Human Pancreatic Ductal Adenocarcinoma Research Articles

Brg1 controls stemness and metastasis of pancreatic cancer through regulating hypoxia pathway.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease. We previously reported that chromatin remodeler Brg1 is essential for acinar cell-derived PDAC formation in mice. However, the functional role of Brg1 in established PDAC and its metastasis remains unknown. Here, we investigated the importance of Brg1 for established PDAC by using a mouse model with a dual recombinase system. We discovered that Brg1 was a critical player for the cell survival and growth of spontaneously developed PDAC in mice. In addition, Brg1 was essential for metastasis of PDAC cells by inhibiting apoptosis in splenic injection and peritoneal dissemination models. Moreover, cancer stem-like property was compromised in PDAC cells by Brg1 ablation. Mechanistically, the hypoxia pathway was downregulated in Brg1-deleted mouse PDAC and BRG1-low human PDAC. Brg1 was essential for HIF-1α to bind to its target genes to augment the hypoxia pathway, which was important for PDAC cells to maintain their stem-like properties and to metastasize to the liver. Human PDAC cells with high BRG1 expression were more susceptible to BRG1 suppression. In conclusion, Brg1 plays a critical role for cell survival, stem-like property and metastasis of PDAC through the regulation of hypoxia pathway, and thus could be a novel therapeutic target for PDAC.

Resistance to Gemcitabine in Pancreatic Cancer Is Connected to Methylglyoxal Stress and Heat Shock Response

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor prognosis. Gemcitabine is the first-line therapy for PDAC, but gemcitabine resistance is a major impediment to achieving satisfactory clinical outcomes. This study investigated whether methylglyoxal (MG), an oncometabolite spontaneously formed as a by-product of glycolysis, notably favors PDAC resistance to gemcitabine. We observed that human PDAC tumors expressing elevated levels of glycolytic enzymes together with high levels of glyoxalase 1 (GLO1), the major MG-detoxifying enzyme, present with a poor prognosis. Next, we showed that glycolysis and subsequent MG stress are triggered in PDAC cells rendered resistant to gemcitabine when compared with parental cells. In fact, acquired resistance, following short and long-term gemcitabine challenges, correlated with the upregulation of GLUT1, LDHA, GLO1, and the accumulation of MG protein adducts. We showed that MG-mediated activation of heat shock response is, at least in part, the molecular mechanism underlying survival in gemcitabine-treated PDAC cells. This novel adverse effect of gemcitabine, i.e., induction of MG stress and HSR activation, is efficiently reversed using potent MG scavengers such as metformin and aminoguanidine. We propose that the MG blockade could be exploited to resensitize resistant PDAC tumors and to improve patient outcomes using gemcitabine therapy.

Induction of cancer neoantigens facilitates development of clinically relevant models for the study of pancreatic cancer immunobiology

Neoantigen burden and CD8 T cell infiltrate are associated with clinical outcome in pancreatic ductal adenocarcinoma (PDAC). A shortcoming of many genetic models of PDAC is the lack of neoantigen burden and limited T cell infiltrate. The goal of the present study was to develop clinically relevant models of PDAC by inducing cancer neoantigens in KP2, a cell line derived from the KPC model of PDAC. KP2 was treated with oxaliplatin and olaparib (OXPARPi), and a resistant cell line was subsequently cloned to generate multiple genetically distinct cell lines (KP2-OXPARPi clones). Clones A and E are sensitive to immune checkpoint inhibition (ICI), exhibit relatively high T cell infiltration, and have significant upregulation of genes involved in antigen presentation, T cell differentiation, and chemokine signaling pathways. Clone B is resistant to ICI and is similar to the parental KP2 cell line in terms of relatively low T cell infiltration and no upregulation of genes involved in the pathways noted above. Tumor/normal exome sequencing and in silico neoantigen prediction confirms successful generation of cancer neoantigens in the KP2-OXPARPi clones and the relative lack of cancer neoantigens in the parental KP2 cell line. Neoantigen vaccine experiments demonstrate that a subset of candidate neoantigens are immunogenic and neoantigen synthetic long peptide vaccines can restrain Clone E tumor growth. Compared to existing models, the KP2-OXPARPi clones better capture the diverse immunobiology of human PDAC and may serve as models for future investigations in cancer immunotherapies and strategies targeting cancer neoantigens in PDAC.

Irbesartan overcomes gemcitabine resistance in pancreatic cancer by suppressing stemness and iron metabolism via inhibition of the Hippo/YAP1/c-Jun axis

BackgroundChemoresistance is the main reason for the poor prognosis of pancreatic ductal adenocarcinoma (PDAC). Thus, there is an urgent need to screen out new targets and compounds to reverse chemotherapeutic resistance.MethodsWe established a bio-bank of human PDAC organoid models, covering a representative range of PDAC tumor subtypes. We screened a library of 1304 FDA-approved compounds to identify candidates efficiently overcoming chemotherapy resistance. The effects of the compounds were evaluated with a CellTiter-Glo-3D assay, organoid apoptosis assay and in vivo patient-derived xenograft (PDX), patient-derived organoid (PDO) and LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) genetically engineered mouse models. RNA-sequencing, genome editing, sphere formation assays, iron assays and luciferase assays were conducted to elucidate the mechanism.ResultsHigh-throughput drug screening of chemotherapy-resistant PDOs identified irbesartan, an angiotensin ‖ type 1 (AT1) receptor antagonist, which could synergistically enhance the ability of chemotherapy to kill PDAC cells. In vitro and in vivo validation using PDO, PDX and KPC mouse models showed that irbesartan efficiently sensitized PDAC tumors to chemotherapy. Mechanistically, we found that irbesartan decreased c-Jun expression by inhibiting the Hippo/YAP1 pathway and further overcame chemotherapy resistance in PDAC. We also explored c-Jun, a potential target of irbesartan, which can transcriptionally upregulate the expression of key genes involved in stemness maintenance (SOX9/SOX2/OCT4) and iron metabolism (FTH1/FTL/TFRC). More importantly, we observed that PDAC patients with high levels of c-Jun expression demonstrated poor responses to the current standard chemotherapy regimen (gemcitabine plus nab-paclitaxel). Moreover, patients with PDAC had significant survival benefits from treatment with irbesartan plus a standard chemotherapy regimen in two-center retrospective clinical cohorts and patients with high c-Jun expression exhibited a better response to combination chemotherapy.ConclusionsIrbesartan could be used in combination with chemotherapy to improve the therapeutic efficacy in PDAC patients with high levels of c-Jun expression. Irbesartan effectively inhibited chemotherapy resistance by suppressing the Hippo/YAP1/c-Jun/stemness/iron metabolism axis. Based on our findings, we are designing an investigator-initiated phase II clinical trial on the efficacy and safety of irbesartan plus a standard gemcitabine/nab-paclitaxel regimen in the treatment of patients with advanced III/IV staged PDAC and are hopeful that we will observe patient benefits.

Sirtuin 6 is required for the integrated stress response and resistance to inhibition of transcriptional cyclin-dependent kinases.

Pancreatic ductal adenocarcinoma (PDAC) is classified into two key subtypes, classical and basal, with basal PDAC predicting worse survival. Using in vitro drug assays, genetic manipulation experiments, and in vivo drug studies in human patient-derived xenografts (PDXs) of PDAC, we found that basal PDACs were uniquely sensitive to transcriptional inhibition by targeting cyclin-dependent kinase 7 (CDK7) and CDK9, and this sensitivity was recapitulated in the basal subtype of breast cancer. We showed in cell lines, PDXs, and publicly available patient datasets that basal PDAC was characterized by inactivation of the integrated stress response (ISR), which leads to a higher rate of global mRNA translation. Moreover, we identified the histone deacetylase sirtuin 6 (SIRT6) as a critical regulator of a constitutively active ISR. Using expression analysis, polysome sequencing, immunofluorescence, and cycloheximide chase experiments, we found that SIRT6 regulated protein stability by binding activating transcription factor 4 (ATF4) in nuclear speckles and protecting it from proteasomal degradation. In human PDAC cell lines and organoids as well as in murine PDAC genetically engineered mouse models where SIRT6 was deleted or down-regulated, we demonstrated that SIRT6 loss both defined the basal PDAC subtype and led to reduced ATF4 protein stability and a nonfunctional ISR, causing a marked vulnerability to CDK7 and CDK9 inhibitors. Thus, we have uncovered an important mechanism regulating a stress-induced transcriptional program that may be exploited with targeted therapies in particularly aggressive PDAC.

Silver Nanomaterials Applied in the Field of Diagnosis and Treatment

Nanomaterials have great application prospect in biological medicine due to their strong permeability, repair and regeneration. In particular, nano silver has excellent antibacterial ability and no toxic effect, which makes it widely used. The specific content of this paper introduces a method of using silver nanoparticles to generate silver ions, and then make it react with hydrogen peroxide, through the colorimetric device to react with different color changes corresponding to different concentrations, through this biosensor to determine the concentration of such as cholesterol lactic acid and other substances.And when detecting miRNA,AuNPs were further modified by complementing the DNA (cDNA) of target mirnas. AgNPs could not be attracted to negatively charged DNA due to electrostatic repulsion by immobilized modified gold nanoparticles on the electrode surface. After analyzing the peak of silver stripping, miRNA can be accurately detected.Furthermore, chitosan coated silver nanoparticles were designed, and polyethylene glycol was used as a stabilizer. Finally, its good blood compatibility and antibacterial ability, as well as its cytotoxicity to cervical cancer cells were demonstrated by characterization.Finally, the accumulation and changes of reactive oxygen species (ROS) and reactive nitrogen (RNS) induced by silver nano are introduced, and it is confirmed by measurement that they are important factors in apoptosis of human pancreatic ductal adenocarcinoma.

Activation of the integrated stress response (ISR) pathways in response to Ref-1 inhibition in human pancreatic cancer and its tumor microenvironment.

Pancreatic cancer or pancreatic ductal adenocarcinoma (PDAC) is characterized by a profound inflammatory tumor microenvironment (TME) with high heterogeneity, metastatic propensity, and extreme hypoxia. The integrated stress response (ISR) pathway features a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) and regulate translation in response to diverse stress conditions, including hypoxia. We previously demonstrated that eIF2 signaling pathways were profoundly affected in response to Redox factor-1 (Ref-1) knockdown in human PDAC cells. Ref-1 is a dual function enzyme with activities of DNA repair and redox signaling, responds to cellular stress, and regulates survival pathways. The redox function of Ref-1 directly regulates multiple transcription factors including HIF-1α, STAT3, and NF-κB, which are highly active in the PDAC TME. However, the mechanistic details of the crosstalk between Ref-1 redox signaling and activation of ISR pathways are unclear. Following Ref-1 knockdown, induction of ISR was observed under normoxic conditions, while hypoxic conditions were sufficient to activate ISR irrespective of Ref-1 levels. Inhibition of Ref-1 redox activity increased expression of p-eIF2 and ATF4 transcriptional activity in a concentration-dependent manner in multiple human PDAC cell lines, and the effect on eIF2 phosphorylation was PERK-dependent. Treatment with PERK inhibitor, AMG-44 at high concentrations resulted in activation of the alternative ISR kinase, GCN2 and induced levels of p-eIF2 and ATF4 in both tumor cells and cancer-associated fibroblasts (CAFs). Combination treatment with inhibitors of Ref-1 and PERK enhanced cell killing effects in both human pancreatic cancer lines and CAFs in 3D co-culture, but only at high doses of PERK inhibitors. This effect was completely abrogated when Ref-1 inhibitors were used in combination with GCN2 inhibitor, GCN2iB. We demonstrate that targeting of Ref-1 redox signaling activates the ISR in multiple PDAC lines and that this activation of ISR is critical for inhibition of the growth of co-culture spheroids. Combination effects were only observed in physiologically relevant 3D co-cultures, suggesting that the model system utilized can greatly affect the outcome of these targeted agents. Inhibition of Ref-1 signaling induces cell death through ISR signaling pathways, and combination of Ref-1 redox signaling blockade with ISR activation could be a novel therapeutic strategy for PDAC treatment.

YAP/BRD4-controlled ROR1 promotes tumor-initiating cells and hyperproliferation in pancreatic cancer.

Tumor-initiating cells are major drivers of chemoresistance and attractive targets for cancer therapy, however, their identity in human pancreatic ductal adenocarcinoma (PDAC) and the key molecules underlying their traits remain poorly understood. Here, we show that a cellular subpopulation with partial epithelial-mesenchymal transition (EMT)-like signature marked by high expression of receptor tyrosine kinase-like orphan receptor 1 (ROR1) is the origin of heterogeneous tumor cells in PDAC. We demonstrate that ROR1 depletion suppresses tumor growth, recurrence after chemotherapy, and metastasis. Mechanistically, ROR1 induces the expression of Aurora kinase B (AURKB) by activating E2F through c-Myc to enhance PDAC proliferation. Furthermore, epigenomic analyses reveal that ROR1 is transcriptionally dependent on YAP/BRD4 binding at the enhancer region, and targeting this pathway reduces ROR1 expression and prevents PDAC growth. Collectively, our findings reveal a critical role for ROR1high cells as tumor-initiating cells and the functional importance of ROR1 in PDAC progression, thereby highlighting its therapeutic targetability.

Lauric Acid Overcomes Hypoxia-Induced Gemcitabine Chemoresistance in Pancreatic Ductal Adenocarcinoma.

Although gemcitabine (GEM) is widely used in chemotherapy for pancreatic ductal adenocarcinoma (PDA), drug resistance restricts its clinical effectiveness. To examine the mechanism of GEM resistance, we established two GEM-resistant cell lines from human PDA cells by continuous treatment with GEM and CoCl2-induced chemical hypoxia. One resistant cell line possessed reduced energy production and decreased mitochondrial reactive oxygen species levels, while the other resistant cell line possessed increased stemness. In both cell lines, ethidium bromide-stained mitochondrial DNA levels decreased, suggesting mitochondrial DNA damage. Inhibition of hypoxia-inducible factor-1α in both cell lines did not restore the GEM sensitivity. In contrast, treatment of both cell types with lauric acid (LAA), a medium-chain fatty acid, restored GEM sensitivity. These results suggest that decreased energy production, decreased mitochondrial reactive oxygen species levels, and increased stemness associated with mitochondrial damage caused by GEM lead to GEM resistance, and that hypoxia may promote this process. Furthermore, forced activation of oxidative phosphorylation by LAA could be a tool to overcome GEM resistance. Clinical verification of the effectiveness of LAA in GEM resistance is necessary in the future.

Activation of Vitamin D/VDR Signaling Reverses Gemcitabine Resistance of Pancreatic Cancer Cells Through Inhibition of MUC1 Expression.

Pancreatic ductal adenocarcinoma (PDA) has a poor prognosis due to its therapeutic resistance. Inactivation of vitamin D/vitamin D receptor (VDR) signaling may contribute to the malignant phenotype of PDA and altered expression of oncoprotein mucin 1 (MUC1) may be involved in drug resistance of cancer cells. To determine whether vitamin D/VDR signaling regulates the expression and function of MUC1 and its effect on acquired gemcitabine resistance of pancreatic cancer cells. Molecular analyses and animal models were used to determine the impact of vitamin D/VDR signaling on MUC1 expression and response to gemcitabine treatment. RPPA analysis indicated that MUC1 protein expression was significantly reduced in human PDA cells after treatment with vitamin D3 or its analog calcipotriol. VDR regulated MUC1 expression in both gain- and loss-of-function assays. Vitamin D3 or calcipotriol significantly induced VDR and inhibited MUC1 expression in acquired gemcitabine-resistant PDA cells and sensitized the resistant cells to gemcitabine treatment, while siRNA inhibition of MUC1 was associated with paricalcitol-associated sensitization of PDA cells to gemcitabine treatment in vitro. Administration of paricalcitol significantly enhanced the therapeutic efficacy of gemcitabine in xenograft and orthotopic mouse models and increased the intratumoral concentration of dFdCTP, the active metabolite of gemcitabine. These findings demonstrate a previously unidentified vitamin D/VDR-MUC1 signaling axis involved in the regulation of gemcitabine resistance in PDA and suggests that combinational therapies that include targeted activation of vitamin D/VDR signaling may improve the outcomes of patients with PDA.

Morphology-guided transcriptomic analysis of human pancreatic cancer organoids reveals microenvironmental signals that enhance invasion.

Pancreatic ductal adenocarcinoma (PDAC) frequently presents with metastasis, but the molecular programs in human PDAC cells that drive invasion are not well understood. Using an experimental pipeline enabling PDAC organoid isolation and collection based on invasive phenotype, we assessed the transcriptomic programs associated with invasion in our organoid model. We identified differentially expressed genes in invasive organoids compared with matched noninvasive organoids from the same patients, and we confirmed that the encoded proteins were enhanced in organoid invasive protrusions. We identified 3 distinct transcriptomic groups in invasive organoids, 2 of which correlated directly with the morphological invasion patterns and were characterized by distinct upregulated pathways. Leveraging publicly available single-cell RNA-sequencing data, we mapped our transcriptomic groups onto human PDAC tissue samples, highlighting differences in the tumor microenvironment between transcriptomic groups and suggesting that non-neoplastic cells in the tumor microenvironment can modulate tumor cell invasion. To further address this possibility, we performed computational ligand-receptor analysis and validated the impact of multiple ligands (TGF-β1, IL-6, CXCL12, MMP9) on invasion and gene expression in an independent cohort of fresh human PDAC organoids. Our results identify molecular programs driving morphologically defined invasion patterns and highlight the tumor microenvironment as a potential modulator of these programs.

Cellular Membrane Localization of Innate Immune Checkpoint Molecule CD47 Is Regulated by Radixin in Human Pancreatic Ductal Adenocarcinoma Cells.

In the past decade, immune checkpoint inhibitors have exhibited potent antitumor efficacy against multiple solid malignancies but limited efficacy against pancreatic ductal adenocarcinoma (PDAC). Cluster of differentiation (CD) 47, a member of the immunoglobulin G superfamily, is overexpressed in the surface membrane of PDAC and independently correlates with a worse clinical prognosis. Furthermore, CD47 functions as a dominant macrophage checkpoint, providing a potent "do not eat me" signal to enable cancer cells to evade the innate immune system. Thus, the blockade of CD47 is a promising immunotherapeutic strategy for PDAC. In this study, we determined whether ezrin/radixin/moesin (ERM) family members, which post-translationally modulate the cellular membrane localization of numerous transmembrane proteins by crosslinking with the actin cytoskeleton, contribute to the cellular membrane localization of CD47 in KP-2 cells derived from human PDAC. Immunofluorescence analysis showed that CD47 and ezrin/radixin were highly co-localized in the plasma membrane. Interestingly, gene silencing of radixin but not ezrin dramatically decreased the cell surface expression of CD47 but had little effects on its mRNA level. Furthermore, CD47 and radixin interacted with each other, as determined by a co-immunoprecipitation assay. In conclusion, radixin regulates the cellular membrane localization of CD47 as a scaffold protein in KP-2 cells.

Mitoxantrone and Mitoxantrone-Loaded Iron Oxide Nanoparticles Induce Cell Death in Human Pancreatic Ductal Adenocarcinoma Cell Spheroids

Pancreatic ductal adenocarcinoma is a hard-to-treat, deadly malignancy. Traditional treatments, such as surgery, radiation and chemotherapy, unfortunately are still not able to significantly improve long-term survival. Three-dimensional (3D) cell cultures might be a platform to study new drug types in a highly reproducible, resource-saving model within a relevant pathophysiological cellular microenvironment. We used a 3D culture of human pancreatic ductal adenocarcinoma cell lines to investigate a potential new treatment approach using superparamagnetic iron oxide nanoparticles (SPIONs) as a drug delivery system for mitoxantrone (MTO), a chemotherapeutic agent. We established a PaCa DD183 cell line and generated PANC-1SMAD4 (-/-) cells by using the CRISPR-Cas9 system, differing in a prognostically relevant mutation in the TGF-β pathway. Afterwards, we formed spheroids using PaCa DD183, PANC-1 and PANC-1SMAD4 (-/-) cells, and analyzed the uptake and cytotoxic effect of free MTO and MTO-loaded SPIONs by microscopy and flow cytometry. MTO and SPION-MTO-induced cell death in all tumor spheroids in a dose-dependent manner. Interestingly, spheroids with a SMAD4 mutation showed an increased uptake of MTO and SPION-MTO, while at the same time being more resistant to the cytotoxic effects of the chemotherapeutic agents. MTO-loaded SPIONs, with their ability for magnetic drug targeting, could be a future approach for treating pancreatic ductal adenocarcinomas.

Abstract 5851: Cancer associated fibroblast crosstalk through VEGF increases tumor cell proliferation in human pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a classically heterogeneous tumor for which tumor cells comprise less than 40% of the total tumor mass. To better understand mechanisms of intercellular interactions driving tumor response or resistance to chemotherapy, it is crucial to account for the complex tumor microenvironment (TME). Cancer associated fibroblasts (CAFs) have been implicated as key drivers supporting mechanisms that both promote and restrain tumor growth. Using our model system that investigates interactions between CAFs and cancer cells, we show a novel intercellular interaction through VEGF signaling that drives proliferation. Methods: We interrogate intracellular crosstalk in the PDAC TME using a novel 3-dimensional (3D), patient-matched coculture system of patient-derived organoids (PDO) and CAFs, obtained from patients undergoing surgical resection. Molecular characterization is performed by profiling with a 1200 analyte multiplex ELISA screen, multiparameter flow cytometry, bulk RNA sequencing of CAF and PDO cocultures, imaging mass cytometry (IMC) on patient samples, and qPCR. Results: Secretome profiling of 7 patient-derived CAF lines demonstrated patient-specific heterogeneity in proteins such as: FGF-12, FGF3, and CD79a. Common CAF-derived signaling proteins with the potential to regulate intercellular crosstalk that were seen across all samples include CRP, IL-6R, R-Spondin and others. To better understand how factors change with intercellular interactions, we set up direct CAF-PDO cocultures over 4 days to identify global transcriptional changes using bulk RNAseq. Phenotypically, coculture drives gene expression changes in both PDO subtype (basal vs classical) and CAF subtype (prevalence of iCAFs vs myCAFs). We used these transcriptome data to identify pathways modulated by CAF-PDO crosstalk. Profiling coculture supernatant, we identified upregulation in VEGF secretion. In tandem, when we investigate cellular surface marker changes, we see upregulation of VEGFR2 on the surface of CAFs by flow cytometry. This interaction is accompanied by an increase in PDO proliferation seen in the coculture conditions. Ongoing experiments aim to investigate the impact of this relationship spatially using matched patient tissue. Conclusions: Interactions between cancer cells and CAFs compound the complexity of the biology of the TME and contribute to poor patient outcomes; therefore, we have put forth a model that better represents these interactions through patient matched PDO - CAF cocultures. Using this model, we demonstrate a novel interaction through VEGF that enhances tumor cell proliferation. We introduce a targeted approach to investigating the complex biology of the TME to inform the mechanisms driving cancer biology in individual patients that can also be used to develop novel therapeutic targets. Citation Format: Samantha Guinn, Joseph Tandurella, Jignasha Patel, Richard Burkhart, Jacquelyn Zimmerman, Elizabeth Jaffee. Cancer associated fibroblast crosstalk through VEGF increases tumor cell proliferation in human pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5851.

Abstract 1634: Novel autophagy inhibitor ESK981 potentiates MEK inhibition cytotoxicity in pancreatic cancer

Abstract Background Pancreatic Ductal Adenocarcinoma (PDAC) is extremely deadly with a dismal 5-year survival rate of only 11%. In PDAC, the overactive KRAS-MEK-ERK pathway is critical to maintaining metabolic homeostasis. Important recent work has shown that when MEK or ERK are inhibited, PDAC upregulates and becomes increasingly dependent on autophagy. Autophagy, the recycling of cellular material through lysosomal degradation, has already been previously established to be upregulated in and crucial to PDAC in order survive in its harsh microenvironment and allows PDAC to evade the immune system. Unfortunately, chloroquine (CQ), the only clinical-grade autophagy inhibitor tested in PDAC failed to improve patient survival in combination with chemotherapies in phase II trials. Recently, our lab reported a novel phase I-cleared multi-tyrosine kinase inhibitor ESK981 which disrupted autophagy and had substantial single agent efficacy in prostate cancer models. Given the importance of autophagy in PDAC and the ongoing clinical trials testing autophagy inhibitor CQ in combination with MEK/ERK inhibitors, this study aims to evaluate ESK981 as a therapeutic agent for PDAC, particularly its ability to potentiate MEK inhibitors’ antiproliferative effects in PDAC. Methods and Results In mouse and human PDAC cell lines, we observed an accumulation of LC3A/B as well as P62 when treated with ESK981, suggesting a disruption of autophagic flux. To confirm an inhibition of autophagy, we used the GFP-LC3-RFP-LC3ΔG reporter system in Mia-Paca2 cells and saw that the GFP:RFP ratio was maintained near 1:1. To assess the effects of ESK981 on cell growth, we used Cell-Titer Glo assays which revealed that ESK981 suppresses proliferation of a panel of human and mouse PDAC cell lines at nanomolar concentrations. To further evaluate ESK981’s potential clinical applications, we evaluated ESK981’s ability to synergize with MEK inhibitor Trametinib. Using the GFP-LC3-RFP-LC3ΔG reporter system we saw that Trametinib decreased the GFP:RFP ratio as expected. When cells were pre-treated with ESK981, the ratio was increased, showing that ESK981 was able to block Trametinib-induced autophagic flux. Finally, using Cell-Titer Glo, we determined that ESK981 and Trametinib had a strong synergistic effect on suppressing cell proliferation (BLISS Score >20) on mouse KPC line 7940B. Taken together, ESK981 is a promising autophagy inhibitor that shows strong anti-proliferative effects on both human and mouse PDAC cell lines as a single agent as well as in combination with Trametinib. Citation Format: Caleb Cheng, Jasmine Wisniewski, Bailey Jackson, Yi Bao, Yuanyuan Qiao, Costas A. Lyssiotis, Arul M. Chinnaiyan. Novel autophagy inhibitor ESK981 potentiates MEK inhibition cytotoxicity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1634.

Abstract 3627: Inhibition of tumor cell-autonomous p38 MAPK suppresses IL1α-mediated inflammatory tumor-stromal crosstalk in pancreatic adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDAC) continues to face major therapeutic challenges due to its innate and acquired chemoresistance. Oncogenic, activating KRAS mutations facilitate extensive cytokine-mediated crosstalk between tumor cells and the fibrotic stroma, producing a pro-inflammatory and immunosuppressive tumor microenvironment (TME). We have identified interleukin-1α (IL1α), downstream of KRAS, as a critical mediator of the inflammatory response due to its pleiotropic effects on cancer-associated fibroblast (CAF) activation and immune evasion. However, the regulatory mechanisms of IL1α expression remain incompletely understood. We identified p38 stress-associated MAPK α (p38α) as a central, KRAS-driven regulatory pathway involved in IL1α production within PDAC tumor cells. Methods: Differential gene expression analyses were performed on patient data from The Cancer Genome Atlas (TCGA) was queried for overactive pathways downstream KRAS in “high-IL1A” PDAC cases. Inhibition of p38α was achieved pharmacologically with pexmetinib and genetically with an shRNA lentiviral system in human and murine PDAC cell lines. ChIP-qPCR and co-immunoprecipitation were performed on a human PDAC cell line with and without p38α inhibition. Single-cell RNA sequencing (scRNA-seq) and flow cytometry were performed on tumors from a PDAC murine model, Ptf1aCre/+; LSL-KrasG12D/+; Tgfbr2flox/flox (PKT), treated with or without pexmetinib. PKT mice were treated daily with pexmetinib, gemcitabine and paclitaxel chemotherapy, or combination therapy for downstream analysis and survival studies. Results: Both pharmacologic and genetic inhibition of p38α significantly reduced IL1A transcription and protein levels in human and murine PDAC tumor cell lines. Furthermore, p38α inhibition reduced binding of Sp1 and NF-κB to the IL1A promoter and prevented IL1α-mediated polarization of inflammatory CAFs in vitro. In PKT mice, p38α inhibition reduced tumor cell-specific Il1a and inflammatory CAF gene signatures by scRNA-seq, and favorably altered the infiltrating immune populations by flow cytometry. Lastly, pexmetinib with chemotherapy significantly reduced tumor burden and improved overall survival in a PDAC murine model. Conclusions: These findings provide a new therapeutic opportunity to target the p38α MAPK pathway for suppression of IL1α-mediated stromal activation and combination with chemotherapy to overcome therapeutic resistance by modulating the stromal and immune microenvironment in PDAC. Citation Format: Samara Singh, Austin R. Dosch, Siddharth Mehra, Iago de Castro Silva, Anna Bianchi, Vanessa Tonin Garrido, Zhiqun Zhou, Haleh Amirian, Edmond W. Box, Jashodeep Datta, Nagaraj Nagathihalli, Nipun B. Merchant. Inhibition of tumor cell-autonomous p38 MAPK suppresses IL1α-mediated inflammatory tumor-stromal crosstalk in pancreatic adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3627.

Abstract 1725: Preclinical evaluation of RM-042, an orally bioavailable inhibitor of GTP-RAS, in models of pancreatic ductal adenocarcinoma

Abstract Approximately 95% of pancreatic ductal adenocarcinoma (PDAC) cases are driven by activating mutations in KRAS, accounting for over 45,000 deaths per year in the US. In KRAS-mutant cells, KRAS protein exists predominantly in an active, GTP-bound state, leading to excessive oncogenic signaling via RAF/MEK/ERK and other effector pathways. After decades of anticipation, the first therapeutic inhibitors of this oncoprotein target a specific mutant allele (KRASG12C) and have shown promising results in this rare subset of human PDAC patients (1-2% of cases). While these encouraging clinical results bolster support for targeting RAS in human PDAC, most patients harbor KRASG12D and KRASG12V mutations and remain unserved by targeted therapies. Here we report preclinical studies of RM-042, a first-in-class, potent, oral, small molecule inhibitor of GTP-bound RAS proteins, designed to treat cancers driven by a variety of RAS mutations. RMC-6236, a clinical candidate and RM-042, a tool compound, both act through non-covalent binding to an abundant intracellular chaperone protein, cyclophilin A (CypA), resulting in a binary complex that engages GTP-bound RAS to form a high-affinity, RAS-selective tri-complex that sterically inhibits RAS binding to effectors.We examined the anti-tumor activity of RM-042 in two complementary preclinical model systems of PDAC. Using primary human PDAC explant cultures, we found that RM-042 potently inhibits phospho-ERK expression (a marker of KRAS/RAF/MEK pathway activity), in a concentration-dependent manner and was associated with reduced viability and altered stromal content. Pilot short-term studies in the K-rasLSL.G12D/+, p53LSL.R172H/+, Pdx1Cretg/+ (KPC) mouse model of PDAC found that RM-042 induced initial tumor stabilizations and regressions associated with loss of pERK expression, in association with increased apoptosis and reduced proliferation, at tolerated doses. However, tumors collected at endpoints showed re-established pERK expression, suggesting activation of resistance/adaptation mechanisms. Ongoing studies will determine pharmacokinetic properties of RM-042 in the autochthonous PDAC mouse model, as well as the duration of tumor responses and evaluate a potential survival benefit provided by RM-042. Regulatory network analysis of single cell RNA sequencing performed on these tumors at multiple timepoints will provide a mechanistic understanding of the regulatory proteins that drive responses in each cell type in the tumors. Through this approach, we will assess the regulatory adaptations that occur as tumors initially respond and as they escape. Together, these studies provide complementary evidence from in vivo murine and ex vivo human models for promising anti-tumor activity in response to GTP-RAS inhibition, supporting the inclusion of PDAC patients in ongoing and future clinical trials of RMC-6236. Citation Format: Urszula N. Wasko, Stephen A. Sastra, Carmine F. Palermo, David Wildes, Mallika Singh, Kenneth P. Olive. Preclinical evaluation of RM-042, an orally bioavailable inhibitor of GTP-RAS, in models of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1725.

Abstract 6306: Pharmacological deuteration of SCID mice using the water-isotopologue deuterium oxide (D2O) inhibits tumor growth in bioluminescent models of human malignant melanoma and pancreatic ductal adenocarcinoma

Abstract Since its initial discovery as a natural isotopologue of dihydrogen oxide (1H2O), extensive research has focused on the biophysical, biochemical, and pharmacological effects of deuterated water [2H2O (D2O, also referred to as ‘heavy water’)]. Here, using diverse panels of cultured human malignant melanoma and pancreatic ductal adenocarcinoma (PDAC) cells we have profiled (i) D2O-induced phenotypic anti-proliferative and apoptogenic effects, (ii) redox- and proteotoxicity-directed stress response gene expression, and (iii) phosphoprotein-signaling related to endoplasmic reticulum (ER) and MAP-kinase stress response pathways. Differential RT-qPCR array analysis revealed early modulation of stress response gene expression elicited by D2O (90%; ≤6 h) confirmed by independent RT-qPCR analysis. Immunoblot-analysis revealed rapid (< 6 h) onset of D2O-induced MAP-kinase signaling (p-JNK) together with ER stress response upregulation (p-eIF2α, ATF4, XBP1s, DDIT3/CHOP) attributable to deuteration-induced alteration of hydrogen bonding triggering proteotoxic stress. Next, we tested the chemotherapeutic efficacy of D2O-based drinking water supplementation in bioluminescent murine models of malignancy (A375-luc melanoma and BxPC-3-luc orthotopic PDAC xenografts in SCID mice). Time course of systemic deuteration (30% D2O in drinking water fed continuously for up to 21 days) was established using time-resolved whole-body proton magnetic resonance imaging (MRI) and isotope-ratio mass spectrometry (IR-MS)-based plasma (D/H)-analysis. In both models, D2O supplementation significantly suppressed tumor growth and metastasis with downregulated expression of PCNA/Ki67 while increasing tumor levels of DDIT3/CHOP, HO-1, and p-eIF2α. Taken together, these data demonstrate for the first time that pharmacological induction of systemic deuteration by oral administration of D2O, associated with induction of cellular ER stress, reduces tumor burden and metastasis in murine models of human malignant melanoma and PDAC. Citation Format: Jana Jandova, Jean-Philippe Galons, David L. Dettman, Georg T. Wondrak. Pharmacological deuteration of SCID mice using the water-isotopologue deuterium oxide (D2O) inhibits tumor growth in bioluminescent models of human malignant melanoma and pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6306.

Abstract 1710: Pharmacologic targeting or silencing of TRIP13 reduces progression of pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignancy with a 5-year overall survival rate of < 9%. Thus, there is a need to identify key driver signaling molecules that regulate PDAC progression and to target them for therapeutic intervention. Thyroid receptor-interacting protein 13 (TRIP13), an enzyme of the AAA-ATPase family, participates in spindle assembly checkpoints, double-strand DNA repair, and development of drug resistance. Recently, a small-molecule inhibitor of TRIP13, DCZ0415, was evaluated in experimental models of multiple myeloma and colorectal cancer. However, the expression, oncogenic role, and targeting of TRIP13 in PDAC are unknown. Thus, we examined the expression and role of TRIP13 in PDAC and investigated if targeting it with DCZ0415 had an inhibitory effect on phenotypic features, tumor growth, metastasis, and immune modulation. Methods: Publicly available cancer OMICS datasets (TCGA and CPTAC) for TRIP13 expression were analyzed by using the UALCAN portal, and results were validated with human PDAC samples. To demonstrate the oncogenic role of TRIP13 in PDAC progression, we used silencing with shRNA or pharmacologic targeting by DCZ0415 and performed MTT, colony formation, wound healing, Transwell, and western blot assays in PDAC cell lines, and assessed tumor growth and metastasis in immunocompromised NSG mice. Immunostaining and western blotting were performed to ascertain the immuno-modulatory mechanism of DCZ0415 in an immunocompetent KPC murine model of PDAC. Results: In human PDACs, TRIP13 is overexpressed at the mRNA and protein levels, relative to adjacent normal pancreatic tissues. Silencing of TRIP13 with shRNA or DCZ0415 treatment of PDAC cells reduced their proliferation, migration, and invasion; induced G2/M cell cycle arrest; and, by modulating Bcl-2 family proteins, increased apoptosis. Furthermore, DCZ0415 treatment or silencing of TRIP13 increased E-cadherin and decreased N-cadherin and vimentin, suggesting its involvement in the epithelial-mesenchymal transition process. Genetic manipulation or pharmacologic targeting of TRIP13 also resulted in reduced expression of FGFR4 and pSTAT3, leading to lower levels of β-catenin, cyclin D1, LEF1, and TCF1, indicating inactivation of the Wnt/β-catenin pathway. In a syngeneic PDCA model, DCZ0415 induced an immune response by increasing granzyme B and perforin and by decreasing PD1. DCZ0415 treatment also facilitated immune cell infiltration, as evident from elevated staining of CD3 and CD4 cells. Conclusions: In sum, silencing of TRIP13 or treatment with DCZ0415 reduces PDAC progression by downregulating the FGFR4/STAT3/β-catenin pathway. DCZ0415 treatment also enhances anti-tumor immunity. These findings suggest that DCZ0415 can be developed as a therapeutic/immunostimulatory agent to improve treatment of PDAC patients expressing elevated levels of TRIP13. Citation Format: Farrukh Afaq, Sumit Agarwal, Prachi Bajpai, Hyung-Gyoon Kim, Sameer Al Diffalha, Shajan P. Sugandha, Moh'd Khushman, Sooryanarayana Varambally, Upender Manne. Pharmacologic targeting or silencing of TRIP13 reduces progression of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1710.

Abstract 4140: Oncogenic KRAS inhibition with ADT-007 primes T cell responses in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) has low (<11%) 5-year survival due to the presence of advanced disease at diagnosis. Currently standard of care provides a median survival of 1 year in the majority of patients presenting with advanced disease. Targeting oncogenic KRAS, which is mutated in >90% of human PDAC tumors, provides an opportunity to inhibit a critical pathway and significantly impact standard of care in patients diagnosed with PDAC. Furthermore, evidence indicates that inhibiting-oncogenic KRAS signaling modulates anti-tumor immune responses and demonstrates synergy with immune checkpoint blockade in pre-clinical models. The purpose of this study is to evaluate efficacy and impact on anti-tumor immunity of a novel inhibitor, ADT-007 that demonstrates pan-RAS inhibitory activity. Methods: Female C57BL/6J, C57BL/6J. Ifng/Thy1.1 KI;Cg-Tg-IL2tm1/eGFP/Weav (IFNg-Thy1.1/IL-2-GFP reporter mice), or immune deficient (T/B cells) Rag 1 −/− mice (n=5-10/group) were implanted SQ with 106 mouse derived PDAC cell lines (7160c2 and 2838c3) bearing KRASG12D mutation, and then randomized (day 7), into vehicle (5% DMSO/5% Kolliphor/90% H2O) or ADT-007 treatment, which was administered SQ (peritumoral), BID at 5 mg/kg for 2 - 3 weeks. Tumor growth in vitro (IC50), KRAS-MAPK/AKT signaling, in vivo tumor volumes (tv = L x W2/2), and tumor immune responses were assessed by CellTiter Glo, phosphoflow/western blotting, bi-weekly caliper measurements, and multi-parameter flow cytometry, respectively. Results: ADT-007 inhibited KRAS-MAPK/AKT signaling in human and mouse PDAC cell lines inducing G2-M phase arrest and apoptosis at low nanomolar concentrations. Peritumoral administration of ADT-007 inhibited tumor cell growth in vivo, modulating T cells in the tumor microenvironment (TME), specifically, an increase in tumor infiltrating, CD44+ CD62L− CD4+ and CD8+ T cells and increases in production of TNFα, IFN-γ, IL-2, and granzyme B upon stimulation with PMA/ionomycin. Conclusion: The small molecule inhibitor, ADT-007 blocks oncogenic KRAS signaling and modulates T cell activation and function in the TiME of immune competent, syngeneic mouse models of PDAC. More broadly, these findings indicate that a pan-RAS inhibitor is capable of modulating anti-tumor immune responses extending previous observations that targeted inhibition of oncogenic KRASG12C modulates tumor immunity in animal models of colon and lung cancer. Citation Format: Jeremy B. Foote, Tyler E. Mattox, Adam B. Keeton, Ganji N. Purnachandra, Yulia Maxuitenko, Xi Chen, Jacob Valiyaveettil, Donald J. Buchsbaum, Gary A. Piazza, Bassel F. El-Rayes. Oncogenic KRAS inhibition with ADT-007 primes T cell responses in pancreatic ductal adenocarcinoma. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4140.