Resistant Pancreatic Ductal Adenocarcinoma Research Articles

Uncovering the role of oncogenic HER2 variants as drivers of pancreatic cancer.

772 Background: The therapeutic resistance of pancreatic ductal adenocarcinoma (PDAC) to conventional cytotoxic therapy underscores the need for advances in targeted molecular therapeutics. Previous studies have reported approximately 2% of PDAC patients with HER2 gene amplification. While anti- HER2 cancer therapy has yielded success in other disease sites, it has had minimal reported benefit in PDAC. Multiple HER2 isoforms exist that are generated either by loss of exon 16 ( d16 HER2 ), or through N-terminal truncations ( p95 HER2 ). These isoforms are implicated in differential tumor behavior and response to anti-HER2 therapy in the breast but have not been investigated in PDAC. Herein, we hypothesize that these isoforms may underlie the observed resistance to HER2 therapy in PDAC. Methods: To test for the presence of HER2 structural variants within human PDAC, a tissue microarray was constructed of fifty-one primary tumor samples and two metastatic samples. Multiplex immunohistochemistry (mIHC) was performed for staining of the extracellular N-terminal and intracellular C-terminal HER2 domains. We then utilized our previously described HER2 + cancer rainbow (Crainbow) mouse model in which human wild-type HER2 ( WT HER2 ), d16 HER2 , and p95 HER2 were expressed in the same mouse along with fluorescent protein reporters. HER2 Crainbow mice were crossed with PDX1-Cre recombinase to initiate recombination and expression of each fluorescently labelled HER2 isoform during pancreas development. PDX1-Cre / HER2 Crainbow mice were followed for up to one year and sacrificed to lineage trace each isoform and register HER2 lineages with histopathology. Results: In our human data, four of fifty-three patient samples (three primary and one metastatic) had moderate-to-strong HER2 staining. All samples identified on mIHC as HER2 positive stained primarily for the p95 variant of HER2 , with absence of the N-terminal domain and preservation of the intracellular domain. In our transgenic mice, 65% of the mouse cohort developed pre-malignant low grade pancreatic intraepithelial neoplasia (PanIN) by eight weeks. At one year, 58.3% of mice demonstrated advanced high grade PanIN, and 25% developed invasive disease. Lineage tracing revealed that while early low grade PanIN began as polyclonal lesions, comprised of WT- , d16- , and p95 HER2 expressing cells, advanced PanIN and invasive disease were unanimously monoclonal with one dominant isoform arising. WT HER2 was represented abundantly in low grade disease, but high grade and invasive disease were predominantly driven by d16- and p95-HER2. Conclusions: Our data implicates the oncogenic d16- and p95 HER2 variants, rather than WT HER2 , as the primary drivers of HER2+ PDAC tumorigenesis. In breast p95 HER2 has been described as a known cause of Trastuzumab resistance. Therefore, future efforts will be aimed at characterizing the mechanisms driving therapeutic resistance in p95-HER2 PDAC tumors.

Preclinical development of the lipid nanoparticle–based mRNA vaccine with multiple natural T cell epitopes for pancreatic cancer.

752 Background: Immunotherapy has recently emerged as a promising avenue in cancer treatment; however, its effectiveness on pancreatic ductal adenocarcinoma (PDAC) is hindered by the lack of effector T cells in the tumor microenvironment. One of the strategies to overcome the resistance of PDAC to immunotherapy involves identifying immunogenic tumor-associated and specific antigens for the development of personalized cancer vaccines. The mRNA platform has become an attractive platform for antigen presentation. The delivery of mRNA cancer vaccines via lipid nanoparticles (LNPs) enables intracellular delivery and controlled release of the payload, offering improved vaccine efficacy. However, mRNA vaccines presenting human T cell epitopes often lack a preclinical model for antitumor immune efficacy testing. Methods: A syngeneic mouse model of PDACs was established by implanting KPC tumor cells into the livers by hemispleen surgery. mRNA vaccine expressing five 25-mer peptide regions were synthesized. Two mouse CD8+ T cell epitopes were predicted. Their MHC binding was tested by the T2 binding assay. CD8+ T cells specific for these two epitopes were examined by ELISpot assays and were quantified by ImmunoSpot ELISpot Reader to identify changes in IFN-g secretion. Results: Using mass spectrometry analysis, we successfully identified natural HLA class I and class II-binding epitopes in the neoplastic tissues of human PDACs. For the design of mRNA vaccine, we chose five 25-mer peptide regions that each contains both class I and class II epitopes punctuated by AAY and GPGPG linkers and followed by an MITD sequence, which all aim to improve the efficiency of antigen presentation to dendritic cells. Using NetMHC, we predicted H2Kb or H2Db-binding epitopes LYPFPLAL and SMVQMTFL, within these five human peptide regions. The T2 binding assay showed a lack of evident binding of both peptides to the T2-Kb or T2-Db cells. However, the ELISpot assay showed that LYPFPLAL, but not SMVQMTFL, can induce the IFN-g expression from CD8+ T cells derived from splenocytes of the KPC tumor mice vaccinated by a peptide vaccine containing both peptides. Therefore, this result confirmed that LYPFPLAL is a mouse CD8+ T cell epitope and can be used to monitor antigen-specific CD8+ T cell response in response to the mRNA vaccine treatment in the syngeneic mouse model of PDACs. Conclusions: Antitumor efficacy of our above-designed mRNA vaccine can be tested in the syngeneic mouse hemispleen model of KPC tumors. Mouse LYPFPLAL epitope-specific CD8+ T cell response can serve as a surrogate marker for the antitumor efficacy of this mRNA vaccine in mouse models for PDAC.

Elevated protein lactylation promotes immunosuppressive microenvironment and therapeutic resistance in pancreatic ductal adenocarcinoma.

Metabolic reprogramming shapes tumor microenvironment (TME) and may lead to immunotherapy resistance in pancreatic ductal adenocarcinoma (PDAC). Elucidating the impact of pancreatic cancer cell metabolism in the TME is essential to therapeutic interventions. "Immune cold" PDAC is characterized by elevated lactate levels resulting from tumor cell metabolism, abundance of pro-tumor macrophages, and reduced cytotoxic T cell in the TME. Analysis of 18F-FDG uptake in patients showed that increased global protein lactylation in PDAC correlates with worse clinical outcomes in immunotherapy. Inhibition of lactate production in pancreatic tumors via glycolysis or mutant-KRAS inhibition reshaped the TME, thereby increasing their sensitivity to immune checkpoint blockade (ICB) therapy. In pancreatic tumor cells, lactate induces K63 lactylation of Endosulfine alpha (ENSA-K63la), a crucial step that triggers STAT3-CCL2 signaling. Consequently, elevated CCL2 secreted by tumor cells facilitates tumor-associated macrophage (TAM) recruitment to the TME. High levels of lactate also drive transcriptional reprogramming in TAMs via ENSA-STAT3 signaling, promoting an immunosuppressive environment. Targeting ENSA-K63la or CCL2 enhances the efficacy of ICB therapy in murine and humanized pancreatic tumor models. In conclusion, elevated lactylation reshapes the TME and promotes immunotherapy resistance in PDAC. Therapeutic approach targeting ENSA-K63la or CCL2 has shown promise in sensitizing pancreatic cancer immunotherapy.

Targeting MXD1 sensitises pancreatic cancer to trametinib

BackgroundThe resistance of pancreatic ductal adenocarcinoma (PDAC) to trametinib therapy limits its clinical use. However, the molecular mechanisms underlying trametinib resistance in PDAC remain unclear.ObjectiveWe aimed to illustrate the mechanisms...

IRE1 is a Promising Therapeutic Target in Pancreatic Cancer.

Pancreatic cancer (PC) is one of the most aggressive malignancies, characterized by an increasing incidence and unfavorable prognosis. Despite recent advances, surgical resection combined with chemotherapy remains the only potentially curative therapeutic option. Therefore, it is of paramount importance to identify novel therapeutic targets and develop effective treatment strategies. Pancreatic ductal adenocarcinoma (PDAC), the most prevalent form of PC, originates from exocrine cells and is subjected to both intrinsic and extrinsic cellular stresses, including oncogene activation, loss of tumor suppressors, a hypoxic and immunosuppressive tumor microenvironment (TME), and chemotherapy, causing an accumulation of misfolded proteins within the endoplasmic reticulum (ER). The loss of ER proteostasis activates the unfolded protein response (UPR), an intracellular sensing-signaling network that enables cancer cells to alleviate ER stress and restore cellular proteostasis. The key UPR sensor Inositol-Requiring Enzyme 1 (IRE1) is an ER membrane protein that activates the transcription factor X-Box Protein 1 Spliced (XBP1s) through its cytoplasmic kinase-RNase module, promoting protein folding, secretion capacity, and proteasomal degradation of misfolded proteins. Additionally, it regulates IRE1-dependent decay (RIDD) of various mRNA and functions through scaffold interactions. In this review, we synthesize current evidence on the cell-autonomous and cell-non-autonomous roles of IRE1 in tumor initiation, progression, metastasis, and drug resistance in PDAC and outline key research directions to investigate IRE1 as a potential therapeutic target.

FOXM1-Driven CKS1B Upregulation Promotes Pancreatic Cancer Progression and Therapeutic Resistance.

Pancreatic ductal adenocarcinoma (PDAC) remains a highly lethal malignancy with limited treatment options. Investigating novel therapeutic targets and understanding mechanisms of chemoresistance are crucial for improving patient outcomes. This study investigated the role of CKS1B in PDAC carcinogenesis, stemness and chemoresistance, and explores the underlying mechanisms driving its upregulation. The findings may provide novel therapeutic insights and potential strategies for the treatment of PDAC. Methods: CKS1B expression was analyzed in PDAC tissues and cell lines, its impact on cell proliferation, migration, apoptosis, stemness and chemosensitivity were evaluated by using in vitro and in vivo models, and its underlying mechanistic connection to transcription factor FOXM1 was explored by using molecular biology methods. Results: CKS1B was significantly upregulated in PDAC tissues and correlated with poor patient survival. CKS1B promoted PDAC cell proliferation, migration, and inhibited apoptosis. Expression of CKS1B enhanced the stemness properties of pancreatic cancer. CKS1B knockdown sensitized PDAC cells to the treatment of gemcitabine and oxaliplatin. Mechanistically, CKS1B is transcriptionally regulated by FOXM1, establishing a novel FOXM1-CKS1B signaling axis that regulates carcinogenesis, proliferation, migration, stemness, apoptosis, and drug resistance in PDAC. Conclusions: Our findings strongly suggest that CKS1B plays a critical role in PDAC progression, stemness and chemoresistance. Targeting the FOXM1-CKS1B axis represents a promising therapeutic strategy for PDAC patients.

Olaparib and Radiotherapy Induce Type I Interferon- and CD8+ T Cell-Dependent Sensitization to Immunotherapy in Pancreatic Cancer.

PARP inhibitors sensitize pancreatic ductal adenocarcinoma (PDAC) to radiation by inducing DNA damage and replication stress. These mechanisms also have the potential to enhance radiation-induced type I interferon (T1IFN)-mediated antitumoral immune responses. We hypothesized that the PARP inhibitor olaparib would also potentiate radiation-induced T1IFN to promote antitumor immune responses and sensitization of otherwise resistant PDAC to immunotherapy. To test this hypothesis, we assessed the effects of olaparib and radiation on T1IFN production and sensitivity to αPD-L1 immunotherapy, as well as on the tumor microenvironment by single-cell RNA sequencing. We found that olaparib enhanced T1IFN production after radiation and had superior therapeutic efficacy in immunocompetent models. Olaparib and radiation treatment sensitized PDAC tumors to αPD-L1, resulting in decreased tumor burden and a 33% complete response rate. Combination treatment provided durable immune responses as shown by tumor rejection upon tumor rechallenge of previously cured mice. Furthermore, single-cell RNA sequencing analysis revealed that combination treatment induced an immunogenic tumor microenvironment characterized by interferon (IFN) responses in both PDAC and myeloid cell populations, macrophage polarization, and increased CD8+ terminal effector T-cell frequency and activity, findings which were confirmed by IHC and flow cytometry. Furthermore, CD8+ T cells and T1IFN signaling were required for therapeutic efficacy as host depletion of CD8+ T cells or the T1IFN receptor diminished treatment responses. Overall, our results indicate that olaparib enhances radiation-induced T1IFN-mediated immune signaling and subsequently an adaptive immune response, thus sensitizing pancreatic cancer to αPD-L1 therapy, supporting an ongoing clinical trial of this therapy in patients with PDAC.

ATR inhibition potentiates FOLFIRINOX cytotoxic effect in models of pancreatic ductal adenocarcinoma by remodelling the tumour microenvironment.

In pancreatic ductal adenocarcinoma (PDAC), the dense stroma rich in cancer-associated fibroblasts (CAFs) and the immunosuppressive microenvironment confer resistance to treatments. To overcome such resistance, we tested the combination of FOLFIRINOX (DNA damage-inducing chemotherapy drugs) with VE-822 (an ataxia-telangiectasia and RAD3-related inhibitor that targets DNA damage repair). PDAC spheroid models and organoids were used to assess the combination effects. Tumour growth and the immune and fibrotic microenvironment were evaluated by immunohistochemistry, single-cell analysis and spatial proteomics in patient-derived xenograft (PDX) and orthotopic immunocompetent KPC mouse models. The FOLFIRINOX and VE-822 combination had a strong synergistic effect in several PDAC cell lines, whatever their BRCA1, BRCA2 and ATM mutation status and resistance to standard chemotherapy agents. This was associated with high DNA damage and inhibition of DNA repair signalling pathways, leading to increased apoptosis. In immunocompetent and PDX mouse models of PDAC, the combination inhibited tumour growth more effectively than FOLFIRINOX alone. This was associated with tumour microenvironment remodelling, particularly decreased proportion of fibroblast activated protein-positive CAFs and increased anti-tumorigenic immune cell infiltration and interaction. The FOLFIRINOX and VE-822 combination is a promising strategy to improve FOLFIRINOX efficacy and overcome drug resistance in PDAC.

MiR-210 Mediated Hypoxic Responses in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one among the most lethal malignancies due to its aggressive behavior and resistance to conventional therapies. Hypoxia significantly contributes to cancer progression and therapeutic resistance of PDAC. microRNAs (miRNAs/miRs) have emerged as critical regulators of various biological processes. miR-210 is known as the "hypoxamir" due to its prominent role in cellular responses to hypoxia. In this study, we investigated the multifaceted role of miR-210 in PDAC using miR-210 knockout (KO) cellular models to elucidate its functions under hypoxic conditions. Hypoxia-inducible factor-1α (HIF1-α), a key transcription factor activated in response to low oxygen levels, upregulates miR-210. miR-210 maintains cancer stem cell (CSC) phenotypes and promotes epithelial-mesenchymal transition (EMT), which is essential for tumor initiation, metastasis, and therapeutic resistance. Our findings demonstrate that miR-210 regulates the expression of CSC markers, such as CD24, CD44, and CD133, and EMT markers, including E-cadherin, Vimentin, and Snail. Specifically, depletion of miR-210 reversed EMT and CSC marker expression levels in hypoxic Panc-1 and MiaPaCa-2 PDAC cells. These regulatory actions facilitate a more invasive and treatment-resistant PDAC phenotype. Understanding the regulatory network involving miR-210 under hypoxic conditions may reveal new therapeutic targets for combating PDAC and improving patient outcomes. Our data suggest that miR-210 is a critical regulator of HIF1-α expression, EMT, and the stemness of PDAC cells in hypoxic environments.

Abstract PR005: Modulating the immunosuppressive pancreas tumor microenvironment through intratumoral delivery of cytokine-encoding mRNAs

Abstract Pancreatic ductal adenocarcinoma (PDAC) is an extremely aggressive cancer, with a mere 13% average 5-year survival rate. The hallmark desmoplastic stromal response in PDAC leads to poor vascularization, drug delivery challenges, and impaired cytotoxic immune cell infiltration, contributing to de novo therapy resistance. Our previous research has shown therapeutic induction of cellular senescence with RAS pathway targeting therapies can induce cytokine and chemokine production through the senescence-associated secretory phenotype (SASP) that can effectively activate anti-tumor T cell immunity and responses to anti-PD-1 immune checkpoint blockade. To build on these findings as well as overcome limitations associated with senescence-inducing therapy toxicity and induction of pro-tumorigenic SASP cytokines, here we leveraged mRNA technology to deliver specific SASP cytokines and chemokines we have found to stimulate immune responses directly into the suppressive PDAC TME as an immune oncology platform. We created an in vitro transcription pipeline for mRNA production and multiplexing, and demonstrate we can intratumorally deliver multiple mRNAs simultaneously into the TME of transplanted and autochthonous PDAC mouse models, stimulating the local production of cytokines normally absent in PDAC. We further characterized a novel combination of five cytokines and chemokines that can effectively recruit and activate both innate and adaptive immune responses against PDAC. Through repeated bi-weekly dosing, this mRNA combination also promotes tumor destruction and improves survival outcomes. Considering the recent success of off-the-shelf neoantigen mRNA vaccines in early human PDAC patient trials, we have now developed mRNAs that encode PDAC-specific antigens. By integrating our cytokine mRNA platform with antigen mRNAs, we observe a significant increase in intratumoral antigen-presenting dendritic cell populations and a substantially enhanced T-cell mediated anti-tumor immune response in orthotopic transplant models of PDAC. Overall, this study not only unveils pivotal insights into the cytokines absent in PDAC that are crucial for promoting anti-tumor immunity, but also pioneers an innovative immunotherapy approach. This platform has the potential to be integrated with existing immunotherapy modalities, addressing the longstanding challenge of therapy resistance in PDAC. Citation Format: Chaitanya Naimesh Parikh, Kelly De Marco, Boyang Ma, Katherine Murphy, Loretah Chibaya, Lin Zhou, Youwei Qiao, Griffin Kane, Li Li, Wen Xue, Marcus Ruscetti. Modulating the immunosuppressive pancreas tumor microenvironment through intratumoral delivery of cytokine-encoding mRNAs [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: RNAs as Drivers, Targets, and Therapeutics in Cancer; 2024 Nov 14-17; Bellevue, Washington. Philadelphia (PA): AACR; Mol Cancer Ther 2024;23(11_Suppl):Abstract nr PR005.

Epigenetic Activation of the CMTM6-IGF2BP1-EP300 Positive Feedback Loop Drives Gemcitabine Resistance in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant tumor with a dismal prognosis. Gemcitabine-based chemotherapy has emerged as a first-line treatment for PDAC. However, the development of gemcitabine resistance often results in therapeutic failure. In order to uncover the underlying mechanisms of gemcitabine resistance, gemcitabine-resistant PDAC cell lines and patient-derived xenograft (PDX) models are established and subjected to RNA sequencing. It is found that CMTM6 is closely related to gemcitabine resistance in PDAC. Multi-omics analysis revealed that EP300-mediated H3K27ac modification is involved in the transcriptional activation of CMTM6, which maintains IGF2BP1 expression by preventing its ubiquitination. The m6A reader IGF2BP1 stabilizes the EP300 and MYC mRNAs by recognizing m6A modifications, forming a positive feedback loop that enhances tumor stemness and ultimately contributes to PDAC resistance. The combined application of the EP300 inhibitor inobrodib and gemcitabine exerts a synergistic effect on PDAC. Overall, these findings reveal that the EP300-CMTM6-IGF2BP1 positive feedback loop facilitates gemcitabine resistance via epigenetic reprogramming and the combined use of inobrodib and gemcitabine represents a promising strategy for overcoming chemoresistance in PDAC, warranting further investigation in clinical trials.

The Role of Dicer Phosphorylation in Gemcitabine Resistance of Pancreatic Cancer.

Dicer, a cytoplasmic type III RNase, is essential for the maturation of microRNAs (miRNAs) and is implicated in cancer progression and chemoresistance. Our previous research demonstrated that phosphorylation of Dicer at S1016 alters miRNA maturation and glutamine metabolism, contributing to gemcitabine (GEM) resistance in pancreatic ductal adenocarcinoma (PDAC). In this study, we focused on the role of Dicer phosphorylation at S1728/S1852 in GEM-resistant PDAC cells. Using shRNA to knock down Dicer in GEM-resistant PANC-1 (PANC-1 GR) cells, we examined cell viability through MTT and clonogenic assays. We also expressed phosphomimetic Dicer 2E (S1728E/S1852E) and phosphomutant Dicer 2A (S1728A/S1852A) to evaluate their effects on GEM resistance and metabolism. Our results show that phosphorylation at S1728/S1852 promotes GEM resistance by reprogramming glutamine metabolism. Specifically, phosphomimetic Dicer 2E increased intracellular glutamine, driving pyrimidine synthesis and raising dCTP levels, which compete with gemcitabine's metabolites. This metabolic shift enhanced drug resistance. In contrast, phosphomutant Dicer 2A reduced GEM resistance. These findings highlight the importance of Dicer phosphorylation in regulating metabolism and drug sensitivity, offering insights into potential therapeutic strategies for overcoming GEM resistance in pancreatic cancer.

Hypoxic and acidic tumor microenvironment-driven AVL9 promotes chemoresistance of pancreatic ductal adenocarcinoma via the AVL9-IκBα-SKP1 complex

Background & AimsGemcitabine combined with albumin-paclitaxel (AG) is a crucial therapeutic option for pancreatic ductal adenocarcinoma (PDAC). However, the response to chemotherapy is relatively poor, with rapid development of resistance. The aim of this study was to explore the mechanism of resistance to AG and to develop strategies that can sensitize the AG regimen. MethodsWe utilized organoid models, patient-derived xenografts (PDX), and genetically engineered mouse models (GEMM) in our study. Chromatin-Immunoprecipitation (Ch-IP), double luciferase assay, Co-immunoprecipitation (Co-IP), and far-western blotting analysis were performed to investigate the mechanism. The AVL9 inhibitors were identified through protein structure analysis and molecular docking analysis, and their efficacy was verified in PDX, PDOX, and KPC models. ResultsThrough multi-strategy screening, we identified AVL9 as a key target for AG resistance in PDAC. Its tumor-promoting effects were confirmed in our clinical cohorts. Mechanistically, HIF-1α, a hypoxia-related transcription factor, drives the expression of AVL9. AVL9 acts as a scaffold that facilitates the binding of IκBα to SKP1, leading to enhanced ubiquitination and degradation of IκBα, which further activates the NF-κB pathway. The potential AVL9-targeting inhibitor, Edotecarin, was shown to reverse AG chemo-resistance in PDAC. ConclusionAVL9 expression is driven by HIF1α in PDAC. The physical interaction of AVL9, IκBα, and SKP1 provides a novel molecular mechanism for the abnormal activation of the NF-κB pathway. Therefore, the AVL9-targeting drug Edotecarin could be a promising therapeutic strategy for sensitizing PDAC to AG.

NFAT5 governs cellular plasticity-driven resistance to KRAS-targeted therapy in pancreatic cancer.

Resistance to KRAS therapy in pancreatic ductal adenocarcinoma (PDAC) involves cellular plasticity, particularly the epithelial-to-mesenchymal transition (EMT), which poses challenges for effective targeting. Chronic pancreatitis, a known risk factor for PDAC, elevates TGFβ levels in the tumor microenvironment (TME), promoting resistance to KRAS therapy. Mechanistically, TGFβ induces the formation of a novel protein complex composed of SMAD3, SMAD4, and the nuclear factor NFAT5, triggering EMT and resistance by activating key mediators such as S100A4. Inhibiting NFAT5 attenuates pancreatitis-induced resistance to KRAS inhibition and extends mouse survival. Additionally, TGFβ stimulates PDAC cells to secrete CCL2, recruiting macrophages that contribute to KRAS bypass through paracrine S100A4. Our findings elucidate the role of TGFβ signaling in EMT-associated KRAS therapy resistance and identify NFAT5 as a druggable target. Targeting NFAT5 could disrupt this regulatory network, offering a potential avenue for preventing resistance in PDAC.

The interaction between UBR7 and PRMT5 drives PDAC resistance to gemcitabine by regulating glycolysis and immune microenvironment

Pancreatic ductal adenocarcinoma (PDAC) is a common malignant tumor of the digestive tract. Although gemcitabine and other therapeutic agents are effective in patients with advanced and metastatic pancreatic cancer, drug resistance has severely limited their use. However, the mechanisms of gemcitabine resistance in pancreatic cancer are poorly understood. In this study, ATAC-seq, ChIP-seq, and RNA-seq were performed to compare chromatin accessibility and gene expression in a patient-derived tumor xenograft (PDX) model of pancreatic cancer with or without gemcitabine resistance. Analyzing these sequencing data, we found a dramatic change in chromatin accessibility in the PDX model of gemcitabine-resistant tissues and identified a key gene, UBR7, which plays an important role in mediating gemcitabine resistance. Further research found that depletion of UBR7 significantly increased pancreatic carcinogenesis and the immunosuppressive microenvironment. Mechanistically, depleted UBR7 increased the stability of PRMT5, thereby promoting glycolysis in pancreatic cancer cells. Finally, an inhibitor that blocks PRMT5 (DS-437) significantly reduced gemcitabine resistance in pancreatic cancer caused by UBR7 depletion. In conclusion, our results illustrate that the UBR7-PRMT5 axis is a key metabolic regulator of PDAC and a promising target for the clinical treatment of gemcitabine resistance in PDAC.

Pirfenidone antagonizes TGF-β1-mediated gabapentin resistance via reversal of desmoplasia and the ‘cold’ microenvironment in pancreatic cancer

Owing to the desmoplastic stroma constituted by cancer-associated fibroblasts (CAFs), few immune cells infiltrate the pancreatic ductal adenocarcinoma (PDAC). Gabapentin can impede the production of ketoacids by CAFs to support cancer cells. However, in our study, we discovered a dose-dependent increase in transforming growth factor β1 (TGF-β1) levels in cancer cells in response to gabapentin. This reverse increase of TGF-β1 contributes to 'Gabapentin-resistance', leading to the antitumor effects on PDAC cell lines are negatively negotiated in the presence of pancreatic stellate cells. Pirfenidone synergistically inhibited the growth and apoptosis resistance of PDAC when combined with Gabapentin. In a mouse orthotopic PDAC model, Fe3+-mediated coordination nanodrugs, which contain gabapentin, pirfenidone and the natural polyphenol (EGCG), efficiently promoted the infiltration of naïve CD8+ T cells (CD44lowCD62Lhigh) and the accumulation of inflammatory CAFs (α-SMAlowIL-6high). This led to a nearly two-fold increase in survival compared to the control. Furthermore, we identified a new subpopulation as Hmox1highiCAFs following treatment with our nanodrugs. Hmox1highiCAFs overexpressed the Cxcl10 receptor (Sdc4) and facilitated functional CD8+ T-cell infiltration through the Tnfsf9-Tnfrsf9 axis. Overall, our nanodrugs reshape the phenotype of CAFs and enhance functional CD8+ T-cell infiltration into tumors, holding the potential to be a safe and promising therapy for PDAC.

Exploring epigenetic dynamics unveils a super-enhancer-mediated NDRG1-β-catenin axis in modulating gemcitabine resistance in pancreatic cancer

Chemoresistance remains a formidable challenge in pancreatic ductal adenocarcinoma (PDAC) treatment, necessitating a comprehensive exploration of underlying molecular mechanisms. This work aims to investigate the dynamic epigenetic landscape during the development of gemcitabine resistance in PDAC, with a specific focus on super-enhancers and their regulatory effects. We employed well-established gemcitabine-resistant (Gem-R) PDAC cell lines to perform high-throughput analyses of the epigenome, enhancer connectome, and transcriptome. Our findings revealed notable alterations in the epigenetic landscape and genome architecture during the transition from gemcitabine-sensitive to -resistant PDAC cells. Remarkably, we observed substantial plasticity in the activation status of super-enhancers, with a considerable proportion of these cis-elements becoming deactivated in chemo-resistant cells. Furthermore, we pinpointed the NDRG1 super-enhancer (NDRG1-SE) as a crucial regulator in gemcitabine resistance among the loss-of-function super-enhancers. NDRG1-SE deactivation induced activation of WNT/β-catenin signaling, thereby conferring gemcitabine resistance. This work underscores a NDRG1 super-enhancer deactivation-driven β-catenin pathway activation as a crucial regulator in the acquisition of gemcitabine-resistance. These findings advance our understanding of PDAC biology and provide valuable insights for the development of effective therapeutic approaches against chemoresistance in this malignant disease.

IL15RA-STAT3-GPX4/ACSL3 signaling leads to ferroptosis resistance in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) is a highly malignant disease with a poor prognosis, and the lack of effective treatment methods accounts for its high mortality. Pancreatic stellate cells (PSCs) in the tumor microenvironment play an important role in the development of PDAC. Previous studies have reported that patients with PDAC are more vulnerable to ferroptosis inducers. To investigate the relationship between PSCs and pancreatic cancer cells, a coculture system is used to further reveal the influence of PSCs on ferroptosis resistance in PDAC using many in vitro and in vivo experiments. Our results show that PSCs promote ferroptosis resistance in pancreatic cancer cells. We further demonstrate that IL15 secretion by PSCs activates the IL15RA-STAT3-GPX4/ACSL3 axis. The simultaneous upregulation of GPX4 and ACSL3 prevents lipid peroxidation and ultimately protects pancreatic cancer cells from ferroptosis both in vitro and in vivo. This study demonstrates that PSCs protect pancreatic cancer cells in a paracrine manner and may indicate a novel strategy for the treatment of PDAC.

G3BP2 promotes tumor progression and gemcitabine resistance in PDAC via regulating PDIA3-DKC1-hENT in a stress granules-dependent manner.

Pancreatic ductal adenocarcinoma (PDAC) is distinguished by its aggressive malignancy, limited treatment avenues and a tendency towards chemotherapy resistance, underscoring the critical need for advanced research to uncover new therapeutic approaches. Stress granules (SGs) that is implicated in cellular self-protection mechanism, along with its associated family molecules have shown pro-cancer effects and are closely related to tumor chemotherapy resistance. In this study we investigated the relationship between Ras GTPase-activating protein-binding proteins 2 (G3BP2), a core component of SGs, and the malignancy of PDAC as well as its resistance to the chemotherapy drug gemcitabine. Analyzing TCGA dataset revealed that the expression of G3BP1 and G3BP2 was significantly upregulated in PDAC compared with adjacent normal pancreatic tissues, and the high expression of G3BP2 rather than G3BP1 was significantly associated with poorer overall survival (OS) in PDAC patients. We demonstrated that knockdown of G3BP2 inhibited the proliferation and invasion of PANC-1 and CFPAC-1 cells in vitro and in vivo. By analyzing the differentially expressed genes in G3BP2 knockdown and overexpressed PANC-1 cells, we identified DKC1 that was associated with RNA stability and regulation as the target of G3BP2. We demonstrated that G3BP2 bound to PDIA3 mRNA and recruited them into SGs, increasing the stability of PDIA3 mRNA and attenuating its translation efficiency, thereby promoting DKC1 expression. Furthermore, DKC1 could bind to hENT mRNA and inhibited its expression, which enhanced gemcitabine resistance of PDAC. Therefore, we propose a novel mechanism wherein G3BP2 facilitates PDAC's resistance to chemotherapy by modulating PDIA3-DKC1-hENT in a SGs-dependent way, suggesting G3BP2 SGs a protentional therapeutic target for the treatment in PDAC.

Abstract C034: MUC1 positive extracellular vesicles play an important role on gemcitabine resistance in PDAC

Abstract Introduction Pancreatic ductal adenocarcinoma (PDAC) is among the deadliest epithelial malignancies. The high death rate caused by PDAC results from complex factors, including difficulties in diagnosing early-stage disease, increased metastatic potential, and resistance to traditional therapies. Mucin1 (MUC1) is one of the transmembrane mucins typically expressed on the apical surface of epithelial cells but is not apically restricted and upregulated dramatically in pancreatic cancer cells. Our study showed that MUC1CT positive EVs promote tumor cell proliferation and migration in vitro, in vivo tumor growth, and metastasis. Moreover, MUC1 increases glucose metabolism in pancreatic cancer cell to resistance gemcitabine though stabilizing HIF-1α. However, the role of MUC1 in gemcitabine resistance has not been well studied and understood. Methods We used CRISPR-Cas9 to knockout MUC1 in pancreatic cancer cells to investigate MUC1 effects on chemotherapy resistance. EVs were isolated from MUC1 positive cells and MUC1 knockout cells by a sequence of centrifugation and ultracentrifugation steps. Proteomics and liquid chromatography-mass spectrometry-based metabolomics were performed to identify specific proteins and metabolites in MUC1-associated EVs. To evaluate the cargo of MUC1+ EVs and MUC1- EVs on chemotherapy resistance, 13C- labeled glucose with metabolomics was applied to trace glucose-related metabolites when the cells were treated with or without gemcitabine and EVs. Results We identified that MUC1 increased gemcitabine resistance in pancreatic cancer cells. MUC1 positive EVs could increase gemcitabine resistance in MUC1 knockout cells. Unique protein cargo in MUC1-positive EVs was enriched in DNA replication and DNA repair pathways. Phosphogluconate dehydrogenase (PGD) was packaged in MUC1-positive EVs, which helped glucose-derived ribose to increase MUC1 knockout cell gemcitabine resistance. Metabolomics in EVs showed that pyrimidine and purine nucleotides biosynthetic pathway-related metabolites are enriched in MUC1-positive EVs. Uridine is one of the important metabolites in MUC1-positive EVs. Our data showed that the pentose phosphate pathway (PPP) and pyrimidine metabolism were involved in MUC1- positive EV-induced gemcitabine resistance. Citation Format: Ying Huang, Dezhen Wang, Chunbo He, Bo Tu, Kamiya Mehla, Pankaj K. Singh, Michael A. Hollingsworth. MUC1 positive extracellular vesicles play an important role on gemcitabine resistance in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C034.