Pancreatic Ductal Adenocarcinoma Cancer Cells Research Articles

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.

Auranofin repurposing for lung and pancreatic cancer: low CA12 expression as a marker of sensitivity in patient-derived organoids, with potentiated efficacy by AKT inhibition

BackgroundThis study explores the repurposing of Auranofin (AF), an anti-rheumatic drug, for treating non-small cell lung cancer (NSCLC) adenocarcinoma and pancreatic ductal adenocarcinoma (PDAC). Drug repurposing in oncology offers a cost-effective and time-efficient approach to developing new cancer therapies. Our research focuses on evaluating AF's selective cytotoxicity against cancer cells, identifying RNAseq-based biomarkers to predict AF response, and finding the most effective co-therapeutic agents for combination with AF.MethodsOur investigation employed a comprehensive drug screening of AF in combination with eleven anticancer agents in cancerous PDAC and NSCLC patient-derived organoids (n = 7), and non-cancerous pulmonary organoids (n = 2). Additionally, we conducted RNA sequencing to identify potential biomarkers for AF sensitivity and experimented with various drug combinations to optimize AF's therapeutic efficacy.ResultsThe results revealed that AF demonstrates a preferential cytotoxic effect on NSCLC and PDAC cancer cells at clinically relevant concentrations below 1 µM, sparing normal epithelial cells. We identified Carbonic Anhydrase 12 (CA12) as a significant RNAseq-based biomarker, closely associated with the NF-κB survival signaling pathway, which is crucial in cancer cell response to oxidative stress. Our findings suggest that cancer cells with low CA12 expression are more susceptible to AF treatment. Furthermore, the combination of AF with the AKT inhibitor MK2206 was found to be particularly effective, exhibiting potent and selective cytotoxic synergy, especially in tumor organoid models classified as intermediate responders to AF, without adverse effects on healthy organoids.ConclusionOur research offers valuable insights into the use of AF for treating NSCLC and PDAC. It highlights AF's cancer cell selectivity, establishes CA12 as a predictive biomarker for AF sensitivity, and underscores the enhanced efficacy of AF when combined with MK2206 and other therapeutics. These findings pave the way for further exploration of AF in cancer treatment, particularly in identifying patient populations most likely to benefit from its use and in optimizing combination therapies for improved patient outcomes.

Abstract 4574: Next generation nuclear export blocker KPT8602 synergizes with KRASG12D inhibitor MRTX1133 resulting in improved antitumor effects against pancreatic ductal adenocarcinoma

Abstract Introduction: A novel KRAS inhibitor, MRTX1133, specifically targeting the G12D isoform, prevalent in pancreatic ductal adenocarcinoma (PDAC), holds promise for a substantial PDAC patient population. Nevertheless, given the clinical experience with sotorasib (a KRASG12C inhibitor) in lung cancer, it's anticipated that resistance to the KRASG12D inhibitor will eventually emerge in most PDAC patients. This underscores the need to identify strategies that can enhance the efficacy of KRAS inhibitors in PDAC. Emerging evidence suggests that the nuclear export protein exportin 1 (XPO1) is a druggable vulnerability in KRAS mutant cancers. XPO1 plays a pivotal role in shuttling various critical tumor suppressors, genome surveillance proteins, and transcription factors out of the nucleus and is frequently overexpressed in various cancers, including PDAC. In this study, we employed both in vitro and in vivo models to assess the potential of KPT8602, an investigational and next-generation selective XPO1 inhibitor, to synergize with the KRASG12D inhibitor MRTX1133 and sensitize MRTX1133-resistant PDAC cells. Methods: We examined the cytotoxic and molecular effects of KRASG12D inhibitor MRTX1133 in combination with nuclear export blocker KPT8602 in KRASG12D mutant 2D and 3D cellular/spheroid models. The preclinical antitumor efficacy of the combination was evaluated in a KRASG12D mutant PDAC cell derived xenograft (CDX) model. Results: KPT8602 synergized with MRTX1133 in vitro yielding suppressed proliferation of KRASG12D mutant PDAC cells. Similar synergistic effects were also seen in 3D cultures of KRASG12D mutant PDAC cancer cell lines and patient derived primary tumor cells where the combination suppressed spheroid formation and reduced spheroid viability. Also, the combination inhibited the clonogenic potential of KRASG12D mutant PDAC cells. In addition, we developed a PDAC cell line resistant to MRTX1133 and observed that such MRTX1133-resistant cell line was acutely sensitive to treatment with KPT8602. Mechanistically, KPT8602 and MRTX1133 combination led to reduction in protein expression of KRAS downstream effectors and cell cycle markers. Furthermore, a combined administration of KPT8602 and MRTX1133 at sub-optimal doses resulted in remarkable tumor growth inhibition in a KRASG12D mutant HPAC CDX mice model. Moreover, KPT8602 as a maintenance therapy also prevented tumor relapse. Conclusion: This is the first study demonstrating that nuclear export blocker KPT8602 can synergize with KRASG12D inhibitor MRTX1133 and has the potential to target MRTX1133-resistant PDAC cells. This study provides a rationale for combining MRTX1133 with KPT8602 for the treatment of PDAC patients with KRASG12D mutant tumors. Citation Format: Husain Yar Khan, Amro Aboukameel, Md Hafiz Uddin, Sahar F. Bannoura, Khaled Keffri, Medha Jasti, Rachael Virga, Sarah Motorwala, Khalil Choucair, Tarik Hadid, Mohammad Najeeb Al-Hallak, Eliza Beal, Miguel Tobon, Rafic Beydoun, Greg Dyson, Steve Kim, Misako Nagasaka, Philip A. Philip, Basel El-Rayes, Herbert Chen, Anthony Shields, Ramzi Mohammad, Boris Pasche, Asfar Azmi. Next generation nuclear export blocker KPT8602 synergizes with KRASG12D inhibitor MRTX1133 resulting in improved antitumor effects against 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 4574.

Abstract 1583: Neutrophil pro-tumorigenic role in progression and therapy resistance in pancreatic ductal adenocarcinoma

Abstract Pancreatic cancer (PC) continues to be a challenge for today’s therapeutics, with a meager 5-year survival rate of 12%. Due to the pancreas’ location, patients often receive diagnosis late, leaving them with limited treatment options. Often, they fail to respond to chemotherapy regimens or develop resistance to those therapies. Tumor-promoting inflammation is a hallmark of cancer, contributing to tumor cells’ survival and proliferation. Infiltrating leukocytes and pro-inflammatory cytokines released into the tumor microenvironment (TME) often cause this inflammation. My lab has previously linked increases in pancreatic ductal adenocarcinoma (PDAC) progression and chemotherapy resistance to inflammation and the CXCR2 pathway. In the present study, we examined the role of neutrophil recruitment in PDAC progression using patient samples and KRAS-dependent KC and KPC murine models. Our data suggest a positive association between tumor-associated neutrophils (TANs) and PDAC progression. In vitro, we observed neutrophil-PDAC interaction enhancing the survival of both neutrophils and PDAC cell lines. Moreover, this survival was mediated by cellular aggressiveness and therapy resistance. Concerning survival, we saw increased expression of anti-apoptotic genes in the neutrophils upon PDAC cancer cell conditioned media treatment. We also observed modulated expression of immunoregulatory molecules. Our data suggest that increased recruitment of TANs supports PDAC progression and survival, and PDAC cell-neutrophil interaction modulates neutrophil survival and immunoregulatory phenotype. Citation Format: Reegan Sturgeon, Esther Johnson, Caitlin Molczyk, Paran Goel, Parker Tinsley, Lauren Abrahams, Michael Maher, Rakesh K. Singh. Neutrophil pro-tumorigenic role in progression and therapy resistance 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 1583.

NDRG1 overcomes resistance to immunotherapy of pancreatic ductal adenocarcinoma through inhibiting ATG9A-dependent degradation of MHC-1

AimsPancreatic ductal adenocarcinoma (PDAC) is a deadly disease that is resistant to immune checkpoint blockade (ICB) therapies. Emerging evidence suggests that NDRG1 may be an important target for the development of new therapies for PDAC. Herein, we investigated the novel roles of NDRG1 and Combretastatin A-4 (CA-4) in the treatment of PDAC ICB resistance. MethodsEnrichment of MHC class I was detected by RNA sequence and verified by RT-qPCR and immunoblotting in NDRG1-knockdown human pancreatic cancer cell lines. The protein degradation mode was found by stimulation with various inhibitors, and the autophagy degradation pathway was found by immunoprecipitation and immunolocalization. The roles of NDRG1 and MHC-I in immunotherapy were investigated by orthotopic solid tumors, histology, immunohistochemistry, multiplex immunofluorescence staining and flow cytometry. ResultsHere, we identified a previously undescribed role of NDRG1 in activating major histocompatibility complex class 1 (MHC-1) expression in pancreatic ductal adenocarcinoma (PDAC) cells through lysosomal-autophagy-dependent degradation. In mouse models of PDAC, either tumor cell overexpression or pharmacologic activation of NDRG1 leads to MHC-1 upregulation in tumor cells, which in turn promotes the infiltration and activity of CD8 + T cells, enhances anti-tumor immunity, and overcomes resistance to ICB therapy. Moreover, combination therapy of CA-4 and ICB overcomes the drug resistance of pancreatic cancer to ICB therapy. In PDAC patients, NDRG1 expression correlates with high MHC-1 expression and better survival. ConclusionOur results reveal NDRG1 in PDAC cancer cells as a tumor suppressor and suggest that pharmaceutically targeting NDRG1 is a promising way to overcome pancreatic cancer resistance to immunotherapy and provides a potential therapeutic strategy for the treatment of pancreatic cancer patients.

THU507 Tumor Activin A Mediates Cachexia Primarily Through White Adipose Tissue Hydrolysis In Pancreatic Ductal Adenocarcinoma

Abstract Disclosure: S. Yu: None. Y. Luan: None. R. Dong: None. A. Abazarikia: None. S. Kim: None. Pancreatic ductal adenocarcinoma (PDAC) commonly harbors mutations in KRAS and TP53 genes and its overall 5-year survival rate is only 10% in the US. Among risk factors associated with the dismal PDAC prognosis, cachexia accounts for about 30% of PDAC-related death and is present in nearly 80% of PDAC patients. Significant adipose tissue loss followed by skeletal muscle loss characterizes PDAC-related cachexia in humans and mice. Accumulated evidence indicates that activin A, a homodimer of inhibin βA that is encoded by the INHBA gene, is a recognized key cachexia mediator as supported by mitigation of weight loss by activin A antagonists in PDAC-bearing mice. However, much remains unclear regarding the primary source cells of activin A and the mechanisms by which activin A mediates cachexia in PDAC. To address the unknowns, we employed CRISPR/Cas9 system for Inhba gene knock-down (KD) in mouse KPC (KrasG12D and Trp53R172H) PDAC cells and generated orthotopic mice using Inhba KD or control KPC cells. Sham group which received the same operation except for cell injection was included. Body weight was measured on a daily basis, and blood and white adipose tissues (WATs) and skeletal muscle tissues were harvested on post-surgery day 12. We found that, when compared to the Sham group, injection of control KPC cells significantly elevated serum activin A levels up to 6 ng/mL and temporally decreased the average body weight in the orthotopic mice (Ctrl group), whereas Inhba gene KD significantly reduced serum activin A levels by ∼ 2 ng/mL and prevented weight loss in the orthotopic mice (KD group). When compared to the Sham and KD groups, notable reductions in organ weights of epididymal, mesenteric or subcutaneous WATs were found in the Ctrl group, however, weights for gastrocnemius and plantaris or soleus muscle tissues were statistically similar between the Ctrl and KD groups at post-surgery day 12. Correspondingly, the average serum free fatty acids levels were higher in the Ctrl group than the KD group. Histological observations support that dramatic adipocyte size reductions in WATs were prevented by serum activin A suppression. Immunofluorescence analysis showed that WATs from the Ctrl group had considerably higher protein expression of hormone-sensitive or adipose triglyceride lipase than the KD and Sham groups. Furthermore, phosphorylation of SMAD3 and phosphorylated PKA substrate which regulate adipocyte lipolysis were intensely stained in WATs from the Ctrl group when compared to the KD and Sham groups. Collectively, our findings identify PDAC cancer cells as the major source of activin A in PDAC and suggest systemic activin A as a potential mediator of PDAC-related adipose tissue loss. The current findings warrant further investigation to address clinical relevance. Presentation: Thursday, June 15, 2023

Exploring Covalent Bond Formation at Tyr-82 for Inhibition of Ral GTPase Activation.

Ral RAS GTPases are directly activated by KRAS through a trimeric complex with a guanine exchange factor. Ral is considered undruggable and lacks an accessible cysteine for covalent drug development. Previously we had reported an aryl sulfonyl fluoride fragment that formed a covalent bond at Tyr-82 on Ral and created a deep and well-defined pocket. Here, we explore this pocket further through design and synthesis of several fragment derivatives. The fragment core is modified by introducing tetrahydronaphthalene or benzodioxane rings to enhance affinity and stability of the sulfonyl fluoride reactive group. The deep pocket in the Switch II region is also explored by modifying the aromatic ring of the fragment that is ensconced into the pocket. Compounds 19 (SOF-658) and 26 (SOF-648) formed a single robust adduct specifically at Tyr-82, inhibited Ral GTPase exchange in buffer and in mammalian cells, and blocked invasion of pancreatic ductal adenocarcinoma cancer cells. Compound 19 (SOF-658) was stable in buffer, mouse, and human microsomes suggesting that further optimization could lead to small molecules to probe Ral activity in tumor models.

Abstract 3938: In vivo genome-wide CRISPR screen in pancreatic ductal adenocarcinoma defines HSPE1 as a potential oncogene by acting through G2/M cell cycle arrest

Abstract Superior knowledge of cancer biology has enabled unprecedented innovations in therapies targeting mutated driver genes. Despite the attempt of targeting cancer-inducing genes such as KRAS, the life expectancy of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) has poorly improved. We performed an unbiased genome wide in-vivo loss-of-function CRISPR screen to discover novel oncogenes and identified heat-shock protein HSPE1, whose function is still unknown in PDAC. By decreasing HSPE1 expression with shRNA and CRISPR technology, we detected a slowdown in cell growth of PDAC cancer cells. HSPE1 knock-out cells injected in mice displayed a drastic reduction in tumor volume. We exploited this vulnerability by disrupting HSPE1 function by using KHS101, a validated HSPD1-HSPE1 complex inhibitor. Several PDAC cell lines cultured in vitro were sensitive upon KHS101 treatment and in vivo administration of KHS101 reduced tumorigenesis. Compiling the negative fitness scores from DepMap database revealed a co-dependency between HSPE1 and PLK1, a protein involved in regulating G2/M checkpoint. Indeed, we detected a protein-level decrease of PLK1 in our experimental model of PDAC cells having either a drug-induced HSPE1 deficiency by KHS101 exposure or a HSPE1 gene-expression reduction by shRNA. We found that the cell cycle was disrupted through G2/M arrest in PDAC cells treated with KHS101. Our findings highlight a new role underlying PDAC tumorigenesis for HSPE1 and could unlock a new area of research towards precision medicine. Citation Format: Julien Boudreault, Ni Wang, Gang Yan, Meiou Dai, Sophie Poulet, Girija Daliah, Jean-Jacques Lebrun. In vivo genome-wide CRISPR screen in pancreatic ductal adenocarcinoma defines HSPE1 as a potential oncogene by acting through G2/M cell cycle arrest. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3938.

Tumor Cell Derived Lnc-FSD2-31:1 Contributes to Cancer-Associated Fibroblasts Activation in Pancreatic Ductal Adenocarcinoma Progression through Extracellular Vesicles Cargo MiR-4736.

Pancreatic ductal adenocarcinoma (PDAC) presents with high mortality and short overall survival. Cancer-associated fibroblasts (CAFs) act as refuge for cancer cells in PDAC. Mechanisms of intracelluar communication between CAFs and cancer cells need to be explored. Long noncoding RNAs (lncRNAs) are involved in the modulation of oncogenesis and tumor progression of PDAC; however, specific lncRNAs and their mechanism of action have not been clarified clearly in tumoral microenvironment. This work aims to identify novel lncRNAs involved in cellular interaction between cancer cells and CAFs in PDAC. To this end, differentially expressed lncRNAs between long-term and short-term survival PDAC patients are screened. Lnc-FSD2-31:1 is found to be significantly increased in long-term survival patients. This work then discovers that tumor-derived lnc-FSD2-31:1 restrains CAFs activation via miR-4736 transported by extracellular vesicles (EVs) in vitro and in vivo. Mechanistically, EVs-derived miR-4736 suppresses autophagy and contributes to CAFs activation by targeting ATG7. Furthermore, blocking miR-4736 suppresses tumor growth in genetically engineered KPC (LSL-KrasG12D/+, LSL-Trp53R172H/+, and Pdx-1-Cre) mouse model of PDAC. This study demonstrates that intratumoral lnc-FSD2-31:1 modulates autophagy in CAFs resulting in their activation through EVs-derived miR-4736. Targeting miR-4736 may be a potential biomarker and therapeutic target for PDAC.

Targeting the bicarbonate transporter SLC4A4 overcomes immunosuppression and immunotherapy resistance in pancreatic cancer

Solid tumors are generally characterized by an acidic tumor microenvironment (TME) that favors cancer progression, therapy resistance and immune evasion. By single-cell RNA-sequencing analysis in individuals with pancreatic ductal adenocarcinoma (PDAC), we reveal solute carrier family 4 member 4 (SLC4A4) as the most abundant bicarbonate transporter, predominantly expressed by epithelial ductal cells. Functionally, SLC4A4 inhibition in PDAC cancer cells mitigates the acidosis of the TME due to bicarbonate accumulation in the extracellular space and a decrease in lactate production by cancer cells as the result of reduced glycolysis. In PDAC-bearing mice, genetic or pharmacological SLC4A4 targeting improves T cell-mediated immune response and breaches macrophage-mediated immunosuppression, thus inhibiting tumor growth and metastases. In addition, Slc4a4 targeting in combination with immune checkpoint blockade is able to overcome immunotherapy resistance and prolong survival. Overall, our data propose SLC4A4 as a therapeutic target to unleash an antitumor immune response in PDAC.

GJB3 promotes pancreatic cancer liver metastasis by enhancing the polarization and survival of neutrophil.

Connexins are membrane expressed proteins, which could assemble into hexamers to transfer metabolites and secondary messengers. However, its roles in pancreatic cancer metastasis remains unknown. In this study, by comparing the gene expression patterns in primary pancreatic cancer patients primary and liver metastasis specimens, we found that Gap Junction Protein Beta 3 (GJB3) significantly increased in Pancreatic ductal adenocarcinoma (PDAC) liver metastasis. Animal experiments verified that GJB3 depletion suppressed the hepatic metastasis of PDAC cancer cells. Further, GJB3 over expression increased the neutrophil infiltration. Mechanistic study revealed that GJB3 form channels between PDAC tumor cells and accumulated neutrophil, which transfer cyclic adenosine monophosphate (cAMP) from cancer to neutrophil cells, which supports the survival and polarization. Taken together, our data suggesting that GJB3 could act as a potential therapeutic target of PDAC liver metastasis.

Niraparib-induced STAT3 inhibition increases its antitumor effects.

Recently, poly(ADP-ribosyl)ation polymerase inhibitors (PARPis), which induce synthetic lethality of tumor cells with DNA damage repair defects, have emerged as a promising therapy for ovarian, breast, and pancreatic cancer. Although the PARPi Olaparib is limited to treating cancer patients with DNA repair deficiencies, the PARPi Niraparib is FDA approved to treat ovarian cancer patients regardless of their status in DNA repair pathways. Despite differences in the affinity to PARP enzymes, the rationale behind the clinical use of Niraparib in patients without DNA repair deficiencies is still lacking. Moreover, only Olaparib has been approved for pancreatic ductal adenocarcinoma (PDAC) patients with BRCA mutations, accounting for only 5-7% of total PDACs. It remains unclear whether Niraparib could be beneficial to PDACs without BRCA mutations. We found that Niraparib inhibits ovarian and PDAC tumor cell growth, regardless of BRCA mutational status, more effectively than Olaparib. Unlike Olaparib, which is known to activate STAT3, Niraparib inhibits STAT3 activity in ovarian and PDAC cancer cell lines and patient tumors. Moreover, Niraparib regulates the expression of several STAT3 downstream genes involved in apoptosis. Overexpression of a constitutively activated STAT3 mutant rescues Niraparib-induced cancer cell apoptosis. Our results suggest that Niraparib inhibits pSTAT3 by interfering with SRC tyrosine kinase. Collectively, our studies provide a mechanism underlying Niraparib's ability to induce tumor cell apoptosis without BRCA mutations, suggesting the potential use of Niraparib for treating PDAC patients regardless of BRCA status.

Cystathionine β-synthase overexpression drives metastatic dissemination in pancreatic ductal adenocarcinoma via inducing epithelial-to-mesenchymal transformation of cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of all cancer types with a constant rise in global incidence. Therefore, better understanding of PDAC biology, in order to design more efficient diagnostic and treatment modalities, is a priority.Here we found that the expression levels of cystathionine β-synthase (CBS), a transsulfuration enzyme, is markedly elevated in metastatic PDAC cells compared to cell lines isolated from non-metastatic primary tumors. On human immunohistochemical samples from PDAC patients we also found higher CBS staining in cancerous ductal cells compared to in non-tumor tissue, which was further elevated in the lymph node metastasis of the same patients. In mice, orthotopically injected CBS-silenced T3M4 cells induced fewer liver metastases compared to control cells indicating important roles for CBS in PDAC cancer cell invasion and malignant transformation. Wound healing and colony formation assays in cell culture confirmed that CBS-deficient metastatic T3M4 and non-metastatic BxPC3 primary tumor cells migrate slower and have impaired anchorage-independent growth capacities compared to control T3M4 cells. CBS silencing in T3M4 cells lowered WNT5a and SNAI1 gene expression down to levels that were observed in BxPC3 cells as well as resulted in an increase in E-cadherin and a decrease in Vimentin signals in mouse tumors when injected orthotopically. These observations suggested a primary role for the epithelial to mesenchymal transformation of cancer cells in CBS-mediated tumor aggressiveness. Under normal conditions, STAT3, an upstream regulator of Wnt signaling pathways, was less phosphorylated and more oxidized in shCBS T3M4 and BxPC3 compared to control T3M4 cells, which is consistent with decreased transcriptional activity at lower CBS levels due to less protection against oxidation. Sulfur metabolome analyses suggested that this CBS-mediated protection against oxidative modifications is likely to be related to persulfide/sulfide producing activities of the enzyme rather than its canonical function to produce cystathionine for cysteine synthesis. Taken together, CBS overexpression through regulation of the EMT plays a significant role in PDAC cancer cell invasion and metastasis.

SMAD2/3 mediate oncogenic effects of TGF-β in the absence of SMAD4

TGF-β signaling is involved in pancreatic ductal adenocarcinoma (PDAC) tumorigenesis, representing one of the four major pathways genetically altered in 100% of PDAC cases. TGF-β exerts complex and pleiotropic effects in cancers, notably via the activation of SMAD pathways, predominantly SMAD2/3/4. Though SMAD2 and 3 are rarely mutated in cancers, SMAD4 is lost in about 50% of PDAC, and the role of SMAD2/3 in a SMAD4-null context remains understudied. We herein provide evidence of a SMAD2/3 oncogenic effect in response to TGF-β1 in SMAD4-null human PDAC cancer cells. We report that inactivation of SMAD2/3 in SMAD4-negative PDAC cells compromises TGF-β-driven collective migration mediated by FAK and Rho/Rac signaling. Moreover, RNA-sequencing analyses highlight a TGF-β gene signature related to aggressiveness mediated by SMAD2/3 in the absence of SMAD4. Using a PDAC patient cohort, we reveal that SMAD4-negative tumors with high levels of phospho-SMAD2 are more aggressive and have a poorer prognosis. Thus, loss of SMAD4 tumor suppressive activity in PDAC leads to an oncogenic gain-of-function of SMAD2/3, and to the onset of associated deleterious effects.

ID1 marks the tumorigenesis of pancreatic ductal adenocarcinoma in mouse and human

Pancreatic Ductal Adenocarcinoma (PDAC) is a deadly disease that has an increasing death rate but no effective treatment to now. Although biological and immunological hallmarks of PDAC have been frequently reported recently, early detection and the particularly aggressive biological features are the major challenges remaining unclear. In the current study, we retrieved multiple scRNA-seq datasets and illustrated the genetic programs of PDAC development in genetically modified mouse models. Notably, the transcription levels of Id1 were elevated specifically along with the PDAC development. Pseudotime trajectory analysis revealed that Id1 was closely correlated with the malignancy of PDAC. The gene expression patterns of human PDAC cells were determined by the comparative analysis of the scRNA-seq data on human PDAC and normal pancreas tissues. ID1 levels in human PDAC cancer cells were dramatically increased compared to normal epithelial cells. ID1 deficiency in vitro significantly blunt the invasive tumor-formation related phenotypes. IPA analysis on the differentially expressed genes suggested that EIF2 signaling was the core pathway regulating the development of PDAC. Blocking EFI2 signaling remarkably decreased the expression of ID1 and attenuated the tumor-formation related phenotypes. These observations confirmed that ID1 was regulated by EIF2 signaling and was the critical determinator of PDAC development and progression. This study suggests that ID1 is a potential malignant biomarker of PDAC in both mouse models and human and detecting and targeting ID1 may be a promising strategy to treat or even rescue PDAC.

Potential of Gold Nanoparticle in Current Radiotherapy Using a Co-Culture Model of Cancer Cells and Cancer Associated Fibroblast Cells.

Many cancer therapeutics are tested in vitro using only tumour cells. However, the tumour promoting effect of cancer associated fibroblasts (CAFs) within the tumour microenvironment (TME) is thought to reduce cancer therapeutics' efficacy. We have chosen pancreatic ductal adenocarcinoma (PDAC) as our tumor model. Our goal is to create a co-culture of CAFs and tumour cells to model the interaction between cancer and stromal cells in the TME and allow for better testing of therapeutic combinations. To test the proposed co-culture model, a gold nanoparticle (GNP) mediated-radiation response was used. Cells were grown in co-culture with different ratios of CAFs to cancer cells. MIA PaCa-2 was used as our PDAC cancer cell line. Co-cultured cells were treated with 2 Gy of radiation following GNP incubation. DNA damage and cell proliferation were examined to assess the combined effect of radiation and GNPs. Cancer cells in co-culture exhibited up to a 23% decrease in DNA double strand breaks (DSB) and up to a 35% increase in proliferation compared to monocultures. GNP/Radiotherapy (RT) induced up to a 25% increase in DNA DSBs and up to a 15% decrease in proliferation compared to RT alone in both monocultured and co-cultured cells. The observed resistance in the co-culture system may be attributed to the role of CAFs in supporting cancer cells. Moreover, we were able to reduce the activity of CAFs using GNPs during radiation treatment. Indeed, CAFs internalize a significantly higher number of GNPs, which may have led to the reduction in their activity. One reason experimental therapeutics fail in clinical trials relates to limitations in the pre-clinical models that lack a true representation of the TME. We have demonstrated a co-culture platform to test GNP/RT in a clinically relevant environment.

Abstract 2119: KRAS and fungi cooperate to drive IL33 secretion and type 2 immunity in the pancreatic cancer tumor microenvironment

Abstract Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest cancers, the five-year survival rate of PDAC patient is around ten percent. It is fourth largest cause of cancer related death and is projected to become the second deadliest cancer by 2030. PDAC patients generally present with advanced stage, metastatic disease that is mostly resistant to chemotherapy, radiation, and immunotherapy. A significant finding is that mutant KrasG12D (Kras*) is the primary oncogenic driver of PDAC development, and therefore efforts have been devoted to target KRAS. Here, we show in pancreatic ductal adenocarcinoma (PDAC), oncogenic KrasG12D (Kras*) increases the expression of IL33, a damage-associated molecular pattern (DAMP) molecule, member of IL1 cytokine family protein and localized in the PDAC cell nucleus, which upon secretion, chemoattracts the type 2 immunocytes. Type 2 immune cells—TH2, eosinophils and innate lymphoid cells (ILC)2, stimulate tumor growth by secreting pro-tumorigenic cytokines such as IL4, IL5 and IL13. However, the mechanisms by which type 2 immune cells traffic to the tumor microenvironment (TME) are unknown. Cancer cell-specific deletion of IL33 leads to a significant reduction in TH2 and ILC2 recruitment and lead to tumor regression. Further, we discovered that the release of IL33 requires interactions between cancer cells and the intratumoral mycobiome. PDA tumors harbors a ~3000-fold higher fungal load compared to normal pancreas in both mouse and humans. However, the mechanism of fungal mediated cytokine release is still unknown and represents a target for limiting IL33 release, and lessening PDAC tumor burden. Genetic deletion of IL33 alone or fungal depletion leads to a decrease in TH2 and ILC2 infiltration along with robust tumor regression. Moreover, repopulation of the PDAC TME with fungal species such as Alternaria and Malassezia lead to a re-infiltration of type 2 immunocytes that reinvigorates tumor growth. Notably, transplantation of tumor-derived ILC2s into mice accelerates PDAC growth. Mechanistically fungus activates dectin-1 pathway in PDAC cancer cells, mediating the release of IL33. Consistent with the murine data, IL33 expression is upregulated in approximately 20% of human PDAC, which is mainly restricted to cancer cells. Collectively, our work shows that the intratumoral mycobiome stimulates PDAC to secrete IL33, which plays a leading role in facilitating the recruitment of type 2 immunocytes that promote tumor progression. Citation Format: Aftab Alam, Eric Levanduski, Maulasri Bhatta, Sharon Senchanthisai, Sheila Jani Sait, Jianmin Wang, Scott I. Abrams I. Abrams, Prasenjit Dey. KRAS and fungi cooperate to drive IL33 secretion and type 2 immunity in the pancreatic cancer tumor microenvironment [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 2119.

Abstract 6285: MicroRNA-217 affects tumor microenvironment and enhances the chemosensitivity of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies and an inherently chemoresistant tumor. Dense fibrotic stroma is a major obstacle for drug delivery to the tumor cells in PDAC. Pancreatic stellate cells (PSCs) are the major stromal cells responsible for fibrotic microenvironment. MicroRNA-217 (miR-217) is known mainly as a tumor suppressor. However, the detailed roles of miR-217 on pathophysiology of PDAC remain unclarified. Methods: Expressions levels of miR-217 were measured in surgically resected human PDAC samples. Using Lipofectamine RNAiMAX, miR-217 or negative control miR (NC) were transfected in human pancreatic cancer cells, Panc-1, MIApaca-2, BxPC-3 and murine PAN02 cells, and chemosensitivities to gemcitabine and paclitaxel were investigated by MTS assay. Human hPSC-1 (obtained from Riken cell bank, Japan) was cultured in 20%FBS+DMEM and the changes of phenotypes and expression levels of miR-217 after the stimulation with TGF-β1 was measured by fluorescein microscope and digital PCR, respectively. Results: The expression level of miR-217 was markedly downregulated in PDAC tissues than in noncancerous pancreatic tissues (P<0.001, n=9). Overexpression of miR-217 significantly inhibited the proliferation of Panc-1, MIApaca-2 and BxPC-3 (P<0.001) and migration of Panc-1, MIApaca-2 and PAN02 (P<0.05). Lipofection of miR-217 further reduced the number of PAN02 exposed to 1-5nM gemcitabine (P<0.0001) and 0.01-0.1uM paclitaxel (P=0.0085) which resulted in the reduced the IC-50 as compared with NC. Human PSC-1 stimulated with TGF-β1 (10ng/ml) significantly reduced miR-217 expression compared to nascent cells. The stimulation with TGF-β1 upregulated the expression of cancer associated fibroblast (CAF) markers, α-smooth muscle actin (SMA) and vimentin. However, the expression levels of these markers were significantly reduced by the kipofection of miR-217. Conclusion: MiR-217 affects both cancer and stromal cells in PDAC which may regulate chemosensitivity in PDAC. Restoring miR-217 levels in tumor cells and/or PSCs could be a future therapeutic strategy. Citation Format: Yuki Kaneko, HIdeyuki Ohzawa, Yuki Kimura, Kohei Tamura, Yurie Futoh, Kazuya Takahashi, Akira Saito, Mineyuki Tojo, Hideyo Miyato, Naohiro Sata, Joji Kitayama. MicroRNA-217 affects tumor microenvironment and enhances the chemosensitivity 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 6285.

Abstract SY24-03: Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy

Abstract Background: Cancer immunotherapies can lead to durable and sometimes curative responses in patients with otherwise treatment-refractory disease, and as such have revolutionized our approach to cancer therapy. Still, there are many roadblocks that must be overcome to make immunotherapies effective across a broader spectrum of patients and cancer types. Immune checkpoint blockade (ICB) therapies that are effective in some malignancies through reactivation of cytotoxic T cell immunity have failed in immunologically “cold” tumors such as pancreatic ductal adenocarcinoma (PDAC) due to poor T cell infiltration and weak antigen presentation necessary for effective anti-tumor T cell responses. We and others have demonstrated that induction of cellular senescence and the senescence associated secretory phenotype (SASP), which leads to production of immunomodulatory cytokines, chemokines, and growth factors, can be a powerful way to not only enhance T cell infiltration into tumors but also reactivate a different type of innate, antigen-independent Natural Killer (NK) cell immunity that can mediate tumor control. Recently, we found that a combination of two RAS pathway targeting therapies- the MEK inhibitor trametinib and CDK4/6 inhibitor palbociclib (T/P)- could induce senescence in genetically engineered mouse models of KRAS mutant lung adenocarcinoma and PDAC (Ruscetti*, Leibold*, Bott* et al. Science 2018; Ruscetti*, Morris*, Mezzadra* et al. Cell 2020). Strikingly, whereas therapy-induced senescence led to NK cell-mediated tumor regressions and long-term survival benefit in KRAS mutant lung cancer, it had little effect on NK cell immunity and tumor progression in PDAC. Here we set out to understand how the pancreas tumor microenvironment (TME) suppresses NK immunity and develop strategies to harness NK cell surveillance for immunotherapy in PDAC. Results and Discussion: We took advantage of genetically similar Kras mutant, p53 altered PDAC and lung cancer cell lines that could be transplanted into different organs of C57BL/6 mice to study whether and how the pancreas TME may impact senescence-driven immune responses. Syngeneic KRAS mutant lung and PDAC tumor cells transplanted into the pancreas of C57BL/6 mice displayed transcriptional repression and reduced chromatin accessibility of SASP transcriptional activators (NF-KB, IRFs) and factors necessary for NK cell activity (IL-15, IL-18) and chemotaxis (CCL2, CCL7, CCL8, CXCL9, CXCL10) following T/P-induced senescence, and did not undergo anti-tumor NK immune surveillance as compared to tumors propagated in the lungs and liver. Transcriptomic analysis identified induction of EZH2 and epigenetic repression of its targets, including key SASP factors and regulators, specifically in tumor cells grown in the pancreas TME. Genetic or pharmacological inhibition of EZH2 enhanced proinflammatory SASP signaling and resulted in NK cell migration and anti-tumor cytotoxicity in murine and human PDAC lines and transplanted and genetically engineered PDAC mouse models. Remarkably, tumor-intrinsic EZH2 knockdown in combination with T/P treatment led to complete tumor regressions in some PDAC-bearing mice that were reversed following NK cell depletion or suppression of key SASP factors. Pharmacological inhibition of EZH2 methyltransferase activity also enhanced NK cell infiltration and tumor control following therapy-induced senescence in transplanted and genetically engineered PDAC mouse models. Importantly, we found that an EZH2 repression signature was associated with enhanced NK cell infiltration and SASP induction in human PDAC patient samples. These results demonstrate that EZH2 mediates transcriptional repression of the proinflammatory SASP in the pancreas TME, and that EZH2 blockade in combination with senescence-inducing therapies could be a powerful means to reactivate absent NK cell surveillance in PDAC to achieve immune-mediated tumor responses. In the future we plan to test these therapeutic interventions in combination with anti-PD-1/PD-L1 ICB therapy as a means to further activate both NK and T cell immunity to fully eradicate pancreatic cancer. This work highlights the importance of understanding the impact of the resident tumor microenvironment on anti-tumor immune responses to come up with new paradigms to potentiate immunotherapy in this and other immunologically “cold” tumor settings that are historically unresponsive to ICB therapy. Citation Format: Marcus Ruscetti. Modulating cellular senescence to reinstate natural killer cell immunity for pancreatic cancer immunotherapy [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 SY24-03.

Partial ligand shielding nanoparticles improve pancreatic ductal adenocarcinoma treatment via a multifunctional paradigm for tumor stroma reprogramming

The dense stroma that acts as a physical and biological barrier in the tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) leads to the failure of chemotherapeutic drug delivery. Cancer-associated fibroblasts (CAFs) and extracellular matrix (ECM) mainly constitute the refuge for cancer cells in PDAC. Herein, a CAF targeting drug delivery system (TDDS) based on RBC vesicles partial protection (RBC-Fn-NP) was established and investigated for reprogramming stroma, as well as enhancing tumor penetration and antitumor efficacy in PDAC. RBC vesicles were firstly used for partial protection of peptide from external influences. The exposed FnBPA5 peptide showed high affinity with both CAFs and the major components as collagen I and relaxed-fibronectin of ECM. Retinoic acid (RA) could disturb Golgi of CAFs, resulting in the reduction of protein secretion from the headstream. As expected, the strategy of RBC vesicles protected FnBPA5 targeting and RA-induced protein reduction was confirmed to reprogram the dense stroma and improve the penetration of Doxorubicin (Dox) in PDAC. RBC-Fn-NP inhibited tumor growth in both Pan02-orthotopic bearing model and Pan02-subcutaneous mice model. Hence, these partial ligand shielding nanoparticles offer a multifunctional and efficient approach to overcome penetration barriers and enhance the antitumor efficacy of chemotherapy in PDAC. Statement of significanceA partial ligand shielding nanoparticle platform (RBC-Fn-NP), which has the function of an RBC vesicle “shell” and thetargeting properties of a “core” to achieve superior therapeutic effects against PDAC, was established. The targeted ligand was modified on the surface of the nanoparticles instead of the RBC membranes. Three-dimensional PDAC stroma-rich spheroids were established to evaluate the penetration and tumor stroma remodeling. The targeting properties of FnBPA5 peptide, the effect of RA-induced Golgi disruption on the reduction of protein secretion, and the incomplete “camouflage” of the RBC vesicles were confirmed both in vitro and in vivo. As expected, our nanoplatform may provide a promising strategy for remolding dense stroma and enhancing the permeability in PDAC.