Clinical Pancreatic Ductal Adenocarcinoma Research Articles

SLC7A11 and the glutathione pathway as novel prognostic markers in resectable pancreatic ductal adenocarcinoma: A metabolomics study of clinical specimens

Background/objectivesDespite the poor prognosis associated with pancreatic ductal adenocarcinoma (PDAC), there remains a lack of clarity regarding the metabolic pathways and their significant impact on its phenotype. Therefore, we aimed to utilize metabolomics to capture changes in clinical PDAC tissues and elucidate the significant metabolic pathways close to its phenotypes. MethodsThis basic research was retrospectively validated using database research, immunohistochemistry, and protein analysis based on the findings obtained from metabolomics using clinical tissues collected from prospectively registered patients with PDAC. mRNA expression analysis using a database and protein analysis using archived clinical specimens was performed to validate the candidate pathways identified using metabolomics. Between-group comparisons were analyzed using paired t-tests and log-rank test, and Kaplan–Meier curves illustrated survival times. ResultsPatients subjected to metabolomics revealed a significant increase in glutathione disulfide levels in PDAC tissues when compared to normal pancreatic tissues. The Cancer Genome Atlas database analysis revealed significant changes in glutathione pathway-related mRNAs in PDAC compared to that in the normal pancreas. Protein analysis of previously resected specimens demonstrated a significant increase in SLC7A11 expression in PDAC tissues. The abundance ratio of SLC7A11 isoforms was associated with the post-operative prognosis in resectable PDAC. ConclusionGlutathione disulfide levels were significantly increased in clinical PDAC metabolomics. Additionally, increased mRNA and protein expression in SLC7A11 was observed in PDAC. Furthermore, the SLC7A11 isoform abundance ratio may be a valuable prognostic marker in patients with resectable PDAC.

Abstract A061: A novel genetically engineered mouse model of Myc-driven pancreatic cancer recapitulates phenotypic heterogeneity, metastasis, and therapy resistance seen in clinical populations

Abstract One of the most aggressive cancers in the US is pancreatic ductal adenocarcinoma (PDAc), whose incidence is expected to rise in the coming years. The standard-of-care treatments have improved over the past few decades, but they still primarily involve harsh chemotherapy cocktails and have only marginally increased overall survival, to a 5-year survival rate of 12 percent. c-MYC (MYC), a potent transcription factor, is frequently dysregulated in PDAc. In PDAc and other cancers, MYC plays a significant role in tumor progression, and its dysregulation has been linked to tumor aggressiveness and therapeutic resistance. In various mouse models of cancer, it has been demonstrated that oncogenic MYC expression controls components of the tumor microenvironment (TME). MYC expression in PDAc has been associated with a desmoplastic immune suppressive TME, though the precise mechanism is still unknown. In this study, we introduce a novel genetically modified mouse model (GEMM) of PDAc that can be used to simulate the disease's heterogeneity and investigate how MYC controls the tumor’s immune and stromal microenvironments. In the pancreas of our KMCERT2 model, both mutant Kras and lowly deregulated Myc are expressed through inducible Cre-driven mechanisms. Our research demonstrates how deregulated MYC cooperates with oncogenic KRASG12D in the adult pancreas to promote PDAc, faithfully reproducing the inter- and intra-tumoral heterogeneity observed in clinical PDAc populations. Currently, an embryonic KrasG12D- and p53 loss/mutant-driven PDAc model (KPC) is used for the majority of murine studies of PDAc. To learn more about the mechanisms underlying observed phenotypes in our KMCERT2 model, RNA- and DNA sequencing were performed on microdissected autochthonous tumor specimens as well as KMCERT2 tumor-derived cell lines. In contrast to the KPC model, our inducible KMCERT2 model of PDAc exhibits genetic alterations, such as CDKN2A loss, comparable to human disease. A distinct loss of nuclear p53 expression is also observed. Intriguingly, multiplexed immunohistochemistry analysis of immune cell composition showed that MYC-driven tumors have a higher density of antigen-presenting cells (APCs) when compared to spontaneous KPC tumors. Standard-of-care medications like gemcitabine and FOLFIRINOX are frequently ineffective against human PDAc. Orthotopic therapeutic studies using our KMCERT2 tumor-derived cell lines show a variety of responses to these therapies, with both resistant and sensitive tumor models, allowing us to use this model to better understand the mechanisms causing therapeutic resistance and to test new therapies. Additionally, we observe consistent liver metastasis in both spontaneous and orthotopic transplant settings. Together, these studies investigate the function of dysregulated MYC expression in murine PDAc metastatic behavior, immune phenotypes, and therapeutic response. It also offers mouse models of PDAc that are both spontaneous and orthotopic, recapitulating the complex and highly metastatic nature of the human disease. Citation Format: Isabel A. English, Patrick J. Worth, Kevin A. MacPherson, Michael B. Heskett, Vidhi Shah, Jackie L. Phipps, Courtney Betts, Shamilene Sivagnanam, Carl Pelz, Amy S. Farrell, Brittany L. Allen-Petersen, Meghan M. Joly, Motoyuki Tsuda, Colin J. Daniel, Jenny Eng, Xiaoyan Wang, Ashley L. Kiemen, Laura D. Wood, Terry K. Morgan, Mary C. Thoma, Jennifer M. Chu, Paul T. Spellman, Lisa M. Coussens, Ellen M. Langer, Rosalie C. Sears. A novel genetically engineered mouse model of Myc-driven pancreatic cancer recapitulates phenotypic heterogeneity, metastasis, and therapy resistance seen in clinical populations [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr A061.

DDX18 drives tumor immune escape through transcription-activated STAT1 expression in pancreatic cancer.

Pancreatic ductal adenocarcinoma (PDAC) resists to current treatments due to its inherent tumor heterogeneity, therapy-resistant cancer stem/initiating cells survival, and immune evasion in the immunosuppressive tumor microenvironment (TME). Here, the results show that clinical PDAC and adjacent tissues undergo distinct chromatin remodeling. Multiple omics analysis revealed DEAD-box RNA helicase 18 (DDX18), a carcinogenic gene with similar H3K4me3 profile, is up-regulated and correlates with poor survival in PDAC patients. We validated that DDX18 deposits on the STAT1 promoter region and counteracts H3K27me3 deposition on the STAT1 promoter sequence by modulating the formation of the PRC2 complex to up-regulate the expression of STAT1, which results in the up-regulation of PD-L1 expression, T lymphocyte accumulation and overactivation in the highly desmoplastic and immunosuppressive pancreatic TME. DDX18-STAT1 axis inhibition also affects stemness of cancer cells, epithelial-mesenchymal transition (EMT) and disrupts the immunosuppressive TME simultaneously, producing sustained remissions of aggressive PDAC by synergizing with anti-PD-L1 therapy. Combining DDX18 inhibition with anti-PD-L1 immunochemotherapy to treat PDAC patients will pave a new way for clinical treatment of patients with PDAC. This study found that clinical PDAC and adjacent pancreatic tissues undergo distinct chromatin remodeling featured by the upregulation of DEAD-box RNA helicase 18 (DDX18). We further validated that DDX18 deposits on the STAT1 promoter region and counteracts H3K27me3 deposition on the STAT1 promoter by modulating the formation of the PRC2 complex to up-regulate the expression of STAT1. DDX18-STAT1 axis enhances the stemness of cancer cells, the upregulation of PD-L1 expression, T lymphocyte accumulation and overactivation in the highly desmoplastic and immunosuppressive pancreatic TME.

Survival outcomes of neoadjuvant therapy followed by radical resection versus upfront surgery for stage I-III pancreatic ductal adenocarcinoma: a retrospective cohort study.

Neoadjuvant therapy remains controversial in treating resectable pancreatic ductal adenocarcinoma (PDAC) patients. This study aims to assess the impact of neoadjuvant therapy on survival in patients with PDAC according to their clinical stage. Patients with resected clinical Stage I-III PDAC from 2010 to 2019 were identified in the Surveillance, Epidemiology, and End Results database. A propensity score matching method was utilized within each stage to reduce potential selection bias between patients who underwent neoadjuvant chemotherapy followed by surgery and patients who underwent upfront surgery. An overall survival (OS) analysis was performed using the Kaplan-Meier method and a multivariate Cox proportional hazards model. A total of 13674 patients were included in the study. The majority of the patients (N=10715, 78.4%) underwent upfront surgery. Patients receiving neoadjuvant therapy followed by surgery had significantly longer OS than those with upfront surgery. Subgroup analysis revealed that the neoadjuvant chemoradiotherapy group's OS is comparable to neoadjuvant chemotherapy. In clinical Stage IA PDAC, there was no difference in survival between the neoadjuvant treatment and upfront surgery groups before or after matching. In Stage IB-III patients, neoadjuvant therapy followed by surgery improved OS before and after matching compared to upfront surgery. The results revealed the same OS benefits using the multivariate Cox proportional hazards model. Neoadjuvant therapy followed by surgery could improve OS over upfront surgery in Stage IB-III PDAC but did not provide a significant survival advantage in Stage IA PDAC.

Scaffolding Protein Connector Enhancer of Kinase Suppressor of Ras 1 (CNKSR1) Regulates MAPK Inhibition Responsiveness in Pancreas Cancer via Crosstalk with AKT Signaling.

The CNKSR1 scaffold, identified within an RNAi screen as a novel mediator of resistance to MEK inhibition in pancreas cancer, connects the MAPK pathway and AKT signaling and may be adopted as a biomarker to select patients for combined MEK AKT blockade.

Liposomal co-delivery system encapsulating celastrol and paclitaxel displays highly enhanced efficiency and low toxicity against pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is the most common pancreas cancer and difficult to treat. Various combination strategies have been utilized to improve the poor therapeutic effect and serious systemic toxicity of clinical PDAC treatments. Here, we report a new combination therapy strategy against PDAC using a liposomal carrier co-loading with anti-inflammatory agent celastrol (CLT) and chemotherapeutic drug paclitaxel (PTX). First, the synergistic effect of CLT and PTX was verified and it is discovered that this effect peaks at 1:2 (CLT/PTX, mol/mol). Then, the co-delivery system CP-Lip with high loading capabilities (97.48% ± 1.14 for CLT, 86.29% ± 3.03 for PTX), sustained release profile and favorable long-circulating feature was fabricated. Tumor-specific accumulation of CP-Lip was significantly improved compared with that of free drug. Moreover, CP-Lip showed comparable cytotoxicity with free CLT and PTX (CLT/PTX) on Pan02 cells in vitro and significantly improved anti-tumor efficacy in vivo with a ∼4-fold increase of tumor inhibition rate compared to single drug treatment in mice model. Furthermore, safety evaluation found no significant damages induced by CP-Lip. Therefore, CLT-PTX combined treatment has good clinic potential, and the simple and effective CP-Lip system fabricated here offers a promising candidate for future development.

An Engineered Paper-Based 3D Coculture Model of Pancreatic Cancer to Study the Impact of Tissue Architecture and Microenvironmental Gradients on Cell Phenotype.

The spatial configuration of cells in the tumor microenvironment (TME) affects both cancer and fibroblast cell phenotypes contributing to the clinical challenge of tumor heterogeneity and therapeutic resistance. This is a particular challenge in stroma-rich pancreatic ductal adenocarcinoma (PDAC). Here, a versatile system is described to study the impact of tissue architecture on cell phenotype using PDAC as a model system. This fully human system encompassing both primary pancreatic stellate cells and primary organoid cells using the TRACER platform to allow the creation of user-defined TME architectures that have been inferred from clinical PDAC samples and are analyzed by CyTOF to characterize cells extracted from the system. High dimensional characterization using CyTOF demonstrates that tissue architecture leads to distinct hypoxia and proliferation gradients. Furthermore, phenotypic markers for both cell types are also graded in ways that cannot be explained by either hypoxia or coculture alone. This demonstrates the importance of using complex models encompassing cancer cells, stromal cells, and allowing control over architecture to explore the impact of tissue architecture on cell phenotype. It is anticipated that this model will help decipher how tissue architecture and cell interactions regulate cell phenotype and hence cellular and tissue heterogeneity.

Machine Learning-Based Desmoplastic Signatures Predict Patient Outcomes in Pancreatic Ductal Adenocarcinoma

Introduction: Pancreatic ductal adenocarcinoma (PDAC) is characterized by desmoplasia, a fibrotic matrix response with unclear impact on prognosis. We profiled clinical PDAC using two novel technologies: a machine learning algorithm to quantify matrix patterning in 436 patients and a spatial protein/cell phenotyping algorithm using CO-Detection by indEXing (CODEX). We integrated these modalities with patient metadata to define a unified, machine learning-based histologic signature for PDAC prognosis. Methods: Trichrome-stained clinical specimens were analyzed by an unsupervised algorithm for ultrastructural analysis (>13,000 images, 147 local/global fiber features), which were processed by uniform manifold approximation and projection (UMAP) and fitted to a minimum spanning tree-based pseudotime model. A custom 31-plex CODEX panel was used to visualize tumor- and stroma-associated cells by UMAP of protein expression. Cell interactions were quantified based on spatial co-localization (k=20 nearest neighbors). Binary classifiers for Poor/Good survivorship and recurrence (two- and one-year thresholds, respectively) were trained in MATLAB. Results: Matrix patterning comprised of two architectural groups (L,R), with group-R patients experiencing earlier median death and relapse by 461d and 95d, respectively. Poorer outcomes were primarily driven by three survival-negative interaction “nodes”: Type-I stromal fibroblasts, endothelial cells, and proliferating tumor cells, collectively forming two cell-pattern signatures (A,B) in which signature-B patients experienced earlier median death by 858d. A unified machine learning model successfully classified binary survivorship and recurrence with area under the curve of 0.943-0.946. Conclusion: Fibrotic architecture and cell patterning are powerful independent predictors for PDAC outcomes, with specific desmoplasia-associated cell phenotypes largely driving differential prognoses.

O-GlcNAcylation promotes pancreatic tumor growth by regulating malate dehydrogenase 1.

Oncogenic Kras-activated pancreatic ductal adenocarcinoma (PDAC) cells highly rely on an unconventional glutamine catabolic pathway to sustain cell growth. However, little is known about how this pathway is regulated. Here we demonstrate that Kras mutation induces cellular O-linked β-N-acetylglucosamine (O-GlcNAc), a prevalent form of protein glycosylation. Malate dehydrogenase 1 (MDH1), a key enzyme in the glutamine catabolic pathway, is positively regulated by O-GlcNAcylation on serine 189 (S189). Molecular dynamics simulations suggest that S189 glycosylation on monomeric MDH1 enhances the stability of the substrate-binding pocket and strengthens the substrate interactions by serving as a molecular glue. Depletion of O-GlcNAcylation reduces MDH1 activity, impairs glutamine metabolism, sensitizes PDAC cells to oxidative stress, decreases cell proliferation and inhibits tumor growth in nude mice. Furthermore, O-GlcNAcylation levels of MDH1 are elevated in clinical PDAC samples. Our study reveals that O-GlcNAcylation contributes to pancreatic cancer growth by regulating the metabolic activity of MDH1.

Abstract 1052: Augmentation of standard chemotherapy response by inhibition of Aurora kinase A in preclinical models of pancreatic cancer

Abstract Background: The prognosis of pancreatic ductal adenocarcinoma (PDAC) is very poor with a 5-year survival rate of 8%. Current standard chemotherapeutic treatment nab-paclitaxel (NPT) plus gemcitabine (Gem) has been able to give an average prognosis of 8.5 months after detection. Aurora Kinase A (AURKA), an enzyme responsible for the regulation of cytokinesis, is mutated in over 55% of PDAC. Increased expression of AURKA inhibits metabolic stress-induced autophagy, thus promoting cell survival. Additionally, amplification of AURKA has been shown to lead to taxane resistance. Through inhibition of AURKA signaling with a small-molecule inhibitor alisertib (MLN8237, ALS), we evaluated an enhancement of NPT+Gem chemotherapy response in PDAC. Methods: In vitro cell proliferation was evaluated in PDAC-associated cell lines by WST-1 assay. Protein expression was measured by Immunoblot analysis. Tumor growth and survival studies were performed using human PDAC cells AsPC-1 in NOD/SCID mice. Intratumoral mechanism of action was determined by IHC and Immunoblot analysis. Results: In AsPC-1 subcutaneous xenografts, chemotherapy regimen (NPT+Gem) and alisertib inhibited tumor growth while their combination exhibited an additive effect. The increase in tumor size was 255 mm3 in controls, 105 mm3 after NPT+Gem, 133 mm3 after ALS and 38 mm3 after NPT+Gem+ALS treatment. In peritoneal dissemination xenografts, animal survival was 23 days in controls that was increased by NPT+Gem (41 days, 78% increase), ALS (25 days, 9% increase) and NPT+Gem+ALS exhibited an additive effect (49 days, 113% increase). NPT+Gem and ALS effect on tumor cell proliferation and apoptosis corresponded with subcutaneous tumor growth data. In vitro proliferation analysis of PDAC epithelial cells (AsPC-1, Panc-1), stromal cells and endothelial cells demonstrated that NPT+Gem and ALS inhibited cell proliferation and their combination treatment had an additive effect. Immunoblot analysis of PDAC cells showed increased levels of cleaved caspase-3 and cleaved PARP-1 in combination therapy pointing to increased levels of apoptosis with NPT+Gem+ALS treatment in all cell lines tested. Conclusion: These findings demonstrate that nab-paclitaxel plus gemcitabine standard chemotherapy response can be enhanced through specific inhibition of aurora kinase A signaling by alisertib in PDAC. The data support the potential of this combinatorial therapeutic strategy for clinical PDAC therapy. Citation Format: David Kronenberger, Margaret A. Schwarz, Md Sazzad Hassan, Urs von Holzen, Roderich E. Schwarz, Niranjan Awasthi. Augmentation of standard chemotherapy response by inhibition of Aurora kinase A in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1052.

Neoadjuvant chemotherapy is associated with improved outcomes in patients with stage 1A and 1B pancreatic cancer undergoing surgery: An NCDB study.

4157 Background: The use of neoadjuvant chemotherapy (NAC) for pancreatic ductal adenocarcinoma (PDAC) has shown clear advantages in locally advanced and borderline resectable disease. The benefit in upfront resectable PDAC is debated. Moreover, in early clinical stages IA/IB, potential benefits including improved R0 resection rate, decreased tumor upstaging, and survival, are not clear. We hypothesize that NAC will be associated with improved outcomes and survival compared to adjuvant therapy in patients with clinical stage IA/IB PDAC. Methods: The National Cancer Database (NCDB) PUFs (2004-2017) were used to perform a retrospective review of patients with clinical stage IA or IB PDAC undergoing surgery. Treatment groups were selected based on timing of chemotherapy. Patients receiving chemotherapy or surgery alone were excluded. Results: We identified 6,613 patients with clinical stage IA or IB PDAC who underwent surgery. The neoadjuvant therapy group (NAT) included 1,533 patients who received neoadjuvant or perioperative chemotherapy, and the adjuvant therapy group (AT) contained 5080 patients who received chemotherapy after surgery. Patients in the NAT had higher rates of T1 and T2 disease and lower rates of T3 pathology compared to the AT (pT1: 18.7% vs 7.8%; pT2: 20.1 vs 18.6%; pT3: 59.3% vs 72.1%, p<0.0001). Additionally, the NAT had significantly higher rates of N0 disease and less N1 pathology (pN0: 54.6% vs 37.5%; pN1: 45.4% vs 62.5%, p<0.0001). The R0 resection rate was higher in the NAT (83.2% vs 62.3%, p=0.0197) and there was less lymphovascular invasion (LVI) compared to the AT (34.8% vs 48.1%, p<0.0001). Using Kaplan Meier estimates, the NAT was associated with improved overall survival (OS) compared to the AT (median OS: 33.4 vs 27.5 months, p<0.0001). On multivariable analysis, R0 resection (HR=0.715, CI: 0.619-0.825, p<0.0001), LVI (HR=1.126, 95% CI: 1.038-1.222, p=0.0043) but not receipt of NAC (HR=0.94, 95% CI: 0.852-1.038, p=0.2229) were independent risk factors for OS. Conclusions: NAC is beneficial in patients with stage IA/IB PDAC undergoing surgical resection as it is associated with improved oncologic outcomes including increased R0 resection rate, decreased tumor upstaging, lymph node metastasis, and LVI. Furthermore, patients receiving NAC were found to have improved survival over those getting adjuvant therapy. Based on these results, we recommend all patients diagnosed with PDAC be considered for NAC prior to surgery. [Table: see text]

Immortalization-upregulated protein promotes pancreatic cancer progression by regulating NPM1/FHL1-mediated cell-cycle-checkpoint protein activity

Immortalization-upregulated protein (IMUP) plays a vital role in cell proliferation and tumor progression. However, its role in pancreatic ductal adenocarcinoma (PDAC) remains unclear. Here, we select IMUP as an alternative gene based on GeneChip analysis of clinical PDAC tissues and transcriptome data from The Cancer Genome Atlas. IMUP expression is upregulated in PDAC tumor tissues. Moreover, high IMUP expression correlates with poor prognosis, while IMUP depletion inhibits PDAC cell proliferation and colony formation capacity in vitro, and decreases xenograft tumor growth in vivo. IMUP downregulation leads to cell-cycle arrest in the S phase. IMUP knockdown increases the expression of four-and-a-half LIM domain protein 1 (FHL1), which regulates the phosphorylation of cell division cycle 25A (CDC25A) by cycle checkpoint kinase 1 (CHK1) and promotes cytoplasmic distribution of CDC25A by interaction with 14–3-3ξ. Furthermore, FHL1 knockdown restores the effects induced by IMUP depletion. Liquid chromatography tandem mass spectrometry and immunoprecipitation analysis further show that IMUP interacts directly with nucleophosmin (NPM1) and enhances its stability. DNA methylation sequencing shows that FHL1 promoter methylation decreases when IMUP is downregulated. Overexpression of NPM1 can increase the methylation level of FHL1, thereby decreasing its expression. Our study provides a novel perspective on IMUP/NPM1/FHL1-mediated cell-cycle arrest by regulating CDC25A phosphorylation in PDAC. These findings may provide a new therapeutic target for PDAC.Graphical abstract

Development of PCR assays to detect signature circulating tumor DNA methylation markers and KRas mutations for pancreatic ductal adenocarcinoma (PDAC).

524 Background: PDAC is a cancer of high mortality. Accurate and cost-effective PCR assays detecting diagnostic and prognostic PDAC markers in blood are desirable for conducting PDAC screening in high-risk populations, evaluating treatments for patients and surveying for post-treatment relapse. Methods: We combined PDAC-diagnosing ctDNA methylation markers with PDAC-driving Kras mutations as targets to develop said PCR tests. Methylation markers were previously validated to classify PDAC plasma at a high accuracy by a targeted methylation sequencing assay. Seven known PDAC driver mutations in KRas exon2 were selected. Taqman-based quantitative methylation-specific PCR (MSP) and ARMS PCR were designed for methylation markers and KRas mutations, respectively, and were validated for technical performances. A step of pre-amplification of input DNA by conventional PCR was included prior to quantitative PCR to improve sensitivity. Over 200 clinical PDAC and control tissue and plasma samples were used to determine their analytical performances in classifying PDAC plasma. Results: All targets were detected at a limit-of-detection of 0.25% or better on standards DNA in PCR. To test clinical plasma samples, methylation markers were first filtered by comparing their levels in PDAC-, para-tumor tissues and whole blood cells. Twenty-four most discriminatory markers for PDAC tissues were selected and used to classify 110 PDAC plasma samples from equal number of normal samples. They were evaluated for their sensitivity at a pre-set specificity of 90%. The 4 best-performing markers were selected to build and cross-validate a general-linear-model (GLM) PDAC classifier, which achieved a sensitivity of 66% at 90% specificity (AUC = 0.829) in cross-validation. Adding status of KRas mutations further improved prediction accuracy by increasing sensitivity to 75% (AUC = 0.89), demonstrating the methylation markers and KRas mutations complement each other in detection PDAC plasma. Conclusions: Our results suggest that a small number of our DNA methylation markers can classify PDAC plasma at a reasonably high accuracy by MSP. Incorporating KRas driver mutations further improved classification accuracy. Together they are promising to be further translated into diagnostics for PDAC early screening, treatment assessment and postoperative surveillance.

Genomic classification of clinically advanced pancreatic ductal adenocarcinoma (PDAC) based on methylthioadenosine phosphorylase (MTAP) genomic loss (MTAP loss).

604 Background: MTAP loss is represented across a wide variety of cancer types including PDAC and is an emerging target for synthetic lethality-based cancer therapies. Preclinically, MTAP loss leads to the accumulation of 2-methylthioadenosine, reduced protein arginine N-methyltransferase 5 (PRMT5) methylation activity and increased vulnerability to targeting of the methionine adenosyltransferase IIα (MAT2A)/ PRMT5 axis. In addition, 9p21 loss, homozygous co-deletion of MTAP/CDKN2A or homozygous deletion of either gene have been associated with an immunologically “cold” tumor microenvironment, primary resistance to anti PD(L)1 immunotherapy (IO) and poor prognosis phenotype (Han G, Nat Commun 2021). We investigated concurrent mutations and immune biomarkers in clinical PDAC samples with MTAP-loss versus -intact status. Methods: From a series of 177705 consecutive cases, we performed comprehensive genomic profiling on 9423 cases of PDAC using an FDA-approved assay (F1CDx) to evaluate all classes of genomic alterations (GA). Tumor mutational burden (TMB) was determined on up to 1.1 Mbp of sequenced DNA and microsatellite instability (MSI) was determined on 114 loci. PD-L1 expression was determined by immunohistochemistry (Dako 22C3). Furthermore, we correlated pertinent findings within a database of 16558 cases of clinically advanced cancer with MTAP loss. Results: 2003 (21.3%) of 9423 PDAC demonstrated MTAP-loss. Similar gender, age and number of GA per tumor were observed between MTAP-loss and -intact groups. Frequencies of TP53, CDKN2A/B, SMAD4, PTEN and ARID1A were significantly higher in MTAP-loss PDAC. However, previously-described biomarkers of IO efficacy (MSI, TMB, CD274 amplification and PD-L1 expression) and resistance ( STK11, KEAP1 and MDM2) were infrequent and similar in both groups. The frequencies of other potentially targetable GA including BRCA1/2, ATM, KRAS G12C, ERBB2, BRAF, FGFR1, NF1 and PIK3CA were also infrequent and similar in both groups of PDAC patients. Amongst a database of 16558 cases of clinically advanced cancer with MTAP loss, 1538 (9.3%) featured co-alterations in MTAP and SMAD4. 52% of the MTAP/SMAD4 co-altered cases were PDAC. Conclusions: MTAP loss is associated with a distinctive concurrent genomic profile in PDAC and represents a potential new synthetic lethality-based opportunity for treatment with PRMT5 and MAT2A inhibitors. Furthermore, MTAP loss may represent an independent negative predictive biomarker for immune checkpoint inhibition in PDAC.[Table: see text]

Sequence-Activated Fluorescent Nanotheranostics for Real-Time Profiling Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC), as one of the most malignant tumors with dense desmoplastic stroma, forms a specific matrix barrier to hinder effective diagnosis and therapy. To date, a paramount challenge is in the search for intelligent nanotheranostics for such hypopermeable tumors, especially in breaking the PDAC-specific physical barrier. The unpredictable in vivo behaviors of nanotheranostics, that is, real-time tracking where, when, and how they cross the physical barriers and are taken up by tumor cells, are the major bottleneck. Herein, we elaborately design sequence-activated nanotheranostic TCM-U11&Cy@P with dual-channel near-infrared fluorescence outputs for monitoring in vivo behaviors in a sequential fashion. This nanotheranostic with a programmable targeting capability effectively breaks through the PDAC barriers. Ultimately, the released aggregation-induced emission (AIE) particle TCM-U11 directly interacts with PDAC cells and penetrates into the deep tissue. Impressively, this fluorescent nanotheranostic intraoperatively can map human clinical PDAC specimens with high resolution. We believe that this unique sequence-activated fluorescent strategy expands the repertoire of nanotheranostics in the treatment of hypopermeable tumors.

Gut-derived lipopolysaccharide remodels tumoral microenvironment and synergizes with PD-L1 checkpoint blockade via TLR4/MyD88/AKT/NF-\u03baB pathway in pancreatic cancer

Lipopolysaccharide (LPS) as an important inflammatory mediator activates the innate/adaptive immune system. The existence of LPS in pancreatic ductal adenocarcinoma (PDAC) has been reported, however, its biological function in PDAC remains unclear. Here, we demonstrated that circulating and tumoral LPS was significantly increased by intestinal leakage in the orthotopic murine PDAC model, and LPS administration promoted T cell infiltration but exhaustion paradoxically in the subcutaneous murine PDAC model. By bioinformatic analysis, Toll-like receptor 4 (TLR4), LPS receptor, was further found to enrich in immune tolerance signaling in PDAC tissues. Then, a significant positive correlation was found between TLR4 and programmed death ligand-1 (PD-L1) in clinical PDAC tissues, as well as serum LPS and tumoral PD-L1. Meanwhile, LPS stimulation in vitro and in vivo obviously upregulated tumor PD-L1 expression, and effectively promoted cancer cells resistance to T cell cytotoxicity. Mechanistically, the activation of TLR4/MyD88/AKT/NF-κB cascade was found to participate in LPS mediated PD-L1 transcription via binding to its promoter regions, which was enhanced by crosstalk between NF-κB and AKT pathways. Finally, PD-L1 blockade could significantly reverse LPS-induced immune escape, and synergized with LPS treatment. Taken together, LPS can remodel tumor microenvironment, and synergize with PD-L1 blockade to suppress tumor growth, which may be a promising comprehensive strategy for PDAC.

Circulating Tissue Polypeptide-Specific Antigen in Pre-Diagnostic Pancreatic Cancer Samples.

Simple SummaryDetecting cancer early significantly increases the chances of successful (surgical) treatment. Pancreatic cancer is one of the deadliest cancer forms, since it is usually discovered at a late and already spread stage. Finding biomarkers showing pancreatic cancer at an early stage is a possible approach to early detection and improved treatment. The aim of our study was to assess the potential of tissue polypeptide specific antigen (TPS) as a biomarker for early pancreatic cancer detection. We studied TPS levels in blood plasma samples from a population-based biobank in Västerbotten, Sweden that were collected before individuals were diagnosed with pancreatic cancer. Although TPS levels are raised at diagnosis, this occurs late, and thus TPS does not seem to hold promise as an early detection marker for pancreatic cancer.Early detection of pancreatic ductal adenocarcinoma (PDAC) is challenging, and late diagnosis partly explains the low 5-year survival. Novel and sensitive biomarkers are needed to enable early PDAC detection and improve patient outcomes. Tissue polypeptide specific antigen (TPS) has been studied as a biomarker in PDAC diagnostics, and it has previously been shown to reflect clinical status better than the ‘golden standard’ biomarker carbohydrate antigen 19-9 (CA 19-9) that is most widely used in the clinical setting. In this cross-sectional case-control study using pre-diagnostic plasma samples, we aim to evaluate the potential of TPS as a biomarker for early PDAC detection. Furthermore, in a subset of individuals with multiple samples available at different time points before diagnosis, a longitudinal analysis was used. We assessed plasma TPS levels using enzyme-linked immunosorbent assay (ELISA) in 267 pre-diagnostic PDAC plasma samples taken up to 18.8 years before clinical PDAC diagnosis and in 320 matched healthy controls. TPS levels were also assessed in 25 samples at PDAC diagnosis. Circulating TPS levels were low both in pre-diagnostic samples of future PDAC patients and in healthy controls, whereas TPS levels at PDAC diagnosis were significantly increased (odds ratio 1.03; 95% confidence interval: 1.01–1.05) in a logistic regression model adjusted for age. In conclusion, TPS levels increase late in PDAC progression and hold no potential as a biomarker for early detection.

HNRNPC impedes m6A-dependent anti-metastatic alternative splicing events in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a malignancy with poor prognosis due to early metastasis. The aberrant N6-methyladenosine (m6A) RNA modification has emerged as an important mechanism in cancer progression and metastasis, but its role in PDAC remained largely unknown. Here, we demonstrated that an m6A regulator, heterogeneous nuclear ribonucleoprotein C (HNRNPC), modulated alternative splicing events to promote PDAC metastasis. In clinical PDAC tissues, high expression of HNRNPC was correlated with metastasis, resulting in poor prognosis in PDAC patients. Knockdown of HNRNPC significantly reduced PDAC cell invasion in vitro and metastasis in vivo. In contrast, overexpression of HNRNPC provoked malignant phenotypes of PDAC cells. Mechanistically, HNRNPC antagonized the anti-metastatic isoform of TAF8 (TAF8L) but increased the pro-metastatic alternative splicing isoform of TAF8 (TAF8S). Mutation of the m6A-site of TAF8 attenuated the interaction between HNRNPC and TAF8 transcript, leading to the decrease of TAF8S. Furthermore, experimental manipulation of the anti-metastasis splicing isoform TAF8L revealed that splice isoform switching of TAF8 is crucial for PDAC metastasis. In conclusion, our findings demonstrate the essentiality of HNRNPC-mediated alternative splicing events that impinges on metastatic PDAC.

Cancer Models to Defeat Therapy Resistance in Pancreatic Ductal Adenocarcinoma

One of the largest hurdles to the efficacy of cancer therapeutics, and a main cause of relapse, is therapy resistance. In response, researchers have developed model systems to better understand therapy resistance. Cancer research employs several model systems that reflect the biology of actual human tumors: in vitro models (2D, 3D cell cultures), in vivo models (PDX, GEMMS, transgenic), proteomic models, and computational or mathematical models. One cancer that has been extensively modeled is pancreatic ductal adenocarcinoma (PDAC). PDAC is the third most common cause of annual cancer deaths in developed countries; as its incidence and mortality rates continue to increase, PDAC is projected to be the second leading cause of cancer deaths by 2030. Although chemotherapy is a pillar of clinical PDAC treatment, its outcome typically leads to multi-drug resistance, drastically restricting the curative effect of drugs for a variety of tumors. Elucidating the underlying mechanisms for resistance through different models is essential for the development of new strategies and therapies. This review provides insight into the range of in vitro and in vivo models of pancreatic cancer used in preclinical research. This paper provides an overview of platforms for cancer research with a focus on those devoted to resistance mechanisms in PDAC and to the primary therapeutic intervention for PDAC, gemcitabine (GEM).

Longitudinal Analysis of Human Pancreatic Adenocarcinoma Development Reveals Transient Gene Expression Signatures.

Previous transcriptome studies of human pancreatic ductal adenocarcinoma (PDAC) compare non-cancerous pancreatic intraepithelial neoplasias (PanIN) with late-stage PDAC obtained from different patients, thus have limited ability to discern network dynamics that contribute to the disease progression. We demonstrated previously that the 10-22 cell line, an induced pluripotent stem cell-like line reprogrammed from late-stage human PDAC cells, recapitulated the progression from PanINs to PDAC upon transplantation into NOD/LtSz-scid/IL2R-gammanull mice. Herein, we investigated the transition from precursor to PDAC using the isogenic model. We analyzed transcriptomes of genetically tagged 10-22 cells progressing from PanINs to PDAC in mice and validated the results using The Cancer Genome Atlas PDAC dataset, human clinical PanIN and PDAC tissues, and a well-established murine PDAC model. We functionally studied candidate proteins using human normal (H6C7) and cancerous (Miapaca2, Aspc1) pancreatic ductal epithelial cell lines. 10-22 cell-derived PDAC displayed the molecular signature of clinical human PDAC. Expression changes of many genes were transient during PDAC progression. Pathways for extracellular vesicle transport and neuronal cell differentiation were derepressed in the progression of PanINs to PDAC. HMG-box transcription factor 1 (HBP1) and BTB domain and CNC homolog 1 (BACH1) were implicated in regulating dynamically expressed genes during PDAC progression, and their expressions inversely correlated with PDAC patients' prognosis. Ectopic expression of HBP1 increased proliferation and migration of normal and cancerous pancreatic cells, indicating that HBP1 may confer the cell dissemination capacity in early PDAC progression. This unique longitudinal analysis provides insights into networks underlying human PDAC progression and pathogenesis. IMPLICATIONS: Manipulation of HBP1, BACH1, and RUN3 networks during PDAC progression can be harnessed to develop new targets for treating PDAC.