Pancreatic Ductal Adenocarcinoma Therapy Research Articles

Antagonizing vasoactive intestinal peptide (VIP) receptors with Muc16CD-directed armored CAR T cells for pancreatic cancer.

2558 Background: Chimeric antigen receptor (CAR) T therapies for pancreatic ductal adenocarcinoma (PDAC) still face significant immunosuppressive obstacles in the tumor microenvironment (TME). We hypothesize that an optimal CAR T cell for PDAC combines targeting an ideal tumor-associated antigen (TAA) and overcomes unique immunosuppression in the PDAC TME. The retained ectodomain of Muc16 (Muc16CD) is a known TAA in ovarian cancer but has yet to be explored in pancreatic cancer. Vasoactive intestinal peptide (VIP) is an emerging checkpoint pathway for T cell function, which expresses VIP receptors (VIPR) and is abundantly expressed by PDAC. In this work, we present a novel armored CAR T cell that targets Muc16CD and antagonizes VIPRs (CAR/VIPRa) to overcome the immunosuppressive PDAC TME. Methods: Patient expression data was assessed based on data generated by the TCGA Research Network. Primary human T cells from healthy donors or PDAC patients were retrovirally transduced to express Muc16CD-directed CARs with or without secretion of novel, potent VIPR antagonist peptides. In vivo, PDAC PDX tumors were engrafted into SCID/Beige mice and treated with CAR T cells. Results: PDAC tumors have significantly increased expression of Muc16 compared to normal pancreas tissue and patients with high Muc16 expression have a significantly decreased overall survival. PDAC patient-derived tumors show robust expression of both Muc16CD and VIP. CAR/VIPRa T cells reveal that VIPR antagonism metabolically reprograms CAR T cells and drives a memory-rich product. CAR/VIPRa T cells are less activated and less exhausted by the manufacturing process, which lends to better viability and a metabolically quiescent phenotype at baseline. These distinct features allow CAR/VIPRa T cells, when antigen-stimulated, to have enhanced activation and expansion with repeated stimulation. To investigate clinical relevance, CAR/VIPRa T cells manufactured from PDAC patient blood are also significantly enriched for memory phenotypes. In vivo, CAR/VIPRa T cells have enhanced expansion, phenotype, infiltration, and persistence, which ultimately reduces PDAC tumor burden. In a patient-derived PDAC preclinical mouse model where CAR T is typically ineffective, CAR/VIPRa T cells significantly reduce tumor burden. Conclusions: This work demonstrates Muc16CD as a clinically relevant TAA target for CAR T therapy in PDAC. Furthermore, antagonizing the previously undescribed VIP checkpoint pathway in CAR T cells produces enhanced phenotypic and functional profiles. Collectively, this data demonstrates that novel CAR/VIPRa T cells create an advantageous cellular therapy product capable of treating PDAC. The long-term goal of this work is translating CAR/VIPRa T cells for the treatment of PDAC and expanding these preclinical findings of cellular therapies for other VIP-abundant tumors.

The rationale of reprogramming TREM2+ macrophages for overcoming the resistance to immune checkpoint inhibitors in pancreatic adenocarcinoma.

e14649 Background: Pancreatic ductal adenocarcinoma (PDAC) resists to immune checkpoint inhibitors as single agents. TAMs expressing the triggering receptor expressed on myeloid cells (TREM2) protein were found to be a dominant macrophage population in PDAC; targeting TREM2 has been found to enhance the efficacy of immune checkpoint inhibitor-based therapies in preclinical studies of other malignancies. Here, we studied the role of targeting TREM2 in PDAC by testing the combination of non-depleting anti-TREM2 antibody and anti-PD-1 antibody in a mouse model of PDAC. We also examined the relationship between TREM2 expression and effector T cells in surgically resected human PDAC collected from a previous clinical trial treated with the neoadjuvant combination therapy of nivolumab (anti-PD-1 antibody), urelumab (anti-CD137 agonist antibody), and the pancreatic cancer GVAX vaccine. Methods: C57BL/6 mice were orthotopically implanted with KPC tumors. Anti-TREM2 was injected intraperitoneally (i.p.) once a week and anti-PD-1 was injected i.p. twice a week. Tumor volume was measured by ultrasound. TREM2 was stained on slides which were previously obtained from surgically resected PDACs following the neoadjuvant combination treatment of nivolumab, urelumab, and GVAX and stained for multiple immune cell markers with a multiplex staining-striping immunohistochemistry (IHC) technique. Results: Tumor growth post-tumor implantation was found to be significantly decreased by the anti-PD-1 and anti-TREM2 combination compared with anti-PD-1 alone, anti-TREM2 alone, or control treatment (p < 0.0001). Survival of tumor-bearing mice was significantly prolonged by the anti-PD-1 and anti-TREM2 combination when compared to anti-PD-1(p < 0.05), anti-TREM2(p < 0.05), and control (p < 0.01). Multiplex IHC analysis of human PDACs following neoadjuvant immunotherapy showed a higher density of granzyme B+CD8+ effector T cells correlated with higher density of TREM2+ cells (p = 0.0357). Conclusions: Our study demonstrates the anti-tumor efficacy of anti-TREM2 antibody in combination with an immune checkpoint inhibitor in mouse PDAC. However, the analysis of human PDAC following neoadjuvant immunotherapy suggests that TREM2 on TAMs may be activated following the activation of effector T cells. Taken together, our results suggest that TREM2+ TAMs should be reprogrammed instead of being depleted in the design of anti-TREM2-based therapy for PDAC.

Gemcitabine-Lipid Conjugate and ONC201 Combination Therapy Effectively Treats Orthotopic Pancreatic Tumor-Bearing Mice.

Gemcitabine (GEM) is a nucleoside analogue approved as a first line of therapy for pancreatic ductal adenocarcinoma (PDAC). However, rapid metabolism by plasma cytidine deaminase leading to the short half-life, intricate intracellular metabolism, ineffective cell uptake, and swift development of chemoresistance downgrades the clinical efficacy of GEM. ONC201 is a small molecule that inhibits the Akt and ERK pathways and upregulates the TNF-related apoptosis-inducing ligand (TRAIL), which leads to the reversal of both intrinsic and acquired GEM resistance in PDAC treatment. Moreover, the pancreatic cancer cells that were able to bypass apoptosis after treatment of ONC201 get arrested in the G1-phase, which makes them highly sensitive to GEM. To enhance the in vivo stability of GEM, we first synthesized a disulfide bond containing stearate conjugated GEM (lipid-GEM), which makes it sensitive to the redox tumor microenvironment (TME) comprising high glutathione levels. In addition, with the help of colipids 1,2-dioleoyl-glycero-3-phosphocholine (DOPC), cholesterol, and 1,2-distearoyl-glycero-3-phosphoethanolamine-poly(ethylene glycol)-2000 (DSPE-PEG 2000), we were able to synthesize the lipid-GEM conjugate and ONC201 releasing liposomes. A cumulative drug release study confirmed that both ONC201 and GEM showed sustained release from the formulation. Since MUC1 is highly expressed in 70-90% PDAC, we conjugated a MUC1 binding peptide in the liposomes which showed higher cytotoxicity, apoptosis, and cellular internalization by MIA PaCa-2 cells. A biodistribution study further confirmed that the systemic delivery of the liposomes through the tail vein resulted in a higher accumulation of drugs in orthotopic PDAC tumors in NSG mice. The IHC of the excised tumor grafts further confirmed the higher apoptosis and lower metastasis and cell proliferation. Thus, our MUC1 targeting binary drug-releasing liposomal formulation showed higher drug payload, enhanced plasma stability, and accumulation of drugs in the pancreatic orthotopic tumor and thus is a promising therapeutic alternative for the treatment of PDAC.

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

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. 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. 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. 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.

Pancreatic body and tail adenocarcinoma: upfront resection versus neoadjuvant therapy, a contemporary analysis.

There is a paucity of data regarding the use of neoadjuvant therapy in pancreatic body or tail ductal adenocarcinomas. Given the differing tumour biology and aggressive nature of pancreatic body or tail adenocarcinomas, patients presenting with these tumours may benefit from upfront resection. A retrospective cohort study was performed analysing patients who underwent distal pancreatectomy for pancreatic ductal adenocarcinoma between January 2013 and June 2022. Patients who underwent upfront resection were compared to those who underwent neoadjuvant therapy. Forty-one patients underwent upfront distal pancreatectomy, while 40 patients underwent neoadjuvant therapy before curative intent resection. Neoadjuvant therapy did not improve overall survival (37 vs. 34 months, p = 0.962) or disease-free survival (13 vs. 15 months, p = 0.414), as compared with upfront resection. There was no significant difference in the rate or R0 resection or post-operative outcomes. No significant improvement in survival was demonstrated for patients undergoing neoadjuvant therapy for pancreatic ductal adenocarcinoma of the pancreatic body or tail when compared to upfront resection. Considering the potential for disease progression given the more aggressive tumour biology of pancreatic body and tail adenocarcinomas, appropriate surgical candidates should be offered upfront resection to provide the best chance of survival and cure.

A Predictive Noninvasive Single-Nucleotide Variation-Based Biomarker Signature for Resectable Pancreatic Cancer: Protocol for a Prospective Validation Study.

Single-nucleotide variations (SNVs; formerly SNPs) are inherited genetic variants that can be easily determined in routine clinical practice using a simple blood or saliva test. SNVs have potential to serve as noninvasive biomarkers for predicting cancer-specific patient outcomes after resection of pancreatic ductal adenocarcinoma (PDAC). Two recent analyses led to the identification and validation of three SNVs in the CD44 and CHI3L2 genes (rs187115, rs353630, and rs684559), which can be used as predictive biomarkers to help select patients most likely to benefit from pancreatic resection. These variants were associated with an over 2-fold increased risk for tumor-related death in three independent PDAC study cohorts from Europe and the United States, including The Cancer Genome Atlas cohorts (reaching a P value of 1×10-8). However, these analyses were limited by the inherent biases of a retrospective study design, such as selection and publication biases, thereby limiting the clinical use of these promising biomarkers in guiding PDAC therapy. To overcome the limitations of previous retrospectively designed studies and translate the findings into clinical practice, we aim to validate the association of the identified SNVs with survival in a controlled setting using a prospective cohort of patients with PDAC following pancreatic resection. All patients with PDAC who will undergo pancreatic resection at three participating hospitals in Switzerland and fulfill the inclusion criteria will be included in the study consecutively. The SNV genotypes will be determined using standard genotyping techniques from patient blood samples. For each genotyped locus, log-rank and Cox multivariate regression tests will be performed, accounting for the relevant covariates American Joint Committee on Cancer stage and resection status. Clinical follow-up data will be collected for at least 3 years. Sample size calculation resulted in a required sample of 150 patients to sufficiently power the analysis. The follow-up data collection started in August 2019 and the estimated end of data collection will be in May 2027. The study is still recruiting participants and 142 patients have been recruited as of November 2023. The DNA extraction and genotyping of the SNVs will be performed after inclusion of the last patient. Since no SNV genotypes have been determined, no data analysis has been performed to date. The results are expected to be published in 2027. This is the first prospective study of the CD44 and CHI3L2 SNV-based biomarker signature in PDAC. A prospective validation of this signature would enable its clinical use as a noninvasive predictive biomarker of survival after pancreatic resection that is readily available at the time of diagnosis and can assist in guiding PDAC therapy. The results of this study may help to individualize treatment decisions and potentially improve patient outcomes. DERR1-10.2196/54042.

Positive feedback regulation between glycolysis and histone lactylation drives oncogenesis in pancreatic ductal adenocarcinoma

BackgroundMetabolic reprogramming and epigenetic alterations contribute to the aggressiveness of pancreatic ductal adenocarcinoma (PDAC). Lactate-dependent histone modification is a new type of histone mark, which links glycolysis metabolite to the epigenetic process of lactylation. However, the role of histone lactylation in PDAC remains unclear.MethodsThe level of histone lactylation in PDAC was identified by western blot and immunohistochemistry, and its relationship with the overall survival was evaluated using a Kaplan-Meier survival plot. The participation of histone lactylation in the growth and progression of PDAC was confirmed through inhibition of histone lactylation by glycolysis inhibitors or lactate dehydrogenase A (LDHA) knockdown both in vitro and in vivo. The potential writers and erasers of histone lactylation in PDAC were identified by western blot and functional experiments. The potential target genes of H3K18 lactylation (H3K18la) were screened by CUT&Tag and RNA-seq analyses. The candidate target genes TTK protein kinase (TTK) and BUB1 mitotic checkpoint serine/threonine kinase B (BUB1B) were validated through ChIP-qPCR, RT-qPCR and western blot analyses. Next, the effects of these two genes in PDAC were confirmed by knockdown or overexpression. The interaction between TTK and LDHA was identified by Co-IP assay.ResultsHistone lactylation, especially H3K18la level was elevated in PDAC, and the high level of H3K18la was associated with poor prognosis. The suppression of glycolytic activity by different kinds of inhibitors or LDHA knockdown contributed to the anti-tumor effects of PDAC in vitro and in vivo. E1A binding protein p300 (P300) and histone deacetylase 2 were the potential writer and eraser of histone lactylation in PDAC cells, respectively. H3K18la was enriched at the promoters and activated the transcription of mitotic checkpoint regulators TTK and BUB1B. Interestingly, TTK and BUB1B could elevate the expression of P300 which in turn increased glycolysis. Moreover, TTK phosphorylated LDHA at tyrosine 239 (Y239) and activated LDHA, and subsequently upregulated lactate and H3K18la levels.ConclusionsThe glycolysis-H3K18la-TTK/BUB1B positive feedback loop exacerbates dysfunction in PDAC. These findings delivered a new exploration and significant inter-relationship between lactate metabolic reprogramming and epigenetic regulation, which might pave the way toward novel lactylation treatment strategies in PDAC therapy.

CHES1 modulated tumorigenesis and senescence of pancreas cancer cells through repressing AKR1B10

Pancreatic ductal adenocarcinoma (PDAC), is characteristic by a heterogeneous tumor microenvironment and gene mutations, conveys a dismal prognosis and low response to chemotherapy and immunotherapy. Here, we found that checkpoint suppressor 1 (CHES1) served as a tumor repressor in PDAC and was associated with patient prognosis. Functional experiments indicated that CHES1 suppressed the proliferation and invasion of PDAC by modulating cellular senescence. To further identify the downstream factor of CHES1 in PDAC, label-free quantitative proteomics analysis was conducted, which showed that the oncogenic Aldo-keto reductase 1B10 (AKR1B10) was transcriptionally repressed by CHES1 in PDAC. And AKR1B10 facilitated the malignant activity and repressed senescent phenotype of PDAC cells. Moreover, pharmaceutical inhibition of AKR1B10 with Oleanolic acid (OA) significantly induced tumor regression and sensitized PDAC cells to gemcitabine, and this combined therapy did not cause obvious side effects. Rescued experiments revealed that CHES1 regulated the tumorigenesis and gemcitabine sensitivity through AKR1B10-mediated senescence in PDAC. In summary, this study revealed that the CHES1/AKR1B10 axis modulated the progression and cellular senescence in PDAC, which might provide revenues for drug-targeting and senescence-inducing therapies for PDAC.

Single dual-specific anti-PD-L1/TGF-β antibody synergizes with chemotherapy as neoadjuvant treatment for pancreatic ductal adenocarcinoma: a preclinical experimental study.

Chemotherapy resistance is an important cause of neoadjuvant therapy failure in pancreatic ductal adenocarcinoma (PDAC). BiTP (anti-PD-L1/TGF-β bispecific antibody) is a single antibody that can simultaneously and dually target transforming growth factor-beta (TGF-β) and programmed cell death ligand 1 (PD-L1). We attempted in this study to investigate the efficacy of BiTP in combination with first-line chemotherapy in PDAC. Preclinical assessments of BiTP plus gemcitabine and nab-paclitaxel were completed through a resectable KPC mouse model (C57BL/6J). Spectral flow cytometry, tissue section staining, enzyme-linked immunosorbent assays, Counting Kit-8, transwell, and Western blot assays were used to investigate the synergistic effects. BiTP combinatorial chemotherapy in neoadjuvant settings significantly downstaged PDAC tumors, enhanced survival, and had a higher resectability for mice with PDAC. BiTP was high affinity binding to targets and reverse chemotherapy resistance of PDAC cells. The combination overcame immune evasion through reprogramming tumor microenvironment via increasing penetration and function of T cells, natural killer cells, and dendritic cells and decreasing the function of immunosuppression-related cells as regulatory T cells, M2 macrophages, myeloid-derived suppressor cells, and cancer-associated fibroblasts. Our results suggest that the BiTP combinatorial chemotherapy is a promising neoadjuvant therapy for PDAC.

Tumor Microenvironment Modulates Invadopodia Activity of Non-Selected and Acid-Selected Pancreatic Cancer Cells and Its Sensitivity to Gemcitabine and C18-Gemcitabine.

Pancreatic ductal adenocarcinoma (PDAC) is a deadly disease with high mortality due to early metastatic dissemination and high chemoresistance. All these factors are favored by its extracellular matrix (ECM)-rich microenvironment, which is also highly hypoxic and acidic. Gemcitabine (GEM) is still the first-line therapy in PDAC. However, it is quickly deaminated to its inactive metabolite. Several GEM prodrugs have emerged to improve its cytotoxicity. Here, we analyzed how the acidic/hypoxic tumor microenvironment (TME) affects the response of PDAC cell death and invadopodia-mediated ECM proteolysis to both GEM and its C18 prodrug. For this, two PDAC cell lines, PANC-1 and Mia PaCa-2 were adapted to pHe 6.6 or not for 1 month, grown as 3D organotypic cultures and exposed to either GEM or C18 in the presence and absence of acidosis and the hypoxia inducer, deferoxamine. We found that C18 has higher cytotoxic and anti-invadopodia activity than GEM in all culture conditions and especially in acid and hypoxic environments. We propose C18 as a more effective approach to conventional GEM in developing new therapeutic strategies overcoming PDAC chemoresistance.

Targeted Nanoparticle-Based Diagnostic and Treatment Options for Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC), one of the deadliest cancers, presents significant challenges in diagnosis and treatment due to its aggressive, metastatic nature and lack of early detection methods. A key obstacle in PDAC treatment is the highly complex tumor environment characterized by dense stroma surrounding the tumor, which hinders effective drug delivery. Nanotechnology can offer innovative solutions to these challenges, particularly in creating novel drug delivery systems for existing anticancer drugs for PDAC, such as gemcitabine and paclitaxel. By using customization methods such as incorporating conjugated targeting ligands, tumor-penetrating peptides, and therapeutic nucleic acids, these nanoparticle-based systems enhance drug solubility, extend circulation time, improve tumor targeting, and control drug release, thereby minimizing side effects and toxicity in healthy tissues. Moreover, nanoparticles have also shown potential in precise diagnostic methods for PDAC. This literature review will delve into targeted mechanisms, pathways, and approaches in treating pancreatic cancer. Additional emphasis is placed on the study of nanoparticle-based delivery systems, with a brief mention of those in clinical trials. Overall, the overview illustrates the significant advances in nanomedicine, underscoring its role in transcending the constraints of conventional PDAC therapies and diagnostics.

Abstract CT214: Paricalcitol addition to chemotherapy in patients with previously untreated metastatic pancreatic ductal adenocarcinoma (PINBALL)

Abstract Background Vitamin D agonism has been shown in preclinical studies to decrease the production of collagen, decrease myeloid derived suppressor cells, and decrease regulatory T cells in pancreatic ductal adenocarcinoma (PDAC). Paricalcitol is a vitamin D analog used for vitamin D replacement for those with secondary hyperparathyroidism. Two individuals with PDAC who had been developed resistance gemcitabine based combination therapy had shown clinical benefit with the addition of paricalcitol. This clinical study was done to study the effect of adding paricalcitol to individuals with metastatic PDAC receiving gemcitabine based combination therapy (NCT04054362, NCT03415854) . Methods Adults with a histologically- or cytologically-confirmed diagnosis of metastatic PDAC and who were previously untreated were eligible. Individuals received gemcitabine based combination- either gemcitabine plus nab-paclitaxel or gemcitabine plus nab-paclitaxel and cisplatin and had to either demonstrate progressive or stable disease. Paricalcitol was given at a fixed dose of 25 mcg IV three days a week. The combination continued until either disease progression or poor tolerance. The primary objective was to determine the clinical benefit of adding paricalcitol to gemcitabine based combination therapy for metastatic PDAC. Secondary objectives included assessing safety and pharmacodynamic through patient biopsies. Results A total of 34 patients were enrolled across two sites. 16 patients received paricalcitol. The overall response rate was 0% ( CR, PR). Disease control rate at 9 weeks was 21.4% (CR+PR+ SD). PFS upon addition of paricalcitol was 1.6 months. OS upon addition of paricalcitol was 4.8 months. Grade 3/4 AE's attributed to paricalcitol include 13% thrombocytopenia; 19% anemia; 6% transaminitis; 6% pleural effusion; 6% ascites- all grade 3. Conclusions The addition of paricalcitol demonstrated evidence of limited clinical benefit in individuals with metastatic PDAC receiving front-line gemcitabine based therapy. Paricalcitol was generally well tolerated. Tumor analysis is ongoing. Addition studies utilizing prospective treatment of paricalcitol for front line treatment of mPDAC are ongoing (NCT03520790). Trial funding provided by SU2C, CRUK, Lustgarten Foundation, and AACR. Citation Format: David Propper, Gayle Jameson, Denise Roe, Betsy Wertheim, Dan Von Hoff, Erkut H. Borazanci. Paricalcitol addition to chemotherapy in patients with previously untreated metastatic pancreatic ductal adenocarcinoma (PINBALL) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr CT214.

Enhancement of in-vivo cellular uptake of Coenzyme Q10 using saponin derivatives in rTALAP transgenic mice model

Pancreatic cancer remains a significant contributor to cancer-related mortality, with pancreatic ductal adenocarcinoma (PDAC) being particularly challenging to treat. Coenzyme Q10 (CoQ10), a hydrophobic antioxidant crucial for cellular energy production, holds promise in PDAC therapy. However, its limited solubility hinders efficient cellular uptake. To overcome this limitation, we developed micelle formulations incorporating CoQ10 and assessed their potential to enhance cellular delivery. In this particular study, we have utilized some of the most common saponins such as Quillaja saponin, Ginsenoside R0, and Ginsenoside Rb1 as a drug carrier in order to enhance the bioavailability and cellular uptake of CoQ10. Micelles' size and shape were characterized using DLS, TEM, and LC-MS/MS. These all saponin micelles showed better encapsulation, zeta potential, and smaller size compared to Pluronic F127 micelles. Moreover, these formulations induced a notable increase in reactive oxygen species (ROS) generation, indicative of potential apoptotic activity. Further investigations revealed that micelle treatments led to modulation of gene expression related to epithelial-mesenchymal transition (EMT) markers, with an increase in E-cadherin expression and a decrease in claudin, snail, slug, and vimentin. Additionally, in-vivo screening studies in transgenic mice demonstrated promising results as anticancer by reducing apoptosis. In conclusion, our findings suggest that CoQ10-loaded micelles, particularly those incorporating Quillaja saponin and ginsenoside derivatives, hold potential as a novel therapeutic approach for PDAC by enhancing cellular uptake, inducing ROS-mediated apoptosis, and modulating EMT markers. These findings contribute to advancing our understanding of CoQ10's role in pancreatic cancer therapy.

Pancreatic cancer: new approaches to drug therapy.

Outcomes in pancreatic ductal adenocarcinoma (PDAC) remain poor due to a variety of biological, clinical, and societal factors. However, in recent years, PDAC has seen 1) increased precision of initial evaluation, 2) increased emphasis on supportive care, 3) deeper understanding of the translation biology of PDAC, especially as pertains to genomic alterations, and 4) foundational combination chemotherapy clinical trials across all disease stages. These advances have led to a wide range of new approaches to drug therapy for PDAC. Currently available drugs are showing added benefit, both by resequencing them with each other and also with respect to other therapeutic modalities. Molecular strategies are being developed to predict response to known therapeutic agents and to identify others. Additionally, a wide range of new drugs for PDAC are under development, including drugs which inhibit critical molecular pathways, drugs which attempt to capitalize on homologous repair deficiencies, immunotherapeutic approaches, antimetabolic agents, and drugs which attack the extracellular matrix which supports PDAC growth. These new approaches offer the promise of improved survival for future PDAC patients.

Abstract 1593: Intercellular network in immune suppressive microenvironment of pancreatic cancer

Abstract Despite constant efforts to improve treatment, the prognosis remains poor, with an overall survival rate of 6% (ranges from 2% to 9%) due to lack of efficient chemotherapy, radiotherapy, and targeted therapy. Immune checkpoint inhibitors (ICIs) has been reported to be effective in various solid cancers, such as melanoma, lung cancer, and renal cell carcinoma. However, pancreatic ductal adenocarcinoma (PDAC) is resistant against ICIs. The responsiveness of immune therapy is mainly determined by immune tumor microenvironment (TME) composed of tumor-infiltrating lymphocytes (TIL) and stromal cells. PDAC possesses unique TME, which is abundant of fibrosis, termed of desmoplasia, and the role of fibrosis in constructing immune suppressive TME remains unclear. First, in this study, to examine the immunological characteristics of TME, we constructed an integrated data base of TIL profiling, RNA-seq and whole exome seq using 31 fresh resected tissues of PDAC. An unsupervised clustering of based on numbers and percentages of 12 TIL types analyzed by flow cytometry showed that PDAC was divided into 2 types of cluster (myeloid cell-, T cell-dominant type), and the prognosis of myeloid type was significantly poorer than T cell-type. Myeloid type contained the high frequency of myeloid cell lineage including monocytes, macrophages and myeloid-derived suppressor cells (MDSC). Only MDSCs were significantly associated with poor prognosis among various immune cell types. Furthermore, gene enrichment analysis identified activated and suppressed pathways in each immune type, and in myeloid cell-dominant type, the immune-related pathways were significantly down-regulated. Then, to understand the characteristics of MDSCs infiltrated into pancreatic cancer tissues, single cell RNA-seq with surgical specimens was performed, and the analysis showed that a specific subtype of MDSCs infiltrated into cancer tissues. In addition, activation status of cancer-associated fibroblasts (CAFs) was related with MDSC recruitment and activation, indicating that the interaction between MDSCs and CAFs is crucial to create MDSC-rich TME. Inhibition of interaction between MDSCs and CAFs is expected to overcome immunosuppressive TME can be a therapy in PDAC. Citation Format: Kazunori Aoki, Hironori Fukuda, Yukihiro Mizoguchi, Kosuke Arai. Intercellular network in immune suppressive microenvironment of 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 1593.

Abstract 6749: A dendritic cell targeted nanosystem to overcome immune suppression and enhance the efficacy of the standard of care for pancreatic cancer

Abstract The development of novel therapies for pancreatic ductal adenocarcinoma (PDAC) remains an urgent unmet medical need, as it was proven to be refractory to most conventional anti-neoplastic therapies. PDAC is characterized by highly dense stroma, immunosuppressive tumor microenvironment (TME), high resistance to chemotherapy, and poor presentation of tumor-associated antigens (TAA) by dendritic cells (DC), which results in low immune cell infiltration to the tumor site. To that end, we designed a DC-targeted nanovaccine (NV) containing PDAC TAA and TLR agonists to modulate the PDAC immune-microenvironment. In recent years, the combination of immunotherapy with chemotherapy has been explored for PDAC therapy to improve efficacy. Therefore, we demonstrate the effect of our NV on the anti-tumor immune-mediated response in PDAC-bearing mice, alone and in combination with Gemcitabine and Nab-Paclitaxel (Gem/NabP). Our DC-targeted NV was formulated by the double emulsion method and loaded with PDAC’s TAA and immune potentiators. We conjugated the NV to a mannose moiety as a targeting moiety for the mannose receptor expressed on DC to increase the NV uptake, which resulted in the enhanced presentation of the encapsulated TAA. Followed by physicochemical characterization and evaluation of preclinical safety of the NV, the stimulation of T cell-specific response against PDAC tumor was assessed in vivo and ex vivo. The NV induced significant changes in PDAC’s TME, such as increased CD8+ T cells, decreased CD4+ T regulatory cells, and induced memory immunity in the tumor site. The combination of NV and Gem/NabP inhibited tumor growth and prolonged survival compared to mice treated with Gem/NabP alone. This could also be related to the immunogenic cell death effect caused by the chemotherapies. In addition to our mice model of PDAC, we also established a patient-derived model, in which we generate 3D-spheroids from the patient’s tumor and activate PBMCs with our NV from the same patient. This unique 3D model enables us to evaluate ex vivo the NV effect in a more clinically relevant manner. The NV proposed herein could induce a strong antigen-specific immune response against PDAC with restricted tumor growth while re-shaping PDAC immunosuppressive TME. Furthermore, our 3D patient-derived PDAC model allows us to assess the ability of our NV to generate specific immune responses in patients and to test other possible drug combinations predicting the clinical outcome. Citation Format: Ron Kleiner, Daniella Vaskovich-Koubi, Rita Acúrcio, Barbara Carreira, Helena Florindo, Ronit Satchi-Fainaro. A dendritic cell targeted nanosystem to overcome immune suppression and enhance the efficacy of the standard of care for 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 6749.

Abstract 2079: Tumor-derived fibrinogen promotes pancreatic ductal adenocarcinoma progression

Abstract Background: Hypercoagulability is a common clinical manifestation in patients with cancers, especially in pancreatic ductal adenocarcinoma (PDAC). Over 20% of PDAC patients with advanced-stage and worse prognosis undergo a venous thromboembolism (VTE) incidence. Fibrinogen (FG) may contribute to VTE due to its high expression in advanced PDAC tumors. Here, we set to determine the biological roles of tumor-derived FG in PDAC. In addition, our previous study showed that Von Willebrand Factor (VWF) A2 domain can effectively bind to FG and reduce platelet clot formation. Therefore, the functional roles of A2 on FG in PDAC were also studied. Methods: The correlation between survival rate and FG expression in PDAC was analyzed using RNAseq profiles of 149 PDAC patient samples from the TCGA database. PDAC subtyping of Baylor College of Medicine (BCM) PDAC patient-derived xenograft (PDX) cohort was performed using RNAseq data according to Moffitt’s criteria. The FG (three chains FGA, FGB, FGG) overexpression (OE) and knock-out (KO) PDAC cell lines were established. The gene expression and biological functions of FG were performed in PDX and cell lines using IHC and immunoblot assays. The FG effect on PDAC progression in vivo was studied in mouse models. Results: TCGA analysis showed that elevated tumor-derived FG gene expression is significantly correlated with worse overall survival and disease-free survival rates in PDAC patients. PDX lines RNAseq analysis clearly separated BCM PDAC PDXs into two subtypes: basal-like and classical PDACs. Basal-like PDXs showed significantly stronger FG staining than that in classical ones. Compared to the classical subtype, the basal-like subtype showed significantly upregulated genes that are associated with the epithelial-mesenchymal transition (EMT), IL6/JAK/STAT3, and TNFα signaling pathways. Accordingly, basal-like subtype PDAC exposed to IL6 or TNFα significantly increased FG and FG receptor expression with activated STAT3 and AKT pathways and elevated EMT markers, whereas classical subtype cells have minimal changes. In addition, we found that the FG-OE cells exhibit enhanced cell proliferation and migration properties whereas FG-KO cells reduced the effects. Consistent with in vitro data, FG-OE PDAC showed significantly higher tumor burdens than that of control PDAC in both orthotopic and subcutaneous PDAC mouse models. Furthermore, A2 protein treatment can effectively block the FG function and dramatically inactivate the FG-induced EMT process and AKT signaling pathway. Conclusions: Tumor-derived fibrinogen is upregulated in a basal-like subtype of PDAC cells and may contribute to tumor growth and metastasis by activating aberrant signaling pathways and promoting EMT. Our findings imply that elevated fibrinogen in PDAC could contribute to tumor progression and the A2 protein which target to fibrinogen may be a promising therapy for basal-like subtype PDAC. Citation Format: Dongliang Liu, Lisa Brubaker, Yichi Niu, Emily LaPlante, George Van Buren, Aleksandar Milosavljevic, Changyi Chen, Chenghang Zong, Miguel Cruz, Qizhi Yao. Tumor-derived fibrinogen promotes pancreatic ductal adenocarcinoma progression [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 2079.

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.

Abstract 4397: Alpha-Ketoglutarate is a master regulator of epigenetic reprogramming in pancreatic ductal adenocarcinoma progression

Abstract Most cases of Pancreatic Ductal Adenocarcinoma (PDAC) are diagnosed after metastasis has occurred, making it crucial to find effective treatments for late-stage disease. Previously, our group found widespread epigenetic reprogramming during PDAC metastasis in the absence of additional driver mutations. These cells develop dependence on glucose metabolism through the oxidative pentose phosphate pathway (oxPPP). Inhibition of oxPPP with 6-aminonicotinamide (6AN) reverses the chromatin changes and restores cells to a less malignant state (McDonald et al. Nat Gen, 2017). To identify a clinically actionable mechanism for restoration, we hypothesized that oxPPP dependence leads to downstream metabolic changes that mediate chromatin state. Using untargeted metabolomic analysis, we found that alpha-ketoglutarate (AKG), a cofactor of lysine and histone demethylases, was downregulated by oxPPP inhibition (p=0.033) in a patient-derived xenograft lung metastasis cell line, A13Lg. Treatment of cells with 6AN and a cell permeable precursor of AKG largely reversed the chromatin changes seen on a western blot. Given that AKG is mainly produced by glutamine metabolism, we inhibited glutaminolysis with glutamine deprivation (Gln(-)) or the glutamine antagonist JHU083. Both treatments decreased AKG (Gln(-) p=0.002, JHU083 p=0.038) and prevented cell growth (Gln(-) p<0.0001, JHU083 p=0.0002). Glutamine-deprived cells showed decreased ability to form colonies (p=0.004), and JHU083 treated cells showed impaired migration in a Boyden chamber assay (p=0.003). Western blots showed a decrease in H3K27 acetylation, a marker of euchromatin, and an increase in H3K9 and H3K27 methylation, markers of heterochromatin, in response to both treatments. Further, homogenized time-resolved fluorescence (HTRF) assays on glutamine-deprived cells showed that H3K9 di- (p=0.0016,) and tri- (p=0.0003) methylation was increased. Adding cell permeable AKG to media without glutamine largely negated both the phenotypic and epigenetic changes. Using CUT&RUN against H3K27Ac, we found that acetylation was depleted at promoters involved in tumor cell proliferation, invasion, and migration. Bulk RNA-sequencing also showed decreased expression of genes involved in these pathways. Epigenetic and phenotypic changes were not as apparent after treatment with CB839, a glutaminase 1 inhibitor, suggesting that broad inhibition of glutamine metabolism is necessary for chromatin restoration. Together, these data suggest that decreasing AKG in PDAC distant metastasis cells, through glutamine deprivation or JHU083, leads to chromatin changes that specify less malignant phenotypes, providing a rationale for targeting glutamine metabolism as a therapy for late-stage PDAC. *APF and MM are co-corresponding authors. Citation Format: Kenna Sherman, Jiaqi Cheng, Weiqiang Zhou, Hongkai Ji, Masahiro Maeda, Andrew P. Feinberg. Alpha-Ketoglutarate is a master regulator of epigenetic reprogramming in pancreatic ductal adenocarcinoma progression [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 4397.

Abstract 4774: Inhibition of pancreatic acinar ductal metaplasia by novel YAP/TAZ inhibitor, CV-4-26

Abstract Current challenges for pancreatic ductal adenocarcinoma (PDAC) treatment include overcoming the disease’s fibrous tumor microenvironment with limited cytotoxicity- in which tissue stiffening is believed to occur even pre-disease onset. Acinar to ductal metaplasia (ADM), the transdifferentiation of acinar cells into duct-like cells, is a predisposing event to PDAC development that is also linked to Hippo signaling, a major pathway involved in fibrosis and inflammation. Hippo activation is initiated by the yes-associated protein (YAP) in conjunction with the PDAZ binding motif (TAZ) and TEA domain (TEAD) transcription factor. Thus, the synthesis of the novel covalent TEAD autopalmitoylation inhibitor, CV-4-26, presented an opportunity to therapeutically target ADM. CV-4-26 binds to the TEAD lipid binding pocket, which, in turns, hinders TEAD palmitoylation, thereby leading to the inhibition of the Hippo pathway. Utilizing our 3D organoid p48Cre/+LSL-KRASG12D/+ (KC) mouse model, we demonstrated pronounced phenotypic and molecular inhibition of ADM by CV-4-26, resulting in a conserved acinar phenotype, as well as downregulation of downstream markers of the YAP/TAZ complex. The compound did not reverse ADM to the acinar state once ducts were visibly formed. Yet, CV-4-26 demonstrated a lack of cytotoxicity (up to 25 µM) as determined by Calcein AM staining. Biomechanical measurements of extracellular matrix stiffness acquired through calculations of fluorescent nanoparticle displacement further displayed decreased moduli as a response to inhibition treatment. Moreover, a 56-fold downregulation of collagen (COL1A1, COL1A2) mRNA, markers for PDAC development, was observed. However, less reduction in the collagens and extracellular matrix stiffness was observed in the ADM reversal treatments, corroborating our morphology and gene expression results. Our data suggest that combining CV-4-26 by ADM inhibition with standard of care therapies for PDAC may enhance the penetration of small molecules through the tumor’s extracellular matrix. Citation Format: Corey M. Perkins, Chen Zhou, Martha Campbell-Thompson, Yating Mao, Kalina R. Atanasova, Jinmai Jiang, Ranjala Ratnayake, Hendrik Luesch, Jamel Ali, Chenglong Li, Thomas D. Schmittgen. Inhibition of pancreatic acinar ductal metaplasia by novel YAP/TAZ inhibitor, CV-4-26 [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 4774.