Pancreatic Ductal Adenocarcinoma Cell Lines Research Articles

Dysregulation of a Subset of Circulating and Vesicle-Associated miRNA in Pancreatic Cancer

Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive neoplasia, characterized by early metastasis, low diagnostic rates at early stages, resistance to drugs, and poor prognosis. There is an urgent need to better characterize this disease in order to identify efficient diagnostic/prognostic biomarkers. Since microRNAs (miRNAs) contribute to oncogenesis and metastasis formation in PDAC, they are considered potential candidates for fulfilling this task. In this work, the levels of two miRNA subsets (involved in chemoresistance or with oncogenic/tumor suppressing functions) were investigated in a panel of PDAC cell lines and liquid biopsies of a small cohort of patients. We used RT-qPCR and droplet digital PCR (ddPCR) to measure the amounts of cellular- and vesicle-associated, and circulating miRNAs. We found that both PDAC cell lines, also after gemcitabine treatment, and patients showed low amounts of cellular-and vesicle-associated miR-155-5p, compared to controls. Interestingly, we did not find any differences when we analyzed circulating miR-155-5p. Furthermore, vesicle-related miR-27a-3p increased in cancer patients compared to the controls, while circulating let-7a-5p, miR-221-3p, miR-23b-3p and miR-193a-3p presented as dysregulated in patients compared to healthy individuals. Our results highlight the potential clinical significance of these analyzed miRNAs as non-invasive diagnostic molecular tools to characterize PDAC.

Highly sensitive ratiometric fluorescent fiber matrices for oxygen sensing with micrometer spatial resolution

AbstractOxygen (O2)-sensing matrices are promising tools for the live monitoring of extracellular O2 consumption levels in long-term cell cultures. In this study, ratiometric O2-sensing membranes were prepared by electrospinning, an easy, low-cost, scalable, and robust method for fabricating nanofibers. Poly(ε-caprolactone) and poly(dimethyl)siloxane polymers were blended with tris(4,7-diphenyl-1,10-phenanthroline) ruthenium(II) dichloride, which was used as the O2-sensing probe, and rhodamine B isothiocyanate, which was used as the reference dye. The functionalized scaffolds were morphologically characterized by scanning electron microscopy, and their physicochemical profiles were obtained by Fourier transform infrared spectroscopy, thermogravimetric analysis, and water contact angle measurement. The sensing capabilities were investigated by confocal laser scanning microscopy, performing photobleaching, reversibility, and calibration curve studies toward different dissolved O2 (DO) concentrations. Electrospun sensing nanofibers showed a high response to changes in DO concentrations in the physiological-pathological range from 0.5 to 20% and good stability under ratiometric imaging. In addition, the sensing systems were highly biocompatible for cell growth promoting adhesiveness and growth of three cancer cell lines, namely metastatic melanoma cell line SK-MEL2, breast cancer cell line MCF-7, and pancreatic ductal adenocarcinoma cell line Panc-1, thus recreating a suitable biological environment in vitro. These O2-sensing biomaterials can potentially measure alterations in cell metabolism caused by changes in ambient O2 content during drug testing/validation and tissue regeneration processes. Graphic abstract

Axon guidance cue SEMA3A promotes the aggressive phenotype of basal-like PDAC

ObjectiveThe dysregulation of the axon guidance pathway is common in pancreatic ductal adenocarcinoma (PDAC), yet our understanding of its biological relevance is limited. Here, we investigated the functional role of...

Anti-pancreatic cancer activity of cassane diterpenoids isolated from the seeds of Caesalpinia sappan mediated by autophagy activation via ROS/AMPK/mTORC1 pathway

Three undescribed cassane diterpenoids, caesalpanins D–F (1–3), and seven known ones were isolated from the seeds of Caesalpinia sappan. Structures and absolute configurations of 1–3 were elucidated based on the extensive spectroscopic analysis, single-crystal X-ray diffraction analysis, and ECD calculations. Structurally, compound 1 was the first example of 18-norcassane diterpenoid and 2 was a rare 20-norcassane diterpenoid having an unusual five-membered oxygen bridge between C-10/C-18. The anti-proliferative activity of 1, 3, and 4–10 against PANC-1 cells (pancreatic ductal adenocarcinoma cell line) was evaluated, and phanginin H (4) was found to exhibit anti-cancer activity with IC50 value of 18.13 ± 0.63 μM. Compound 4 inhibited PANC-1 cell growth by arresting the cell cycle at G2/M phase via regulation of cyclin-dependent kinases, and the self-renewal and metastasis of PANC-1 cells by suppressing cancer cell stemness. Furthermore, compound 4 induced ROS generation and subsequently activated autophagy, which was demonstrated by the formation of autophagic vacuoles and dynamic change of autophagic flux. The induced ROS accumulation resulted in AMPK activation and subsequently regulation of mTORC1 activity and ULK phosphorylation, indicating that 4 triggered autophagy through ROS/AMPK/mTORC1 pathway. These findings suggested that 4 might potentially be an autophagy inducer for the therapy of pancreatic cancer.

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

Background: 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. Methods: 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. Results: We found that C18 has higher cytotoxic and anti-invadopodia activity than GEM in all culture conditions and especially in acid and hypoxic environments. Conclusions: We propose C18 as a more effective approach to conventional GEM in developing new therapeutic strategies overcoming PDAC chemoresistance.

Heterodimerization of T cell engaging bispecific antibodies to enhance specificity against pancreatic ductal adenocarcinoma

BackgroundEGFR and/or HER2 expression in pancreatic cancers is correlated with poor prognoses. We generated homodimeric (EGFRxEGFR or HER2xHER2) and heterodimeric (EGFRxHER2) T cell-engaging bispecific antibodies (T-BsAbs) to direct polyclonal T cells to these antigens on pancreatic tumors.MethodsEGFR and HER2 T-BsAbs were constructed using the 2 + 2 IgG-[L]-scFv T-BsAbs format bearing two anti-CD3 scFvs attached to the light chains of an IgG to engage T cells while retaining bivalent binding to tumor antigens with both Fab arms. A Fab arm exchange strategy was used to generate EGFRxHER2 heterodimeric T-BsAb carrying one Fab specific for EGFR and one for HER2. EGFR and HER2 T-BsAbs were also heterodimerized with a CD33 control T-BsAb to generate ‘tumor-monovalent’ EGFRxCD33 and HER2xCD33 T-BsAbs. T-BsAb avidity for tumor cells was studied by flow cytometry, cytotoxicity by T-cell mediated 51Chromium release, and in vivo efficacy against cell line-derived xenografts (CDX) or patient-derived xenografts (PDX). Tumor infiltration by T cells transduced with luciferase reporter was quantified by bioluminescence.ResultsThe EGFRxEGFR, HER2xHER2, and EGFRxHER2 T-BsAbs demonstrated high avidity and T cell-mediated cytotoxicity against human pancreatic ductal adenocarcinoma (PDAC) cell lines in vitro with EC50s in the picomolar range (0.17pM to 18pM). They were highly efficient in driving human polyclonal T cells into subcutaneous PDAC xenografts and mediated potent T cell-mediated anti-tumor effects. Both EGFRxCD33 and HER2xCD33 tumor-monovalent T-BsAbs displayed substantially reduced avidity by SPR when compared to homodimeric EGFRxEGFR or HER2xHER2 T-BsAbs (∼150-fold and ∼6000-fold respectively), tumor binding by FACS (8.0-fold and 63.6-fold), and T-cell mediated cytotoxicity (7.7-fold and 47.2-fold), while showing no efficacy against CDX or PDX. However, if either EGFR or HER2 was removed from SW1990 by CRISPR-mediated knockout, the in vivo efficacy of heterodimeric EGFRxHER2 T-BsAb was lost.ConclusionEGFR and HER2 were useful targets for driving T cell infiltration and tumor ablation. Two arm Fab binding to either one or both targets was critical for robust anti-tumor effect in vivo. By engaging both targets, EGFRxHER2 heterodimeric T-BsAb exhibited potent anti-tumor effects if CDX or PDX were EGFR+HER2+ double-positive with the potential to spare single-positive normal tissue.

Disruption of the pro-oncogenic c-RAF–PDE8A complex represents a differentiated approach to treating KRAS–c-RAF dependent PDAC

Pancreatic ductal adenocarcinoma (PDAC) is considered the third leading cause of cancer mortality in the western world, offering advanced stage patients with few viable treatment options. Consequently, there remains an urgent unmet need to develop novel therapeutic strategies that can effectively inhibit pro-oncogenic molecular targets underpinning PDACs pathogenesis and progression. One such target is c-RAF, a downstream effector of RAS that is considered essential for the oncogenic growth and survival of mutant RAS-driven cancers (including KRASMT PDAC). Herein, we demonstrate how a novel cell-penetrating peptide disruptor (DRx-170) of the c-RAF–PDE8A protein–protein interaction (PPI) represents a differentiated approach to exploiting the c-RAF–cAMP/PKA signaling axes and treating KRAS–c-RAF dependent PDAC. Through disrupting the c-RAF–PDE8A protein complex, DRx-170 promotes the inactivation of c-RAF through an allosteric mechanism, dependent upon inactivating PKA phosphorylation. DRx-170 inhibits cell proliferation, adhesion and migration of a KRASMT PDAC cell line (PANC1), independent of ERK1/2 activity. Moreover, combining DRx-170 with afatinib significantly enhances PANC1 growth inhibition in both 2D and 3D cellular models. DRx-170 sensitivity appears to correlate with c-RAF dependency. This proof-of-concept study supports the development of DRx-170 as a novel and differentiated strategy for targeting c-RAF activity in KRAS–c-RAF dependent PDAC.

CDK4/6 inhibition sensitizes MEK inhibition by inhibiting cell cycle and proliferation in pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is not sensitive to most chemotherapy drugs, leading to poor chemotherapy efficacy. Recently, Trametinib and Palbociclib have promising prospects in the treatment of pancreatic cancer. This article aims to explore the effects of Trametinib on pancreatic cancer and address the underlying mechanism of resistance as well as its reversal strategies. The GDSC (Genomics of Drug Sensitivity in Cancer) and CTD2 (Cancer Target Discovery and Development) were utilized to screen the potential drug candidate in PDAC cell lines. The dose-increase method combined with the high-dose shock method was applied to induce the Trametinib-resistant PANC-1 and MIA PaCa-2 cell lines. The CCK8 proliferation assay, colony formation assay, flow cytometry, and western blot were conducted to verify the inhibitory effect of Trametinib and Palbociclib. RNA-seq was performed in resistant PDAC cell lines to find the differential expression genes related to drug resistance and predict pathways leading to the reversal of Trametinib resistance. The GDSC and CTD2 database screening revealed that Trametinib demonstrates a significant inhibitory effect on PDAC. We found that Trametinib has a lower IC50 than Gemcitabine in PDAC cell lines. Both Trametinib and Gemcitabine can decrease the proliferation capacity of pancreatic cells, induce cell cycle arrest, and increase apoptosis. Simultaneously, the phosphorylation of the AKT and ERK pathways were inhibited by the treatment of Trametinib. In addition, the RNA-seq of Trametinib-induced resistance PDAC cell lines reveals that the cyclin-dependent kinase (CDK)-RB-E2F regulatory axis and G2/M DNA damage checkpoint might lead the drug resistance. Besides, the combination of Trametinib with Palbociclib could inhibit the proliferation and cell cycle of both resistant cells lines and also restore the sensitivity of drug-resistant cells to Trametinib. Last but not least, the interferon-α and interferon-γ expression were upregulated in resistance cell lines, which might lead to the reversal of drug resistance. The study shows Trametinib has a critical inhibitory effect on PDAC. Besides, the combination of Trametinib with Palbociclib can inhibit the proliferation of PDAC-resistant cells.

Abstract CT105: Early clinical experience with a novel first-in-class G-quadruplex experimental anti-cancer drug

Abstract The human genome contains G-rich regions that are over-represented in many cancer gene promoters. These can form higher-order G-quadruplex arrangements under the influence of an appropriate and selective small molecule compound. The resulting ligand-G-quadruplex complex may be resistant to polymerase read-through and thus results in transcriptional down-regulation of target genes. QN-302, based on a tetra-substituted naphthalene diimide chemotype, is a potent and selective G-quadruplex-binding compound that has been designed and initially developed at University College London. It displays single-digit cell growth inhibition in a panel of pancreatic ductal adenocarcinoma (PDAC) cell lines. RNA-seq analysis shows that it is a pan-quadruplex agent, down-regulating the expression of multiple G-quadruplex-promoter cancer genes and pathways in PDAC. It also shows in vivo anti-tumor activity in several animal models for PDAC, with low toxicity observed at therapeutic doses. QN-302 was licensed to Qualigen Therapeutics Inc in January 2022. It was granted Orphan Drug designation by the FDA in December 2022 for PDAC. This was followed by extensive regulatory toxicological and pharmacological studies together with successful scale-up synthesis, purification and formulation. QN-302 was granted IND clearance in August 2023 by the FDA for phase 1 clinical trials in advanced or metastatic solid tumors. This Phase 1a clinical trial is being conducted in two USA cancer centers (START Midwest, Grand Rapids, MI and Honor Health, Scottsdale, AZ) using a protocol based on 28-day once-weekly dose escalation and evaluation of three potential biomarkers, two of which have been suggested by transcriptomic analysis as being indicative of G-quadruplex-induced drug response and accompanying decrease of anti-tumor activity. So far 1 Cohort (3 patients) has been dosed at the lowest dose of 1.62 mg of QN-302 as part of this Phase 1a clinical trial. Patient #1, a male patient of metastatic pancreatic cancer, is currently in Cycle 4 of therapy showing Stable Disease and no AEs or SAEs reported. Patient #2, a female patient of metastatic colorectal cancer, completed Cycle 2 after which she showed progression upon CT Scan before entering Cycle 3 and was taken off study. Patient #3, a male patient of metastatic PDAC, is currently ending Cycle 2, about to enter Cycle 3, showing Stable Disease upon CT scan. All patients dosed showed no AEs, SAEs or side effects. They were also clinically observed to have improved QOL compared to prior SOC while on therapy with QN-302, This presentation will outline this unique approach and summarize safety, tolerability, PK/PD data and any early clinical data as described above including preliminary biomarker data now that clinical evaluation has commenced. The advantages and challenges of QN-302 will also be discussed. Citation Format: Tariq Arshad, Stephen Neidle, Sreenivasa Chandana, Erkut Borazanci. Early clinical experience with a novel first-in-class G-quadruplex experimental anti-cancer drug [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 CT105.

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

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

Sphingosine-1-Phosphate Inhibition Increases Endoplasmic Reticulum Stress to Enhance Oxaliplatin Sensitivity in Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer resistant to current therapies, including oxaliplatin (Oxa). Growing evidence supports the ability of cancers to harness sphingolipid metabolism for survival. Sphingosine-1-phosphate (S1P) is an anti-apoptotic, pro-survival mediator that can influence cellular functions such as endoplasmic reticulum (ER) stress. We hypothesize that PDAC drives dysregulated sphingolipid metabolism and that S1P inhibition can enhance ER stress to improve therapeutic response to Oxa in PDAC. RNA sequencing data of sphingolipid mediators from The Cancer Genome Atlas (TCGA) and Genotype-Tissue Expression Project (GTEx) datasets were analyzed. Murine and human PDAC cell lines were treated with small interfering RNA (siRNA) against sphingosine kinase-2 (SPHK2) or ABC294640 (ABC) and incubated with combinations of vehicle control or Oxa. In an orthotopic syngeneic KPC PDAC model, tumors were treated with either vehicle control, Oxa, ABC, or combination therapy. RNA sequencing analysis revealed multiple significantly differentially expressed sphingolipid mediators (P < 0.05). In vitro, both siRNA knockdown of SPHK2 and ABC sensitized cells to Oxa therapy (P < 0.05), and induced eukaryotic initiation factor 2α (eIF2α) and protein kinase RNA-like endoplasmic reticulum kinase (PERK) phosphorylation, hallmarks of ER stress. In vitro therapy also increased extracellular high mobility group box 1 (HMGB1) release (P < 0.05), necessary for immunogenic cell death (ICD). In vivo combination therapy increased apoptotic markers as well as the intensity of HMGB1 staining compared to control (P < 0.05). Our evidence suggests that sphingolipid metabolism is dysregulated in PDAC. Furthermore, S1P inhibition can sensitize PDAC to Oxa therapy through increasing ER stress and can potentiate ICD induction. This highlights a potential therapeutic target for chemosensitizing PDAC as well as an adjunct for future chemoimmunotherapy strategies.

The Co-Localization of NLRP3 and ASC Specks Does Not Automatically Entail NLRP3 Inflammasome Functionality in PDAC Cell Lines

The NLRP3 inflammasome is an important mediator of the host inflammatory response, and downregulation of inflammation is important in cancer treatment. Here, we investigated four different pancreatic ductal adenocarcinoma (PDAC) cell lines, AsPC-1, BxPC-3, CFPAC-1 and Panc-1, with regards to NLRP3 inflammasome formation and cytokine secretion. ASC specks were observed in all the cell lines investigated, but AsPC-1 was the only cell-line with the co-localization of anti-ASC and anti-NLRP3 and spontaneously formed multiple NLRP3 inflammasomes per cell. The co-localization of NLRP3 and ASC was not accompanied by IL-1β release nor significant IL-18 release. BxPC-3 displayed relatively high expression of the inflammasome-related genes IL1B and CASP1 and had the highest levels of IL1β and IL18 secretion and the highest amount of ASC. The inflammasome-associated genes IL18 and PYCARD were up-regulated in the PDAC primary tumors compared to normal tissue, and high PDAC tumor expression of IL18, CASP1 and PYCARD correlated with low patient survival. We have shown that PDAC cell lines display significant variations in their inflammasome-related gene expression and readouts. We conclude that spontaneous ASC speck formation is possible in PDAC cells and that multiple NLRP3 inflammasomes are formed spontaneously in AsPC-1 cells but that the co-localization of NLRP3 and ASC specks does not automatically entail inflammasome function.

Role of Wnt5b-Ror1 signaling in the proliferation of pancreatic ductal adenocarcinoma cells.

Pancreatic ductal adenocarcinoma (PDAC) is one of the most refractory cancers with the worst prognosis. Although several molecules are known to be associated with the progression of PDAC, the molecular mechanisms underlying the progression of PDAC remain largely elusive. The Ror-family receptors, Ror1 and Ror2, which act as a receptor(s) for Wnt-family ligands, particularly Wnt5a, are involved in the progression of various types of cancers. Here, we show that higher expression of Ror1 and Wnt5b, but not Ror2, are associated with poorer prognosis of PDAC patients, and that Ror1 and Wnt5b are expressed highly in a type of PDAC cell lines, PANC-1 cells. Knockdown of either Ror1 or Wnt5b in PANC-1 cells inhibited their proliferation significantly in vitro, and knockout of Ror1 in PANC-1 cells resulted in a significant inhibition of tumor growth in vivo. Furthermore, we show that Wnt5b-Ror1 signaling in PANC-1 cells promotes their proliferation in a cell-autonomous manner by modulating our experimental setting in vitro. Collectively, these findings indicate that Wnt5b-Ror1 signaling might play an important role in the progression of some if not all of PDAC by promoting proliferation.

Abstract 5957: Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment of pancreatic cancer

Abstract Introduction Transcription regulators, such as cyclin-dependent kinase 9 (CDK9), have been implicated in the super-enhancer driven transcriptional control of oncogenes in multiple cancers including pancreatic cancer. CDK9 has been shown to be employed by cancer cells for the constant production of short-lived oncoproteins such as c-Myc to maintain their survival. Due to its critical role in regulating transcription in cancer cells, CDK9 has emerged as a potential therapeutic target. Here, we report the antitumor activity of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, in preclinical models for pancreatic cancer. Experimental Procedures Fragment-based MolecuLern driven AI workflow methods were used to design a series of ATP-competitive CDK9 specific kinase inhibitors. BLX-3030 was selected as one of the most potent leads with an IC50 of 1.1 nM in a cell-free CDK9 kinase assay. A Sulforhodamine B (SRB) assay was used to evaluate the cell growth inhibitory activity of BLX-3030 in a panel of 10 pancreatic ductal adenocarcinoma (PDAC) and 2 pancreatic neuroendocrine tumor (pNET) cell lines. Western blotting was used to determine the effects of BLX-3030 treatment on c-Myc expression in cancer cells. The combination effects between BLX-3030 and standard of care regimen, gemcitabine (GEM) and nab-paclitaxel (NP), was assessed using the SRB assay. Finally, the in vivo antitumor activity of BLX-3030 alone or in combination with GEM and NP was evaluated in a mouse cell line xenograft model for pancreatic cancer. Results BLX-3030 exhibited potent cell growth inhibitory activity in the panel of pancreatic cancer cell lines with IC50 values ranging from 133.5 nM to 508.1 nM. The potency of BLX-3030 in the cell lines appears to correlate with the expression level of c-Myc in the cell lines. BLX-3030 showed similar activities in PDAC and pNET cell lines with mean IC50 values of 244.5 nM and 194.6 nM, respectively. However, treatment with BLX-3030 for 48 hours did not seem to significantly reduce c-Myc expression in PSN-1 PDAC cells. When combined with GEM and NP, BLX-3030 demonstrated mostly additive effects in PSN-1 cells. Preliminary data from the in vivo study show that BLX-3030 improves the antitumor activity of GEM and NP. Conclusion In summary, BLX-3030 potently inhibited the growth of both PDAC and pNET cells in vitro. The growth inhibitory activity of BLX-3030 in PDAC cell lines correlates with their c-Myc expression levels. BLX-3030 showed an additive effect when combined with standard of care drugs (GEM and NP) in PDAC cell lines. Overall, the novel CDK9 inhibitor, BLX-3030, demonstrated potent activity in pancreatic cancer cell line models and presents a promising preclinical lead for pancreatic cancer. (This work was supported in part by Biolexis Therapeutics and the Seena Magowitz Foundation). Citation Format: Yesenia Barrera-Millan, Zhaoliang Li, Hariprasad Vankayalapati, David J. Bearss, Daniel D. Von Hoff, Haiyong Han. Development of a selective, oral ATP-competitive CDK9 inhibitor, BLX-3030, for treatment 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 5957.

Abstract 955: Double-targeting of pancreatic ductal adenocarcinoma (PDAC): Unveiling the potent combination of NMS-873 and BAY-876

Abstract Pancreatic ductal adenocarcinoma (PDAC) stands out as one of the most aggressive and challenging-to-treat cancers known. Consequently, the development of effective therapeutic strategies holds significant importance in enhancing patient outcomes. In contrast to single-drug treatments, combination therapies offer the potential for improved results by addressing multiple pathways. This approach not only reduces the likelihood of drug resistance but also encourages synergistic effects, all while permitting the use of lower individual drug doses, thus minimizing potential side effects. In this study, we subjected a panel of PDAC cell lines with varying degrees of sensitivity to a high-throughput combinatorial drug screen. Mitochondrial complex 1/p97 inhibitor NMS-873 was tested in combination with 95 other compounds, revealing remarkable synergy when paired with Glut1 inhibitor BAY-876. In order to affirm the validity of these findings, we conducted further experiments involving multi-dose drug combinations within long-term clonogenic assays. These supplementary investigations also demonstrated significant synergistic effects between BAY-876 and NMS-873, as quantified through the calculation of Bliss and Zip scores. Notably, this combination exhibited superior efficacy when confronting PDAC cell lines that are more resistant to NMS-873, highlighting the potential of this combination to overcome therapy resistance. To gain insights into the underlying mechanisms behind these outcomes, we performed a gene enrichment analysis which revealed that the more sensitive cell lines exhibited elevated rates of cell division, while the more resistant ones demonstrated heightened cRNA processing and increased metabolic activity, particularly in protein, phospholipid, and lipid synthesis. The combination of BAY-876 and NMS-873 presents a promising avenue for the targeted treatment of PDAC. This study underscores the remarkable synergy observed in mouse PDAC cell lines with a particular emphasis on the heightened efficacy against cell populations that are more resistant to NMS-873. These results warrant further investigation to elucidate the underlying molecular mechanisms and validate these findings in clinical settings. Citation Format: Emily Rita Eppinger, Christian Schneeweis, Dieter Saur. Double-targeting of pancreatic ductal adenocarcinoma (PDAC): Unveiling the potent combination of NMS-873 and BAY-876 [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 955.

Abstract 5943: Development of a SMART theranostic agent for precision pancreatic cancer therapeutics

Abstract Pancreatic cancer is predicted to emerge as the second leading cause of cancer-related deaths in the United States by 2030. Pancreatic ductal adenocarcinoma (PDAC) - the most common form of pancreatic cancer - is often diagnosed as metastatic disease and has demonstrated the least improvement in overall survival over the last three decades with incidence rising by 0.5% to 1.0% per year due in part to lack of effective and tumor-selective treatment strategies. One approach to reduce host toxicity is the development of novel molecules and innovative therapeutic approaches that are nontoxic to normal cells but can be activated only under tumor-specific conditions for lethality, which we coined as SMART (Selective Method of Activation for Response in Tumors) therapeutic agents. We utilized explicit properties and biomarkers present in PDAC tumors but not in normal tissues to develop a SMART DNA-damaging anti-cancer agent with both therapeutic and diagnostic (theranostic) activities. We evaluated the tumoricidal effect of our SMART agent using long-term survival assays in PDAC cell lines and 3D co-culture spheroids, as well as in tumor xenograft model studies in mice. We also tested the ability of our SMART agent to treat, detect/diagnose (via click chemistry), and identify critical resistance mechanisms (via proteomics) as a single agent or as a chemosensitizer with predictive tumor biomarkers for tumor-selective therapeutic response. We found that our SMART small molecule induces tumor-selective lethal effects as a monotherapy or in combination with DNA repair inhibitors or small molecules known to generate hydrogen peroxide (H2O2) in a tumor-selective manner for activation. Using “click” chemistry, we detected and quantified the incorporation of our SMART small molecule as a DNA-damaging agent via flow cytometry that correlated with therapeutic response, which is a feature that could facilitate the stratification, treatment monitoring, and management of patients with pancreatic cancers. We also found that our SMART agent is less toxic and more effective as a monotherapy than cisplatin in PDAC cell line-derived xenograft studies in mice. Overall, our novel SMART theranostic agent could provide a significant advancement as they are preferentially activated under tumor-specific conditions to induce tumor-selective therapeutic response. Furthermore, the diagnostic component our SMART agent could provide oncologists with essential information to make rapid clinical decisions on treatment strategies. Our SMART agent is also remarkably versatile as it can be utilized as a chemosensitizer or as a chemical probe to identify targetable vulnerabilities to potentiate its therapeutic effects at lower doses. Citation Format: Edward A. Motea, Jarrett J. Smith, Oluwasijibomi Ketiku. Development of a SMART theranostic agent for precision pancreatic cancer therapeutics [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 5943.

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 4588: Combination of MTA-cooperative PRMT5 inhibitor and direct mutant-selective KRAS inhibitors as a novel therapeutic approach for MTAP-deficient pancreatic cancer

Abstract KRAS is mutationally activated in 95% of pancreatic ductal adenocarcinoma (PDAC) patients. Direct KRAS inhibitors are under intense preclinical and clinical development, with two KRASG12C mutant-selective inhibitors (G12Ci) now approved. However, treatment-associated resistance to KRAS inhibitors has been reported in the clinic highlighting an urgent need to identify both additional therapeutic targets and novel combination treatment strategies with KRAS inhibitors. To that end, we performed a CRISPR/Cas9 loss-of-function screen using a library targeting ~2,500 druggable genes in KRASG12C/D-mutant PDAC cell lines. We identified multiple genes that were essential for PDAC cell growth and selected PRMT5, a protein arginine methyltransferase, to assess as a novel therapeutic target for pancreatic cancer. We found that suppression of PRMT5 activity using two distinct first-generation clinical candidate small molecule inhibitors (JNJ-64619178 and GSK3326595) demonstrated single agent activity and reduced PDAC cell growth. We further assessed a mechanistically distinct MTA-cooperative clinical candidate PRMT5 inhibitor, MRTX1719, that is selective for MTAP-deleted tumors, which occur in ~25% of PDAC patients. We determined that MRTX1719 exhibited low nanomolar GI50 activities in MTAP-deficient but not MTAP wild-type KRAS-mutant cell lines. Unexpectedly, PDAC cells treated with MRTX1719 exhibited a paradoxical activation of ERK, suggesting that combined inhibition of PRMT5 and KRAS might be therapeutically advantageous. In support of our hypothesis, we demonstrated that combination treatment with MRTX1719 and mutant selective KRAS inhibitors (G12Ci/MRTX849 and G12Di/MRTX1133) further sensitized KRASG12C/D-mutant PDAC cells to KRAS inhibition in short- and long-term growth assays as well as in vivo xenograft studies. Our ongoing studies are evaluating the consequences of co-targeting PRMT5 and KRAS on gene expression changes using RNA-Seq and cancer cell signaling pathways using RPPA analyses. In summary, our data support concurrent inhibition of PRMT5 and KRAS as a promising therapeutic strategy for MTAP-deficient KRAS-mutant pancreatic cancer. Citation Format: Kristina Drizyte-Miller, Wen-Hsuan Chang, Andrew M. Waters, Clint A. Stalnecker, Junning Wang, Andrew J. Aguirre, Adrienne D. Cox, Channing J. Der. Combination of MTA-cooperative PRMT5 inhibitor and direct mutant-selective KRAS inhibitors as a novel therapeutic approach for MTAP-deficient 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 4588.

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

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

Abstract 5384: The hypoxia-alleviating agent ITPP restores vascular function in zebrafish xenografts of pancreatic cancer

Abstract Pancreatic cancer mostly manifests as pancreatic ductal adenocarcinoma (PDAC) and is characterized by a highly hypoxic microenvironment that is associated with worse patient survival and resistance to therapy. Hypoxia induces angiogenesis and the resulting leaky and unstructured vessels fail to reoxygenate the tumor mass, thus exacerbating hypoxia and its downstream impact on tumorigenesis. Myo-inositol trispyrophosphate (ITPP) is a non-toxic allosteric effector of hemoglobin shown to reduce tumor growth and enhance survival in mice and rat models of different tumor types. Herein the aim is to assess for the first time the ability of ITPP to effectively treat PDAC tumors in vivo in zebrafish xenografts and to investigate its impact on vascular normalization. To that aim, four PDAC cell lines, AsPC1, Capan1, MIA PaCa2, and PANC1 were included, as well as the angiogenic breast cancer cell line, Hs578t. Cells were grown in appropriate conditions and on injection day, labeling was done with deep red. Labeled cells were then injected in the perivitelline space of anesthetized larvae. The following transgenic larvae were used: those having labeled blood vessels only (Tg(kdrl:DsRed)), labeled blood and lymphatic vessels (Tg(fli1:eGFP)), and double transgenics with labeled gata+ erythrocytes (Tg(fli1:eGFP;gata:dsRed)). One day post injection (dpi) xenografts were sorted based on size and treated by immersion for three consecutive days with control medium or ITPP at a concentration selected based on the maximum tolerated dose assay. Treatment was refreshed daily, and at 4dpi live confocal imaging was conducted on select Tg(fli1:eGFP;gata:dsRed) xenografts. All xenografts were then fixed and subjected to whole mount immunofluorescence. Nuclei were stained with DAPI and anti-Caspase3Asp175 was used as a marker for apoptosis. Imaging was performed using the Zeiss LSM confocal microscope and tumor size, apoptosis and mitotic figures were quantified with ImageJ. Vascular function was defined as the presence of gata+ cells within tumor-related vasculature, while vessel density and infiltration were determined in ImageJ by calculating the percentage of GFP area in the tumor. Thus far results from AsPC1-derived xenografts demonstrate that ITPP leads to a significant decrease in mitotic figures (p=0.0132) and an increase in apoptosis (p=0.0025), with no change in tumor size. Preliminary data reveal a trend of increased functional vasculature in both the AsPC1- and Hs578t- derived xenografts upon ITPP treatment. A trend of decreased vasculature infiltration could also be observed in the ITPP-treated Hs578t-derived xenografts. Based on these results, the zebrafish model could be an appropriate alternative for assessing the anti-tumorigenic activity of ITPP in PDAC in a short turnaround time and could potentially enable the visualization of ITPP’s impact on angiogenesis at single cell resolution. Citation Format: Raefa Abou Khouzam, Filipa Amorim, Ayesha Rifath, Husam Nawafleh, Vanda Povoa, Rania Faouzi Zaarour, Jean Marie Lehn, Rita Fior, Perparim Limani, Salem Chouaib. The hypoxia-alleviating agent ITPP restores vascular function in zebrafish xenografts 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 5384.