that loss of PIN1 reduces their proliferation and alters their secretion of paracrine factors that support oncogenic phenotypes. For example, PSCs with loss of PIN1 have reduced expression of HGF and increased expression of SPINT1 and SPINT2, inhibitors of HGF activation. Conditioned media from control PSCs, but not from PSCs lacking PIN1 expression, activates the MET receptor on cancer cell lines, resulting in altered cancer cell phenotypes. In addition, we show that loss of PIN1 in PSCs inhibits TGF-beta induced stellate cells activation into a myofibroblast phenotype. Single cell ATAC-seq analysis demonstrated that a subset of TGF-beta responsive chromatin changes are impaired in the absence of PIN1. Our ongoing work utilizes 2D co-cultures, heterotypic 3D bioprinted tissues, and in vivo mouse models to interrogate the mechanisms by which fibroblast phenotypes and the tumor-stromal crosstalk is impacted by PIN1. Citation Format: Ellen M. Langer, Isabel A. English, Kayleigh M. Kresse, Kevin MacPherson, Brittany L. Allen-Petersen, Colin J. Daniel, Andrew Adey, Rosalie C. Sears. The prolyl isomerase PIN1 plays a critical role in fibroblast plasticity to impact pancreatic cancer [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 LT012.

Lipid droplet storage promotes murine pancreatic tumor growth.

Hypoxia Inducible Lipid Droplet Associated (HILPDA) is frequently overexpressed in tumors and promotes neutral lipid storage. The impact of Hilpda on pancreatic ductal adenocarcinoma (PDAC) tumor growth is not known. In order to evaluate Hilpda‑dependent lipid storage mechanisms, expression of Hilpda in murine pancreatic cells(KPC) was genetically manipulated. Lipid droplet(LD) abundance and triglyceride content invitro were measured, and model tumor growth in nu/nu mice was determined. The results showed that excess lipid supply increased triglyceride storage and LD formation in KPC cells in a HILPDA‑dependent manner. Contrary to published results, inhibition of Adipose Triglyceride Lipase (ATGL) did not ameliorate the triglyceride abundance differences between Hilpda WT and KO cells. Hilpda ablation significantly decreased the growth rate of model tumors in immunocompromised mice. In conclusion, Hilpda is a positive regulator of triglyceride storage and lipid droplet formation in murine pancreatic cancer cells invitro and lipid accumulation and tumor growth invivo. Our data suggest that deregulated ATGL is not responsible for the absence of LDs in KO cells in this context.

Inhibition of de novo pyrimidine synthesis augments Gemcitabine induced growth inhibition in an immunocompetent model of pancreatic cancer.

Leflunomide (Lef) is an agent used in autoimmune disorders that interferes with DNA synthesis. De Novo pyrimidine synthesis is a mechanism of Gemcitabine (Gem) resistance in pancreatic cancer. This study aims to assess the efficacy and changes in the tumor microenvironment of Lef monotherapy and in combination with Gem, in a syngeneic mouse model of pancreatic cancer.Methods: MTS proliferation assays were conducted to assess growth inhibition by Gem (0-20 nM), Lef (0-40 uM) and Gem+Lef in KPC (KrasLSL.G12D/+;p53R172H/+; PdxCretg/+) cells in vitro. An in vivo heterotopic KPC model was used and cohorts were treated with: PBS (control), Gem (75 mg/kg/q3d), Lef (40 mg/kg/d), or Gem+Lef. At d28 post-treatment, tumor burden, proliferation index (Ki67), and vascularity (CD31) were measured. Changes in the frequency of peripheral and intratumoral immune cell subsets were evaluated via FACS. Liquid chromatography-mass spectrometry was used for metabolomics profiling.Results: Lef inhibits KPC cell growth and synergizes with Gem in vitro (P<0.05; Combination Index 0.44 (<1 indicates synergy). In vivo, Lef alone and in combination with Gem delays KPC tumor progression (P<0.001). CTLA-4+T cells are also significantly decreased in tumors treated with Lef, Gem or in combination (Gem+Lef) compared to controls (P<0.05). Combination therapy also decreased the Ki67 and vascularity (P<0.01). Leflunomide inhibits de novo pyrimidine synthesis both in vitro (p<0.0001) and in vivo (p<0.05).Conclusions: In this study, we demonstrated that Gem+Lef inhibits pancreatic cancer growth, decrease T cell exhaustion, vascularity and as proof of principle inhibits de novo pyrimidine synthesis. Further characterization of changes in adaptive immunity are necessary to characterize the mechanism of tumor growth inhibition and facilitate translation to a clinical trial.

Abstract PO-032: Targeting the KRAS dimerization interface enhances T-cell mediated anti-tumor response in vivo

Abstract RAS is the most frequently mutated human oncogene and about 20% of all human cancers harbor mutations in one of three RAS oncogenes (K, N and H-RAS) with pancreatic cancers harboring RAS mutations in &amp;gt;90% of tumors. Therapeutically targeting RAS has been highly challenging and other than allele specific KRAS (G12C) inhibitors, no direct RAS inhibitor has been approved for clinical trial. However, KRAS (G12C) mutations are less abundant in some cancers like pancreatic cancer, thus there remains an unmet need for inhibitors that target the more common RAS mutants. This demand devising novel strategies to identify new approaches to inhibit RAS. Previously, we developed the H/KRAS-specific monobody called NS1 that allosterically inhibit RAS-mediated signaling by targeting the α4-α5 dimerization interface. When used as a genetically encoded intracellular reagent, NS1 inhibited the growth of H/KRAS-mutant human tumor lines both in vitro and in xenograft tumor models. However, these tumor models lack a functional immune system. Here, we evaluated the potency of targeting KRAS dimerization interface with NS1 in an immune-competent murine model of pancreatic cancer. Although human and murine KRAS differ by a single amino acid [Asp (D) to Glu (E)] at position 132 in the NS1 binding region of the allosteric lobe, NS1 bound similarly to human and murine KRAS both in vitro and in vivo. Furthermore, NS1 expression inhibited ERK-MAPK activation, proliferation and anchorage-independent growth of engineered KPC cells derived from pancreatic tumor in the KPC genetic mouse model. Finally, when these engineered KPC cells were injected orthotopically in immune-competent C57/B6 mice, we observed a dramatic decrease in tumor growth kinetics and tumor burden in NS1 expressing cohorts. Similarly, NS1 expression resulted in decreased ERK-MAPK activation and increased apoptosis. NS1 expressing tumors were characterized by increased infiltration of helper CD4+ T cells and macrophage. Thus, inhibition of mutant KRAS by NS1 results in an enhanced anti-tumor immune response in a syngeneic model for pancreatic cancer. These results establish that targeting α4-α5 dimerization interface of RAS can be a viable therapeutic option for targeting KRAS-mutant pancreatic cancer. Citation Format: Imran Khan, Julia Lefler, Catherine Marelia, Mariyam Zuberi, Eric Denbaum, Akiko Koide, Cynthia Timmers, Michael Ostrowski, Shohei Koide, John O’Bryan. Targeting the KRAS dimerization interface enhances T-cell mediated anti-tumor response in vivo [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-032.

Abstract 5160: A GPCR candidate in pancreatic ductal adenocarcinoma: A potential repurposing opportunity

Abstract The 5-year survival of patients with pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer (90%), is very low (~9% all stages). PDAC thus requires new, effective and safe therapies. By comparing transcriptomic data for PDAC (in The Cancer Genome Atlas) with that of normal pancreas (in the GTEx database), we discovered that many G protein-coupled receptors (GPCRs) are increased in expression in PDAC. One such GPCR is the histamine receptor H1 (HRH1). GPCRs regulate metabolism, growth/death and functional activities of normal and cancer cells, including features of the malignant phenotype. GPCRs are the largest family of targets of approved drugs but have been largely ignored in cancer therapy. We found that HRH1 is prominently (~32-fold) overexpressed in PDAC tumors compared to normal pancreatic tissue and that high HRH1 expression is an unfavorable prognostic marker for PDAC patients. HRH1 mRNA is also highly expressed in multiple human PDAC cell lines. Studies with such cell lines revealed that histamine, acting via HRH1, increases intracellular Ca2+, cell proliferation and migration, effects blocked by multiple FDA-approved HRH1 antihistamines. PDAC tumors and cancer cells isolated from KPC mice also have high HRH1 expression compared to normal mouse pancreas. Similar to human PDAC cells, the KPC cells showed histamine/HRH1-promoted increase in intracellular Ca2+. Altogether, our findings identify HRH1 as a prominently overexpressed, functional GPCR in human and mouse PDAC tumors and cells. HRH1 may thus be a novel therapeutic target; its blockade by FDA-approved antihistamines represents a novel potential repurposing approach for the treatment of PDAC. Citation Format: Cristina Salmerón, Krishna Sriram, Alyssa Baird, Paul A. Insel. A GPCR candidate in pancreatic ductal adenocarcinoma: A potential repurposing opportunity [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 5160.

Abstract 5571: Turning up the heat on pancreatic ductal adenocarcinoma via inhibiting vasoactive intestinal peptide signaling

Abstract Pancreatic ductal adenocarcinoma (PDAC) constitutes more than 90% of all pancreatic cancers and has a 5-year survival rate of only 5-7%. A dense stroma, an immunosuppressive tumor microenvironment (TME) and scarce tumor infiltrating T cells, contribute toward the poor responsiveness of PDAC to conventional and FDA-approved checkpoint therapies. In this study, we show that vasoactive intestinal peptide (VIP) signaling is a novel immune checkpoint pathway in PDAC, which when inhibited in combination with anti-PD1 checkpoint inhibitor, significantly decreases tumor burden and enhances T cell infiltration in mouse models of PDAC. VIP is a 28-amino acid neuropeptide secreted at the nerve terminals, pancreas and the GI tract, and is commonly associated with regulating gut motility and blood pressure. Interestingly, VIP is also secreted by immune cells and is known to have immunosuppressive properties such as decreasing T cell proliferation and polarizing T cells towards Th2 phenotype. Intriguingly, according to the cancer genome atlas (TCGA), human exocrine pancreatic cancers expresses the highest level of VIP mRNA expression when compared to other solid malignancies. Similarly, we have generated preliminary data that shows enhanced VIP levels both systemically and locally in mouse models of PDAC and in human PDAC patients. Thus, taking all this into consideration, we hypothesized that enhanced VIP signaling is a potential mechanism of immune escape in PDAC and that inhibiting VIP signaling either alone or in combination with checkpoint inhibitors would significantly enhance treatment response in mouse models of PDAC. We tested the effect of inhibiting VIP signaling by using a VIP receptor antagonist (VIPR antagonist) in orthotopic mouse models of PDAC, where luciferase transfected KPC cells (mouse PDAC cell line), were implanted in the tail of the pancreas. Mice were then treated with VIPR antagonist and/or anti-PD1 seven days after tumor implantation. Our results showed that the combination treatment significantly reduced tumor burden and growth rate, as well as, enhanced the infiltration of both CD4+ and CD8+ T cells into the tumor microenvironment (as per immunohistochemistry of tumor tissues). Furthermore, analysis of tumor infiltrating T cells via Nanostring analysis showed that the combination treatment also significantly enhanced levels of IFN gamma and TNF alpha secreting T cells and costimulatory and effector molecules, including CD27 and FasL. In summary, we have identified that VIP signaling is a novel and targetable immune checkpoint pathway in PDAC, which when inhibited in combination with checkpoint inhibitors, significantly improves treatment response in mouse PDAC models. As the VIP sequence is conserved across different species including mouse and human, and since we have observed VIPR antagonists stimulate proliferation of human T cells, there is an increased potential for clinical translation of VIPR antagonists in the treatment of human PDAC. Citation Format: Sruthi Ravindranathan, Brandon Ware, Mohammad Zaidi, Jingru Zhu, Rohan K. Dhamsania, Shuhua Wang, Yiwen Li, Ishani H. Rao, Susan N. Thomas, Gregory B. Lesinski, Bassel El-Rayes, Edmund K. Waller. Turning up the heat on pancreatic ductal adenocarcinoma via inhibiting vasoactive intestinal peptide signaling [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 5571.

Abstract 5102: Gemcitabine and nab-paclitaxel increase immunosuppressive environment in PDAC

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) recently became the third-deadliest cancer due to its resistance to effective therapies. The tumor microenvironment (TME) in PDAC is thought to contribute to this resistance, with up to 80% of the tumor bulk consisting of stroma including cancer associated fibroblasts (CAFs) and tumor-associated macrophages (TAMs). Gemcitabine (gem) and nab-paclitaxel (nab-p) is standard of care for PDAC with modest efficacy. Immunotherapies, effective in other cancers, are often combined with chemotherapy in an attempt to augment tumor response. We investigated the impact of gem and nab-p on the TME and antigen presentation of PDAC. Methods: A cancer organoid line from KPC (LSL-KrasG12D/+;LSL-Trp53R172H/+;Pdx-1-Cre) mice, was used as a murine model of PDAC. The organoids were treated in vitro with 50uM gem and 5.85uM nab-p for 12-24 hrs and subsequently tested for MHC class I expression via flow cytometry. A co-culture in Matrigel was created using KPC organoids, pro-tumor M2-polarized bone-marrow derived macrophages (BMDMs), and pancreatic stellate cells (PSCs). After 48 hrs the co-culture was treated with gem and nab-p for 24 hrs and collected to assess macrophage and fibroblast polarization markers via flow cytometry. KPC organoids were used to generate allografts in wild-type B6 mice randomized to receive 100 mg/kg gem (i.p.) and 30 mg/kg nab-p (i.v.) or control. After 96 hrs, tumors were processed for flow cytometry analyses. Results: The percentage of MHC I expressing KPC cells increased from baseline following 12 hrs of gem/nab-p treatment (32.7% vs 50.4%, p= 0.12 and declined at 24, 48 and 72 hour post-treatment time points (22.5%, p=0.3, 23.2%, p=0.31, and 13.1%, p=0.06, respectively). In the co-culture, gem and gem/nab-p significantly enhanced immunoregulatory inflammatory CAF (iCAF) subtype over control (17.5 vs 17.6 vs 10%, respectively, p&amp;lt;0.001). A decrease in myofibroblastic CAFs (myCAFs) was also observed. M1 macrophages in the co-culture decreased significantly with gem and gem/nab-p compared to controls (6.7% vs 6.9% vs 13.8%, respectively, p=0.02/p=0.004). There was also a subsequent increase in M2 macrophages. KPC allografts demonstrated no significant change in MHC I levels following a 96-hour gem/nab-p treatment compared to vehicle. The CAF subtypes showed trends similar to the in vitro co-culture, with more iCAFs (14.9% vs 26.3%, p=0.32) and less myCAFs (83.5% vs 71.3%, p=0.31). We were also able to identify an MHC II class of antigen-presenting CAFs (apCAFs), within both our in vitro and in vivo studies at levels less than 1% of all CAFs. However, no change was observed with treatment. Conclusion: Here we demonstrate reduced MHC class I expression, increased M2 TAM polarization, and increased iCAFs, all associated with an immunoregulatory phenotype. These finding suggest that further studies on alternative combinations of immunotherapies are warranted. Citation Format: Hanna R. Rainiero, Philip B. Emmerich, Nathaniel Verhagen, Rebecca Destefanis, Cheri A. Pasch, Linda Clipson, Kristina A. Matkowskyj, Dustin A. Deming. Gemcitabine and nab-paclitaxel increase immunosuppressive environment in PDAC [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 5102.

ZNF251 promotes the progression of lung cancer by activating ERK signaling.

Aberrant activation of ERK signaling is a hallmark of lung cancer. Although constitutively activating mutations of EGFR and KRAS contribute to the hyperactivation of ERK1/2, other mechanisms remain elusive. In this study, the zinc finger protein ZNF251 was found to be upregulated in clinical lung cancer samples, and it promoted the growth of lung cancer cells and the growth of primary lung KPC cells from mouse models (Ad‐Cre, KrasG12D, and P53f/f). In studying the molecular mechanism, ZNF251 was found to inhibit the expression of dual‐specificity phosphatase 6, a negative regulator of ERK activation, by directly binding to its promoter region. Taken together, our data indicate the tumor‐promoting effects of ZNF251 in lung cancer and suggest that ZNF251 is a therapeutic target.

High expression of AMAP1, an ARF6 effector, is associated with elevated levels of PD-L1 and fibrosis of pancreatic cancer

BackgroundNot merely the onset of immune evasion, but other factors, such as acidosis and fibrosis, are also major barriers in cancer therapeutics. Dense fibrosis is a hallmark of pancreatic ductal carcinoma (PDAC), in which hyperactivation of focal adhesion kinase (FAK) in tumor cells was shown to be crucial. Double mutations of KRAS/ TP53 are characteristic to PDAC. We previously showed that high protein expression of ARF6 and its downstream effector AMAP1, as well as processes involved in the ARF6 activation by cell surface tyrosine kinase receptors, are major targets of the KRAS/TP53 mutations to promote PDAC invasion, metastasis, and immune evasion. This notion was recaptured by KPC mouse model of human PDAC (LSL-Kras(G12D/+); LSL-Trp53(R172H/+)); Pdx-1-Cre). Mechanistically, the ARF6-AMAP1 pathway is primarily involved in cellular dynamics of PD-L1, β1-integrins, and E-cadherin; and hence modulates cell-adhesion properties when ARF6 is activated. Here, with an aim to understand whether the ARF6-AMAP1 pathway is critically involved in the elevated levels of PD-L1 and fibrosis of PDAC, we analyzed relationship between AMAP1 and these malignant phenotypes. Moreover, because the ARF6 pathway may closely be related to focal adhesion dynamics and hence to FAK, we also investigated whether AMAP1 employs FAK in fibrosis.MethodsClinical specimens, as well as KPC cells/tumors and their shAMAP1 or shFAK derivatives were analyzed.ResultsElevated levels of PD-L1 and fibrosis correlated with poor outcome of our patient cohort, to be consistent with previous reports; in which high AMAP1 expression statistically correlated with the elevated PD-L1 and fibrosis. To be consistent, silencing of AMAP1 (shAMAP1) in KPC cells resulted in reduced PD-L1 expression and fibrosis in their tumors. On the other hand, shAMAP1 only slightly affected FAK activation in KPC cells, and phosphorylated FAK did not correlate with enhanced fibrosis or with poor outcome of our patients.ConclusionsTogether with our previous data, our results collectively indicated that the ARF6-AMAP1 pathway, empowered by the KRAS/TP53 mutations, is closely associated with elevated PD-L1 expression and fibrosis of human PDACs, to be recaptured in the KPC mouse model. The ARF6 pathway may promote fibrosis independent of FAK.2ZgVZH5hf3HfReDrMoiYXhVideo abstract

Nano-delivery of Gemcitabine Derivative as a Therapeutic Strategy in a Desmoplastic KRAS Mutant Pancreatic Cancer.

Pancreatic ductal adenocarcinoma remains one of the challenging malignancies to treat, and chemotherapy is the primary treatment strategy available to most patients. Gemcitabine, one of the oldest chemotherapeutic drugs approved for pancreatic cancer, has limited efficacy, due to low drug distribution to the tumor and chemoresistance following therapy. In this study, we delivered gemcitabine monophosphate using lipid calcium phosphate nanoparticles, to desmoplastic pancreatic tumors. Monophosphorylation is a critical, rate-limiting step following cellular uptake of gemcitabine and precursor of the pharmacologically active gemcitabine triphosphate. Our drug delivery strategy enabled us to achieve robust tumor regression with a low parenteral dose in a clinically relevant, KRAS mutant, syngeneic orthotopic allograft, lentivirus-transfected KPC cell line-derived model of pancreatic cancer. Treatment with gemcitabine monophosphate significantly increased apoptosis of cancer cells, enabled reduction in the proportion of immunosuppressive tumor-associated macrophages and myeloid-derived suppressor cells, and did not increase expression of cancer stem cell markers. Overall, we could trigger a strong antitumor response in a treatment refractory PDAC model, while bypassing critical hallmarks of gemcitabine chemoresistance.

An Immunocompetent Model of Pancreatic Cancer Resection and Recurrence

BackgroundEven after surgical resection, most patients with localized pancreatic ductal adenocarcinoma (PDAC) succumb to disease recurrence. Current animal models do not recapitulate this pattern of disease recurrence. Our goal was to develop a clinically relevant, immunocompetent model of PDAC resection to study recurrence and evaluate therapy. MethodsPancreatic cancer cells derived from tumors arising in KPC (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) mice were co-injected with stromal cells (pancreatic stellate cells) into the pancreas of immunocompetent mice to simulate the stroma-rich tumors seen in human PDAC. After allowing tumors to form, we resected these localized tumors and followed the mice for tumor recurrence. Circulating tumor cells (CTCs) were isolated, and systemic chemotherapy or immunotherapy was administered following tumor resection. ResultsTumors formed by co-injection of KPC cells and stromal cells demonstrated a dense desmoplastic reaction similar to that seen in human disease. Resection at days 15 and 21 after implantation revealed uniform tumor volumes of 92 ± 19 mm3 on day 15 and 444 ± 54 mm3 on day 21. Histology of resected tumors showed negative margins. Resembling human PDAC, mice that underwent resection showed improved median survival (58 vs 47 days) but most animals developed intra-abdominal recurrence on follow-up. Adjuvant chemotherapy (median survival 69 vs 58 days), but not immunotherapy (median survival 69 vs 65 days) tended towards improved survival as seen in human disease. Circulating tumor cells were reliably identified from mice with and without resection, suggesting utility of this model in studying tumor metastases and recurrence. ConclusionWe describe an immunocompetent animal model that recapitulates human disease in morphology and recurrence patterns. We show that it can be used to evaluate therapy in clinical scenarios associated with surgical resection and may help characterize factors responsible for disease recurrence.

Arid5a orchestrates immune evasion of pancreatic cancer via the Ido1-–mediated tryptophan metabolism

Abstract Although pancreatic ductal adenocarcinoma (PDAC) is known to be “immunologically cold” tumors, in which the presence of immune cells and their activity are limited by the tumor microenvironment, the immunological nature of this cancer is still largely unknown. AT-rich interaction domain-containing protein 5a (Arid5a) was identified as an RNA-binding protein that plays significant roles in the development of inflammatory diseases via the augmentation of IL-6 signaling. However, the role of Arid5a in cancer remains elusive. To clarify the role of Arid5a in cancer, we used the well-established mouse PDAC model KPC cells, bearing KRAS/TP53 mutations. We found that although KPC cells lacking Arid5a (Arid5a-KO) had similar growth rates as wild-type (WT) KPC cells when subcutaneously inoculated into immunodeficient mice, Arid5a-KO cells had remarkably impaired their growth in immunocompetent mice. Here, we show that Arid5a promotes immunosuppressive tumor microenvironment by inducing the infiltration of regulatory T cells and granulocytic myeloid-derived suppressor cells; and inhibiting the activation of CD8+T lymphocytes. Interestingly, we observed that Arid5a-KO KPC cells had significantly lower expression of indoleamine 2,3-dioxygenase 1 (Ido1) compared with WT KPC cells. Mechanistically, we identified that Arid5a stabilizes Ido1 mRNA through binding to its 3′-untranslated region, and augments the production of kynurenine, which is an immunoregulatory metabolite of Ido1-mediated tryptophan catabolism. Our findings indicate Arid5a as a crucial molecule involved in the immune evasion of PDAC which can be exploited for immunotherapy in malignant pancreatic cancer.