Target For Pancreatic Ductal Adenocarcinoma Research Articles

Single-cell transcriptome reveals the heterogeneity of malignant ductal cells and the prognostic value of REG4 and SPINK1 in primary pancreatic ductal adenocarcinoma

Background Pancreatic ductal adenocarcinoma (PDAC) is one of the leading causes of cancer-related deaths, with very limited therapeutic options available. This study aims to comprehensively depict the heterogeneity and identify prognostic targets for PDAC with single-cell RNA sequencing (scRNA-seq) analysis. Methods ScRNA-seq analysis was performed on 16 primary PDAC and three adjacent lesions. A series of analytical methods were applied for analysis in cell clustering, gene profiling, lineage trajectory analysis and cell-to-cell interactions. In vitro experiments including colony formation, wound healing and sphere formation assay were performed to assess the role of makers. Results A total of 32,480 cells were clustered into six major populations, among which the ductal cell cluster expressing high copy number variants (CNVs) was defined as malignant cells. Malignant cells were further subtyped into five subgroups which exhibited specific features in immunologic and metabolic activities. Pseudotime trajectory analysis indicated that components of various oncogenic pathways were differentially expressed along tumor progression. Furthermore, intensive substantial crosstalk between ductal cells and stromal cells was identified. Finally, genes (REG4 and SPINK1) screened out of differentially expressed genes (DEGs) were upregulated in PDAC cell lines. Silencing either of them significantly impaired proliferation, invasion, migration and stemness of PDAC cells. Conclusions Our findings offer a valuable resource for deciphering the heterogeneity of malignant ductal cells in PDAC. REG4 and SPINK1 are expected to be promising targets for PDAC therapy.

E2F transcription factors promote tumorigenicity in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal cancers with limited treatment options, illustrating an urgent need to identify new drugable targets in PDACs. Using the similarities between tumor development and normal embryonic development, which is accompanied by rapid cell expansion, we aimed to identify and characterize embryonic signaling pathways that were reinitiated during tumor formation and expansion. Here, we report that the transcription factors E2F1 and E2F8 are potential key regulators in PDAC. E2F1 and E2F8 RNA expression is mainly localized in proliferating cells in the developing pancreas and in malignant ductal cells in PDAC. Silencing of E2F1 and E2F8 in PANC-1 pancreatic tumor cells inhibited cell proliferation and impaired cell spreading and migration. Moreover, loss of E2F1 also affected cell viability and apoptosis with E2F expression in PDAC tissues correlating with expression of apoptosis and mitosis pathway genes, suggesting that E2F factors promote cell cycle regulation and tumorigenesis in PDAC cells. Our findings illustrate that E2F1 and E2F8 transcription factors are expressed in pancreatic progenitor and PDAC cells, where they contribute to tumor cell expansion by regulation of cell proliferation, viability, and cell migration making these genes attractive therapeutic targets and potential prognostic markers for pancreatic cancer.

PH-responsive targeted nanoparticles release ERK-inhibitor in the hypoxic zone and sensitize free gemcitabine in mutant K-Ras-addicted pancreatic cancer cells and mouse model.

Therapeutic options for managing Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest types of aggressive malignancies, are limited and disappointing. Therefore, despite suboptimal clinical effects, gemcitabine (GEM) remains the first-line chemotherapeutic drug in the clinic for PDAC treatment. The therapeutic limitations of GEM are primarily due to poor bioavailability and the development of chemoresistance resulting from the addiction of mutant-K-RAS/AKT/ERK signaling-mediated desmoplastic barriers with a hypoxic microenvironment. Several new therapeutic approaches, including nanoparticle-assisted drug delivery, are being investigated by us and others. This study used pH-responsive nanoparticles encapsulated ERK inhibitor (SCH772984) and surface functionalized with tumor-penetrating peptide, iRGD, to target PDAC tumors. We used a small molecule, SCH772984, to target ERK1 and ERK2 in PDAC and other cancer cells. This nanocarrier efficiently released ERKi in hypoxic and low-pH environments. We also found that the free-GEM, which is functionally weak when combined with nanoencapsulated ERKi, led to significant synergistic treatment outcomes in vitro and in vivo. In particular, the combination approaches significantly enhanced the GEM effect in PDAC growth inhibition and prolonged survival of the animals in a genetically engineered KPC (LSL-KrasG12D/+/LSL-Trp53R172H/+/Pdx-1-Cre) pancreatic cancer mouse model, which is not observed in a single therapy. Mechanistically, we anticipate that the GEM efficacy was increased as ERKi blocks desmoplasia by impairing the production of desmoplastic regulatory factors in PDAC cells and KPC mouse tumors. Therefore, 2nd generation ERKi (SCH 772984)-iRGD-pHNPs are vital for the cellular response to GEM and denote a promising therapeutic target in PDAC with mutant K-RAS.

Effects of transmembrane serine protease 4 on the survival in patients with pancreatic ductal adenocarcinoma undergoing surgery followed by adjuvant chemotherapy.

The transmembrane serine protease 4 (TMPRSS4) gene is upregulated in various human cancers. However, its biological functions in pancreatic ductal adenocarcinoma remain unclear. We examined the expression of TMPRSS4 in pancreatic ductal adenocarcinoma tissues and its correlation with clinicopathological parameters in patients with pancreatic ductal adenocarcinoma who underwent surgery. The TMPRSS4 expression was immunohistochemically examined in 81 PDAC patients with pancreatic ductal adenocarcinoma. We analyzed the association between the TMPRSS4 expression and clinicopathological factors, the recurrence-free survival (RFS), and the overall survival (OS) and examined the effect of TMPRSS4 expression on cell migration and sensitivity to 5-fluorouracil. The expression rate of TMPRSS4 in the samples was 62.9% (51/81). The TMPRSS4 expression was not correlated with any clinicopathological feature. The five-year overall and recurrence-free survival rates were significantly lower in the TMPRSS4-positive group than in the TMPRSS4-negative group. On a multivariate analysis, TMPRSS4 positivity, poorly differentiated histology, and non-adjuvant chemotherapy predicted a poor OS, while TMPRSS4 positivity and poorly differentiated histology predicted a poor RFS. TMPRSS4-silenced pancreatic ductal adenocarcinoma cells showed higher sensitivity to 5-fluorouracil than did the control siRNA-transfected cells. TMPRSS4 can be considered a prognostic factor and therapeutic target for pancreatic ductal adenocarcinoma.

Examination of Wnt signaling as a therapeutic target for pancreatic ductal adenocarcinoma (PDAC) using a pancreatic tumor organoid library (PTOL).

Pancreatic ductal adenocarcinoma (PDAC) presents at advanced stages and is refractory to most treatment modalities. Wnt signaling activation plays a critical role in proliferation and chemotherapeutic resistance. Minimal media conditions, growth factor dependency, and Wnt dependency were determined via Wnt inhibition for seven patient derived organoids (PDOs) derived from pancreatic tumor organoid libraries (PTOL). Organoids demonstrating response in vitro were assessed in vivo using patient-derived xenografts. Wnt (in)dependent gene signatures were identified for each organoid. Panc269 demonstrated a trend of reduced organoid growth when treated with ETC-159 in combination with paclitaxel or gemcitabine as compared with chemotherapy or ETC-159 alone. Panc320 demonstrated a more pronounced anti-proliferative effect in the combination of ETC-159 and paclitaxel but not with gemcitabine. Panc269 and Panc320 were implanted into nude mice and treated with ETC-159, paclitaxel, and gemcitabine as single agents and in combination. The combination of ETC-159 and paclitaxel demonstrated an anti-tumor effect greater than ETC-159 alone. Extent of combinatory treatment effect were observed to a lesser extent in the Panc320 xenograft. Wnt (in)dependent gene signatures of Panc269 and 320 were consistent with the phenotypes displayed. Gene expression of several key Wnt genes assessed via RT-PCR demonstrated notable fold change following treatment in vivo. Each pancreatic organoid demonstrated varied niche factor dependencies, providing an avenue for targeted therapy, supported through growth analysis following combinatory treatment of Wnt inhibitor and standard chemotherapy in vitro. The clinical utilization of this combinatory treatment modality in pancreatic cancer PDOs has thus far been supported in our patient-derived xenograft models treated with Wnt inhibitor plus paclitaxel or gemcitabine. Gene expression analysis suggests there are key Wnt genes that contribute to the Wnt (in)dependent phenotypes of pancreatic tumors, providing plausible mechanistic explanation for Wnt (in)dependency and susceptibility or resistance to treatment on the genotypic level.

Tumor Stroma as a Therapeutic Target for Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) has a poor prognosis owing to its desmoplastic stroma. Therefore, therapeutic strategies targeting this tumor stroma should be developed. In this study, we describe the heterogeneity of cancer-associated fibroblasts (CAFs) and their diverse roles in the progression, immune evasion, and resistance to treatment of PDAC. We subclassified the spatial distribution and functional activity of CAFs to highlight their effects on prognosis and drug delivery. Extracellular matrix components such as collagen and hyaluronan are described for their roles in tumor behavior and treatment outcomes, implying their potential as therapeutic targets. We also discussed the roles of extracellular matrix (ECM) including matrix metalloproteinases and tissue inhibitors in PDAC progression. Finally, we explored the role of the adaptive and innate immune systems in shaping the PDAC microenvironment and potential therapeutic strategies, with a focus on immune cell subsets, cytokines, and immunosuppressive mechanisms. These insights provide a comprehensive understanding of PDAC and pave the way for the development of prognostic markers and therapeutic interventions.

Glycolysis and tumor progression promoted by the m6A writer VIRMA via m6A-dependent upregulation of STRA6 in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive and lethal malignancies, highlighting the urgent need to elucidate the underlying oncogenic mechanisms. VIRMA is a classic isoform of methyltransferases that participates in epigenetic transcriptomic modification in eukaryotic mRNAs. However, the exact roles of VIRMA in PDAC remain unclear. Here, we identified that VIRMA is highly expressed in PDAC, and histone modifications of the promoter may partly account for this dysregulation. Moreover, VIRMA is closely related to glycolysis and poor prognosis in PDAC. We further determined that STRA6 is a direct downstream target of VIRMA in PDAC by RNA sequencing (RNA-seq) and m6A sequencing (m6A-seq). VIRMA is involved in gene expression regulation via 3’ UTR targeting of STRA6 mRNA. Furthermore, the m6A reader IGF2BP2 was shown to critically contribute to the stability of STRA6 mRNA. We describe the role of VIRMA in promoting signaling via the STRA6/STAT3 axis, which results in increased levels of HIF-1α, a key activator of glycolysis. In vivo and in vitro experiments reveal that the VIRMA-STRA6-STAT3-HIF-1α axis plays an instrumental role in glycolysis and tumor progression in PDAC. In conclusion, we demonstrate that VIRMA can increase glycolysis in PDAC by upregulating STRA6, a cell surface membrane protein that stimulates the STAT3 pathway, thereby activating HIF-1α and leading to pancreatic cancer malignancy. Overall, our data strongly suggest that the VIRMA-STRA6-STAT3-HIF-1α axis is a viable therapeutic target in PDAC.

Nuclear MAST4 Suppresses FOXO3 through Interaction with AKT3 and Induces Chemoresistance in Pancreatic Ductal Carcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is highly malignant, with a 5-year survival rate of less than 10%. Furthermore, the acquisition of anticancer drug resistance makes PDAC treatment difficult. We established MIA-GEM cells, a PDAC cell line resistant to gemcitabine (GEM), a first-line anticancer drug, using the human PDAC cell line-MIA-PaCa-2. Microtubule-associated serine/threonine kinase-4 (MAST4) expression was increased in MIA-GEM cells compared with the parent cell line. Through inhibitor screening, dysregulated AKT signaling was identified in MIA-GEM cells with overexpression of AKT3. MAST4 knockdown effectively suppressed AKT3 overexpression, and both MAST4 and AKT3 translocation into the nucleus, phosphorylating forkhead box O3a (FOXO3) in MIA-GEM cells. Modulating FOXO3 target gene expression in these cells inhibited apoptosis while promoting stemness and proliferation. Notably, nuclear MAST4 demonstrated higher expression in GEM-resistant PDAC cases compared with that in the GEM-sensitive cases. Elevated MAST4 expression correlated with a poorer prognosis in PDAC. Consequently, nuclear MAST4 emerges as a potential marker for GEM resistance and poor prognosis, representing a novel therapeutic target for PDAC.

Genome-wide CRISPR screens identify PKMYT1 as a therapeutic target in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with an overall 5-year survival rate of <12% due to the lack of effective treatments. Novel treatment strategies are urgently needed. Here, PKMYT1 is identified through genome-wide CRISPR screens as a non-mutant, genetic vulnerability of PDAC. Higher PKMYT1 expression levels indicate poor prognosis in PDAC patients. PKMYT1 ablation inhibits tumor growth and proliferation in vitro and in vivo by regulating cell cycle progression and inducing apoptosis. Moreover, pharmacological inhibition of PKMYT1 shows efficacy in multiple PDAC cell models and effectively induces tumor regression without overt toxicity in PDAC cell line-derived xenograft and in more clinically relevant patient-derived xenograft models. Mechanistically, in addition to its canonical function of phosphorylating CDK1, PKMYT1 functions as an oncogene to promote PDAC tumorigenesis by regulating PLK1 expression and phosphorylation. Finally, TP53 function and PRKDC activation are shown to modulate the sensitivity to PKMYT1 inhibition. These results define PKMYT1 dependency in PDAC and identify potential therapeutic strategies for clinical translation.

NNT-AS1 in CAFs-derived exosomes promotes progression and glucose metabolism through miR-889-3p/HIF-1α in pancreatic adenocarcinoma

It is metabolic and signaling crosstalk between stromal cells and tumors in the tumor microenvironment, which influences several aspects of tumor formation and drug resistance, including metabolic reprogramming. Despite considerable findings linking lncRNAs in HIF-1-related regulatory networks to cancer cell, little emphasis has been given to the role in communication between cancer-associated fibroblasts (CAFs) and tumor cells. Previously, we observed that NNT-AS1 was substantially expressed in CAFs cells and CAFs exosomes, and subsequently investigated the influence of CAFs exosomal NNT-AS1 on glucose metabolism, proliferation, and metastasis of pancreatic ductal adenocarcinoma (PDAC) cells. Transmission electron microscopy was used to examine exosomes secreted by PDAC patient-derived CAFs. qRT-PCR was used to evaluate the expression of NNT-AS1, miR-889-3p, and HIF-1. The role of CAFs-derived exosomal NNT-AS1 in PDAC cell progression and metabolism have been identified. Dual luciferase reporter assays examined the binding between NNT-AS1, miR-889-3p, and HIF-1. After PDAC cells co-culture exosomes secreted by CAFs, we found that they alter glucose metabolism, proliferation, and metastasis. In PDAC cells, CAF-derived exosomal lncRNA NNT-AS1 acted as a molecular sponge for miR-889-3p. Furthermore, HIF-1 could be targeted by miR-889-3p and was controlled by NNT-AS1. This study explores the mechanism by which NNT-AS1 influences the interaction of CAFs on glycolytic remodeling, proliferation, and metastasis of tumor cells through regulating miR-889-3p/HIF-1α, which also helps discover new clinical treatment targets for PDAC.

Abstract 5587: Regulator of chromosome condensation 1 (RCC1) as a novel therapeutic target in pancreatic ductal adenocarcinoma

Abstract Background and Introduction: Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal disease with limited treatment options. There is an urgent need for the identification of novel therapeutic targets for PDAC. The transport of molecular cargo between the nucleus and cytoplasm, facilitated by Ran-GTPase, is a critical process that dividing cells rely on to maintain their growth. Elevated nuclear Ran-GTP levels are produced by the activity of the Ran guanine nucleotide exchange factor (GEF) known as Regulator of Chromosome Condensation 1 (RCC1), which is found in the nucleus bound to chromatin. This creates a Ran-GTP gradient across the nuclear membrane, which maintains proper directional transport of proteins and RNA. To sustain proliferative signaling, cancer cells become reliant on high rates of nucleocytoplasmic transport through dysregulation of transport machinery. Methods: We examined the role of RCC1 in the biology of PDAC. The impact of RCC1 modulation on PDAC growth was evaluated using RNA interference and CRISPR-Cas9 in vitro and in vivo using PDAC cell lines and LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx1-Cre (KPC) tumor-derived cells. The broader impact of RCC1 silencing on PDAC-sustaining signaling was evaluated through RNA-sequencing and proteomics. Results: Transcriptomic sequencing of PDAC tissue (n=5,071) as well as analysis of publicly available data from the TCGA, CPTAC and GTEx revealed that RCC1 expression is higher in PDAC tissues compared to normal pancreas. Moreover, PDAC patients with higher RCC1 expression were more likely to have poorer outcomes. RCC1 silencing by RNAi and CRISPR-Cas9 resulted in reduced proliferation in 2D and 3D cultures and attenuated tumor growth in vivo. RCC1 KD decreased migration and colony formation, enhanced apoptosis, and altered the cell cycle in human and KPC mouse PDAC cells. Subcutaneous RCC1 KO cell line-derived xenografts show arrested growth. Subcellular Ran distribution was disrupted upon RCC1 KO, suggesting that nuclear Ran concentration is important for PDAC proliferation. Nuclear and cytosolic proteomic analysis revealed altered subcellular proteome in RCC1 KD KPC tumor-derived cells. Altered cytoplasmic protein pathways include several metabolic pathways, PI3K-AKT, and Hedgehog signaling. Nuclear enriched pathways include cell cycle, mitosis, metabolic processes, and RNA processing. RNA-seq of RCC1 KO cells showed widespread transcriptional alterations. Upstream of RCC1, c-MYC activates the RCC1-RAN axis, and RCC1 KO cells show differential sensitivity to c-MYC inhibitors. Finally, RCC1 KD resulted in the sensitization of PDAC cells to Gemcitabine. Molecular characterization of conditional Rcc1fl/fl KO transgenic model to study the role of RCC1 in PDAC in vivo is ongoing. Conclusions: Overall, our results show that RCC1 is involved in the regulation of PDAC growth and is a potential target for therapy. Citation Format: Sahar F. Bannoura, Husain Y. Khan, Amro Aboukameel, Md Hafiz Uddin, Rund Nimri, Eliza Beal, Seongho Kim, Mohammed Najeeb Al Hallak, Bassel El-Rayes, Philip A. Philip, Boris Pasche, Ramzi Mohammad, Asfar S. Azmi. Regulator of chromosome condensation 1 (RCC1) as a novel therapeutic target in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5587.

Abstract 1581: Colony-stimulating factor-1 receptor as a potential therapeutic target in pancreatic ductal adenocarcinoma

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is associated with poor prognosis, rising incidence and suffers from limited existing treatment options. Currently, salient features of PDAC are being investigated that can potentially predict immunotherapy outcomes, to identify small patient populations that can benefit from such strategies. Preliminary studies show that colony-stimulating factor-1 receptor (CSF1R), a surface receptor tyrosine kinase that drives survival, function, proliferation and differentiation of several myeloid lineage cell types can be a potentially interesting target for combination immunotherapy. Methods: PDAC patients from the TCGA PanCancer Atlas were stratified into CSF1Rhi and CSF1Rlo cohorts and clinically relevant characteristics were compared between the two. The mRNA expression of CSF1R and immunological markers of interest were evaluated and Gene Set Enrichment Analysis (GSEA) was run on the two groups to analyze differential expression of gene sets. Results: mRNA expression z-scores were used to classify 129 patient samples in the TCGA PanCancer Atlas dataset into CSF1Rhi (z-score&amp;gt;1, n=20) and CSF1Rlo (z-score&amp;lt;0, n=109) cohorts. Both have similar disease staging, MSI MANTIS scores, tumor mutation burden, median diagnosis age, sex and race distribution. However, the CSF1Rhi cohort has a distinct, non-conventional PDAC driver gene mutation profile (genomic alteration frequencies in CSF1Rhi vs CSF1Rlo cohorts respectively: a) KRAS: 30% vs 73% b) CDKN2A: 15% vs 57% and c) SMAD4: 5% vs 42%) (p-value&amp;lt;0.01 for each). CSF1R correlates with markers of CD8+ T-cell function (R2 values: CD8A: 0.683, GZMB: 0.508, PRF1: 0.673), CD8+ T-cell infiltration (CXCL9: 0.596, CXCL10: 0.483, CCL5: 0.666) and expression of immune checkpoint genes (PDCD1: 0.602, CD274: 0.544, CTLA4: 0.613). CD8A, GZMB, PRF1, CXCL9, CXCL10, CCL5, PDCD1, CD274, CTLA4 are significantly upregulated in the CSF1Rhi group (log2Fold changes respectively: 2.07, 1.57, 1.48, 2.51, 1.71, 1.75, 1.63, 1.34, 1.80). GSEA against the hallmark gene set database shows that KRAS signaling, Inflammatory response, IL6 JAK STAT3 and Interferon-gamma signaling are significantly upregulated in the CSF1R high patient group (FDR q-value &amp;lt;0.01 for each). Conclusion: Preliminary in silico analysis suggests that PDAC patients with higher levels of CSF1R may be more immune infiltrated. Future studies will evaluate the potential of CSF1R as a biomarker to identify an immune infiltrated subset of PDAC patients ex vivo and the mechanism of immune infiltration in CSF1Rhi PDACs in vivo. Citation Format: Somak Chaudhuri, Dustin A. Deming, Cheri A. Pasch, Katherine A. Johnson, Chelsie K. Sievers, Philip B. Emmerich. Colony-stimulating factor-1 receptor as a potential therapeutic target in pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1581.

Abstract 2087: Targeting lipid metabolism in pancreatic cancer

Abstract Background PIKfyve is a lipid kinase that serves as the sole source of cellular PI(3,5)P2 and PI5P, critical phosphatidylinositols for autophagy and lysosome function. Pancreatic ductal adenocarcinoma (PDAC) is known to upregulate and depend on lysosomal functions such as autophagy to exist in its harsh, nutrient-disrupted microenvironment. Thus, we aimed to establish PIKfyve as a therapeutic target in PDAC. Using a PIKfyve-knockout genetically engineered mouse model of pancreatic cancer (KC and KPC), we determined that PIKfyve was necessary for PDAC development. Given these results, we hypothesized that PIKfyve was critical for maintaining metabolic homeostasis in PDAC and that inhibiting PIKfyve would be an efficacious therapeutic strategy for PDAC. Methods and results We first validated the known role of PIKfyve in regulating autophagy using CRISPRi mediated knockdown, PIKfyve inhibitors Apilimod and ESK981, and a PIKfyve degrader, which all demonstrated that PIKfyve perturbation disrupted autophagic flux and suppressed PDAC cell growth. Little is known about the metabolic impacts of PIKfyve inhibition in PDAC beyond disruption of autophagy. Thus, we paired RNA-seq and a metabolic CRISPR screen to discover that PIKfyve inhibition obligated PDAC cells to upregulate de novo lipid synthesis. Specifically, PDAC cells upregulated fatty acid synthesis genes ACACA and FASN upon PIKfyve inhibition. Additionally, these genes were synthetically critical in the context of PIKfyve inhibition. We further performed metabolomics and lipidomics which revealed accumulation of sphingolipids upon PIKfyve inhibition. Given this, we concluded that PIKfyve regulates lipid homeostasis in PDAC. To further establish PIKfyve as a target in PDAC, we investigated the role of PIKfyve upon inhibition of KRAS, which drives PDAC through MAPK pathway signaling. First, KRAS/MAPK inhibition diminished expression of FASN and ACACA, genes synthetically essential upon PIKfyve inhibition. Additionally, KRAS/MAPK inhibition upregulated autophagy which was attenuated upon concurrent PIKfyve inhibition. Given this metabolic conflict between KRAS/MAPK and PIKfyve, we assessed the effects of dual inhibiting KRAS/MAPK and PIKfyve in a syngeneic orthotopic model. Encouragingly, combination therapy of trametinib (MEK inhibitor) and ESK981 eliminated tumor burden in most of mice, while single treatments only had modest effects. Further, we established a patient-derived xenograft model and showed that while the treatment with individual compounds trametinib, MRTX-1133 (KRASG12D inhibitor), and ESK981 all suppressed tumor growth, the combination of ESK981 and trametinib or ESK981 and MRTX-1133 caused substantial tumor regression in nearly all mice. Taken together, targeting lipid metabolism through inhibition of PIKfyve is a promising therapeutic strategy for PDAC, particularly in combination with the numerous KRAS/MAPK inhibitors in clinical development. Citation Format: Caleb Cheng, Jasmine P. Wisniewski, Ahmet Korkaya, Bailey Jackson, Rahul Mannan, Somnath Mahapatra, Nicholas Rossiter, Rupam Bhattacharyya, Pietro Morlacchi, Sydney Peters, Yuanyuan Qiao, Costas A. Lyssiotis, Arul M. Chinnaiyan. Targeting lipid metabolism in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2087.

Abstract 7246: Enhancing PDAC therapy through in vivo correction of KRAS G12D using enIscB-directed base editor combined with ICB

Abstract In this study, we aim to investigate the therapeutic potential of in vivo genetic therapy using AAV-mediated adenine base editor (ABE) delivery for pancreatic ductal adenocarcinoma (PDAC) carrying the KRASG12D mutation (PDACG12D), and further explore its potential when combined with immune checkpoint blockade (ICB). The KRASG12D mutation is a dominant genetic alteration representing a highly sought-after and effective therapeutic target in PDAC. However, the in vivo genetic therapy for KRASG12D in PDAC has been rarely explored. Programmable base editors have been shown to install genetic mutations precisely and have significant therapeutic value, providing a promising strategy for genetic therapy of PDACG12D. Here, we engineered ABEs derived from various programmable nucleases, considering the protospacer adjacent motif (PAM) or target adjacent motif (TAM) sequence. The hypercompact A8e-enIscB demonstrated the highest editing efficiency in HEK293TG12D and PANC-1 cell lines. Subsequently, A8e-enIscB was packaged into a single-AAV system and delivered to humanized mice subcutaneously transplanted with patient-derived organoidG12D (PDOG12D). We found the delayed tumor progression with BE-based genetic therapy. Additionally, a substantial editing efficiency in tumor tissues and lower off-target editing in normal tissues was observed. By comparing single-cell transcriptome sequencing data between BE-treated and non-treated samples, we observed a reprogrammed immune microenvironment characterized by elevated infiltration of cytotoxic CD8+ T cells and NK cells, as well as decreased recruitment of suppressive PMN-MDSCs, Tregs and TAMs upon correction of KRASG12D. Furthermore, AAV-directed ABE genetic therapy could significantly sensitize PDAC to ICB therapy (anti-PD-1, anti-CTLA4) in humanized PDAC PDO/PDX mouse models. Accordingly, an ongoing work in which the genetic therapy combined with ICB phase IA investigator-initialized trial will be designed to evaluate the safety of this regimen. In summary, our study demonstrates the efficiency and safety of innovative genetic therapy for correcting the KRASG12D mutation in PDAC. Furthermore, we demonstrated the therapeutic potential of combining this genetic therapy with ICB. Citation Format: Chao Yang, Qingxiao Fang, Tianxing Zhou, Yongjie Xie, Jun Yu, Jihui Hao. Enhancing PDAC therapy through in vivo correction of KRAS G12D using enIscB-directed base editor combined with ICB [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7246.

Tumor-associated macrophages drive glycolysis through the IL-8/STAT3/GLUT3 signaling pathway in pancreatic cancer progression

Glycolytic metabolism is a hallmark of pancreatic ductal adenocarcinoma (PDAC), and tumor-associated stromal cells play important roles in tumor metabolism. We previously reported that tumor-associated macrophages (TAMs) facilitate PDAC progression. However, little is known about whether TAMs are involved in regulating glycolysis in PDAC. Here, we found a positive correlation between CD68+ TAM infiltration and FDG maximal standardized uptake (FDG SUVmax) on PET-CT images of PDAC. We discovered that the glycolytic gene set was prominently enriched in the high TAM infiltration group through Gene Set Enrichment Analysis using The Cancer Genome Atlas database. Mechanistically, TAMs secreted IL-8 to promote GLUT3 expression in PDAC cells, enhancing tumor glycolysis both in vitro and in vivo, whereas this effect could be blocked by the IL-8 receptor inhibitor reparixin. Furthermore, IL-8 promoted the translocation of phosphorylated STAT3 into the nucleus to activate the GLUT3 promoter. Overall, we demonstrated that TAMs boosted PDAC cell glycolysis through the IL-8/STAT3/GLUT3 signaling pathway. Our cumulative findings suggest that the abrogation of TAM-induced tumor glycolysis by reparixin might exhibit an antitumor impact and offer a potential therapeutic target for PDAC.

Analysis of single nuclear chromatin accessibility reveals unique myeloid populations in human pancreatic ductal adenocarcinoma.

A better understanding of the pancreatic ductal adenocarcinoma (PDAC) immune microenvironment is critical to developing new treatments and improving outcomes. Myeloid cells are of particular importance for PDAC progression; however, the presence of heterogenous subsets with different ontogeny and impact, along with some fluidity between them, (infiltrating monocytes vs. tissue-resident macrophages; M1 vs. M2) makes characterisation of myeloid populations challenging. Recent advances in single cell sequencing technology provide tools for characterisation of immune cell infiltrates, and open chromatin provides source and function data for myeloid cells to assist in more comprehensive characterisation. Thus, we explore single nuclear assay for transposase accessible chromatin (ATAC) sequencing (snATAC-Seq), a method to analyse open gene promoters and transcription factor binding, as an important means for discerning the myeloid composition in human PDAC tumours. Frozen pancreatic tissues (benign or PDAC) were prepared for snATAC-Seq using 10× Chromium technology. Signac was used for preliminary analysis, clustering and differentially accessible chromatin region identification. The genes annotated in promoter regions were used for Gene Ontology (GO) enrichment and cell type annotation. Gene signatures were used for survival analysis with The Cancer Genome Atlas (TCGA)-pancreatic adenocarcinoma (PAAD) dataset. Myeloid cell transcription factor activities were higher in tumour than benign pancreatic samples, enabling us to further stratify tumour myeloid populations. Subcluster analysis revealed eight distinct myeloid populations. GO enrichment demonstrated unique functions for myeloid populations, including interleukin-1b signalling (recruited monocytes) and intracellular protein transport (dendritic cells). The identified gene signature for dendritic cells influenced survival (hazard ratio=.63, p=.03) in the TCGA-PAAD dataset, which was unique to PDAC. These data suggest snATAC-Seq as a method for analysis of frozen human pancreatic tissues to distinguish myeloid populations. An improved understanding of myeloid cell heterogeneity and function is important for developing new treatment targets in PDAC.

PRMT1 promotes pancreatic cancer development and resistance to chemotherapy

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal types of cancer, and novel treatment regimens are direly needed. Epigenetic regulation contributes to the development of various cancer types, but its role in the development of and potential as a therapeutic target for PDAC remains underexplored. Here, we show that PRMT1 is highly expressed in murine and human pancreatic cancer and is essential for cancer cell proliferation and tumorigenesis. Deletion of PRMT1 delays pancreatic cancer development in a KRAS-dependent mouse model, and multi-omics analyses reveal that PRMT1 depletion leads to global changes in chromatin accessibility and transcription, resulting in reduced glycolysis and a decrease in tumorigenic capacity. Pharmacological inhibition of PRMT1 in combination with gemcitabine has a synergistic effect on pancreatic tumor growth invitro and invivo. Collectively, our findings implicate PRMT1 as a key regulator of pancreatic cancer development and a promising target for combination therapy.

Identification of Tissue miRNA Signatures for Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC), a neoplasm of the gastrointestinal tract, is the most common pancreatic malignancy (90%) and the fourth highest cause of cancer mortality worldwide. Surgery intervention is currently the only strategy able to offer an advantage in terms of overall survival, but prognosis remains poor even for operated patients. Therefore, the development of robust biomarkers for early diagnosis and prognostic stratification in clinical practice is urgently needed. In this work, we investigated deregulated microRNAs (miRNAs) in tissues from PDAC patients with high (G3) or low (G2) histological grade and with (N+) or without (N-) lymph node metastases. miRNA expression profiling was performed by a comprehensive PCR array and subsequent validation by RT-qPCR. The results showed a significant increase in miR-1-3p, miR-31-5p, and miR-205-5p expression in G3 compared to G2 patients (** p < 0.01; *** p < 0.001; *** p < 0.001). miR-518d-3p upregulation and miR-215-5p downregulation were observed in N+ compared to N- patients. A statistical analysis performed using OncomiR program showed the significant involvement (p < 0.05) of two miRNAs (miR-31 and miR-205) in the histological grade of PDAC patients. Also, an expression analysis in PDAC patients showed that miR-31 and miR-205 had the highest expression at grade 3 compared with normal and other tumor grades. Overall, survival plots confirmed that the overexpression of miR-31 and miR-205 was significantly correlated with decreased survival in TCGA PDAC clinical samples. A KEGG pathway analysis showed that all three miRNAs are involved in the regulation of multiple pathways, including the Hippo signaling, adherens junction and microRNAs in cancer, along with several target genes. Based on in silico analysis and experimental validation, our study suggests the potential role of miR-1-3p, miR-31-5p, and miR-205-5p as useful clinical biomarkers and putative therapeutic targets in PDAC, which should be further investigated to determine the specific molecular processes affected by their aberrant expression.

Caffeic acid, a dietary polyphenol, pre-sensitizes pancreatic ductal adenocarcinoma to chemotherapeutic drug

Resistance to chemotherapeutics is an eminent cause that leads to search for options that help in diminution of pancreatic ductal adenocarcinoma (PDAC) by overcoming resistance issues. Caffeic acid (CFA), a polyphenol occurring in many dietary foods, is known to show antidiabetic and anticancer properties potential. To unveil the effect of CFA on PDAC, we carried out this research in PDAC cells, following which we checked the combination effect of CFA and chemotherapeutics and pre-sensitization effects of CFA. Multitudinous web-based approaches were applied for identifying CFA targets in PDAC and then getting their interconnections. Subsequently, we manifested CFA effects by in-vitro analysis showing IC50 concentrations of 37.37 and 15.06 µM on Panc-1 and Mia-PaCa-2, respectively. The combination index of CFA with different drugs was explored which showed the antagonistic effects of combination treatment leading to further investigation of the pre-sensitizing effects. CFA pre-sensitization reduced IC50 concentration of doxorubicin in both PDAC cell lines which also triggered ROS generation determined by 2′,7′-dichlorofluorescin diacetate assay. The differential gene expression analysis after CFA treatment showed discrete genes affected in both cells, i.e. N-Cad and Cas9 in Panc-1 and Pi3K/AkT/mTOR along with p53 in Mia-PaCa-2. Collectively, this study investigated the role of CFA as PDAC therapeutics and explored the mechanism in mitigating resistance of PDAC by sensitizing to chemotherapeutics. Communicated by Ramaswamy H. Sarma

Peptidase inhibitor 16 promotes proliferation of pancreatic ductal adenocarcinoma cells through OASL signaling.

Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive cancer with a poor prognosis and a 5-year survival rate of less than 11%. As a member of the CAP superfamily of proteins, the role of peptidase inhibitor 16 (Pi16) in tumor progression is still unclear. Immunohistochemistry and quantitative RT-PCR methods were used to detect the expression levels of Pi16 protein and mRNA in PDAC patients. CRISPR/Cas9 technology was used to knock out the expression of Pi16 in PDAC cell lines. In vivo and in vitro experiments were used to verify the effect of Pi16 on PDAC proliferation ability. By RNA sequencing, we found that oligoadenylate synthetase L(OASL) can serve as a potential downstream target of Pi16. The expression of Pi16 was higher in PDAC tissues than in matched adjacent tissues. High expression of Pi16 was associated with PDAC progression and poor prognosis. Overexpression of Pi16 could promote the proliferation of PDAC cells in vitro and in vivo. Bioinformatics analysis and coimmunoprecipitation assays showed that Pi16 could bind to OASL. Moreover, the functional recovery test confirmed that Pi16 could promote the proliferation of PDAC via OASL. Our present study demonstrates that Pi16 might participate in the occurrence and development of PDAC by regulating cell proliferation by binding to OASL, indicating that Pi16 might be a promising novel therapeutic target for PDAC.