Pancreatic Ductal Adenocarcinoma Xenografts Research Articles

Oncogenic GALNT5 confers FOLFIRINOX resistance via activating the MYH9/ NOTCH/ DDR axis in pancreatic ductal adenocarcinoma.

Chemotherapy resistance has been a great challenge in pancreatic ductal adenocarcinoma(PDAC) treatments. Current first-line chemotherapy regimens for PDAC include gemcitabine-based regimens such as AG regimen (albumin paclitaxel and gemcitabine), fluorouracil-based regiments such as FOLFIRINOX regimen ((5-fluorouracil5-FU), oxaliplatin, Irinotecan) and platinum-based regimens for patients with BRCA mutations. large amounts of work have been done on exploring the mechanism underlying resistance of gemcitabine-based and platinum-based regimens, while little research has been achieved on the mechanism of FOLFIRINOX regimens resistance. Hence, we identified Polypeptide N-Acetylgalactosaminyltransferase 5, (GALNT5) as a vital regulator and a potential therapeutic target in FOLFIRINOX regimens resistance. Colony formation assays and flow cytometry assays were performed to explore the roles of GALNT5 in cell proliferation and apoptosis in PDAC treated with FOLFIRINOX. IC50 alterations were calculated in GALNT5 knockdown and overexpressed cell lines. RNA-seq followed by GSEA (gene set enrichment analysis) was displayed to explore the potential mechanism. WB (western blotting), real-time PCR, and IF (immunofluorescence) were performed to validate relative pathways. The mouse orthotopic xenograft PDAC model was established to examine GALNT5 functions in vivo. GALNT5 was highly expressed in PDAC tissues and predicted poor prognosis in PDAC. Upregulation of GALNT5 in PDAC cells conferred FOLFIRINOX resistance on PDAC by inhibiting DNA damage. Moreover, GALNT5 interacted with MYH9, thus participating in the activation of the NOTCH pathways, resulting in hampering FOI-induced DNA damage. Functions of GALNT5 promoting FOLFIRINOX resistance were validated in vivo. In this study, we found that aberrantly overexpressed GALNT5 in PDAC took part in the activation of the NOTCH pathway by interacting with MYH9, thus inhibiting the DDR to achieve FOLFIRINOX resistance and causing poor prognosis. We identified GALNT5 as a potential therapeutic target for PDAC patients resistant to FOLFIRINOX chemotherapy.

Unveiling the 4-aminoquinoline derivatives as potent agents against pancreatic ductal adenocarcinoma (PDAC) cell lines

Common antimalarials such as artemisinins, chloroquine and their derivatives also possess potent anti-inflamantory, antiviral and anticancer properties. In the search for new therapeutics to combat difficult-to-treat pancreatic carcinomas, we unveiled that 4-aminoquinoline derivatives, with significant antiplasmodial properties and a great safety profile in vivo, have remarkable anticancer activity against pancreatic ductal adenocarcinoma (PDAC) and considerable efficacy in the xenograft model in vivo. The aim of the present study was to further investigate anticancer properties of these compounds in a drug-repurposing manner. The compounds showed profound cytotoxic effects at nanomolar to low micromolar concentration in 2D cultured cells (in vitro) and in the zebrafish PDAC xenograft model (in vivo). A deeper insight into their mechanisms of cytotoxic action showed these compounds induce apoptosis while increasing reactive oxygen species levels along with autophagy inhibition. Additional investigation of the autophagy modulation proved that tested quinoline derivatives cause P62 and LC3-II accumulation in PDAC cells alongside lysosomal alkalinization. Further, in vivo toxicity studies in the zebrafish model showed low toxicity without developmental side effects of the investigated 4-aminoquinolines, while the applied compounds effectively inhibited tumor growth and prevented the metastasis of xenografted pancreatic cells. Taken together, these results highlight the 4-aminoquinolines as privileged structures that ought to be investigated further for potential application in pancreatic carcinoma treatment.

Abstract B037: Banana Lectin expressing CAR T cells to target Pancreatic Cancer

Abstract To overcome both the heterogenous and dynamic expression of tumor antigen expression in pancreatic ductal adenocarcinoma (PDAC) and the physical barrier provided by the associated fibrotic stroma, we have developed a novel therapy that recognizes and binds to distinct patterns of aberrant glycosylation specifically expressed on tumor cells and their surrounding stroma. Aberrant glycosylation patterns are a hallmark of cancer and are widely present on tumors including PDAC. We therefore incorporated H84T BanLec as part of a chimeric antigen receptor and co-expressed the construct in T cells and showed that H84T BanLec CAR expressing T-cells can effectively target pancreatic tumors. We hypothesized that our novel lectin CAR would enhance the activity of T cells when co-expressed with conventional scFV – derived CARs targeting tumor associated antigens We compared the activity of dual-expressing H84T/HER.2 CAR-T or H84T/PSMA CAR-T cells against single- expressing CARs targeting PDAC. We determined anti-tumor cytotoxicity by measuring residual GFP+ tumor cells in a sequential killing assay using flow cytometry. We observed superior anti-tumor activity using dual expressing CAR T cells compared to T cells expressing either HER.2- or H84T- or PSMA- CAR alone. Similarly, in 3D multicellular spheroids containing tumor and cancer-supportive stromal cells, an IncuCyte live imaging assay shows dual CAR T cells have greater cytotoxicity against PDAC than single CAR expressing T cells (p= 0.02 H84T/HER.2 vs HER.2 CAR; p=0.00003 H84T/PSMA CAR vs PSMA CAR). These results also translate to improved anti- tumor function of dual CAR T cells in PDAC xenograft in vivo models where H84T/HER.2 CAR T cells significantly reduced the tumor volume burden (p=0.015; H84T/HER.2 average=57.1mm3; HER.2 average=116.1mm3; H84T average=130.3mm3). These findings suggest that H84T BanLec CAR T cells provide a tool to target the aberrant glycosylation patterns of malignant cells and their supporting stroma and may contribute to an effective treatment option for PDAC patients. We are now elucidating the underlying mechanism by which H84T BanLec CARs enhance tumor activity and promote T cell infiltration. Citation Format: Katie McKenna, Ada I Ozcan, David Markovitz, Malcolm K Brenner. Banana Lectin expressing CAR T cells to target Pancreatic Cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr B037.

Abstract C032: De novo pyrimidine biosynthesis inhibition synergizes with BCL-XL targeting in pancreatic cancer

Abstract Oncogenic KRAS, the genetic driver of 90% of PDAC, induces a metabolic rewiring characterized, in part, by dependency on de novo pyrimidine biosynthesis. Dihydroorotate dehydrogenase (DHODH) is the enzymatic ‘chokepoint’ of the de novo pyrimidine biosynthesis pathway and an in vivo metabolic liability in pancreatic ductal adenocarcinoma (PDAC). Targeting enzymes in the de novo pyrimidine synthesis pathway such as dihydroorotate dehydrogenase (DHODH) with clinically available inhibitor brequinar (BQ) starved the tumor of nucleotide synthesis and restrained growth of PDAC cells in vitro. However, therapeutic resistance limited BQ long-term effects in PDAC xenografts, which suggests compensatory pathways and that combinatorial strategies are required for enhanced efficacy. Here, we integrated a mass spectrometry (MS)-based quantitative temporal proteomics workflow, LC/MS- based metabolomics and in vitro and in vivo drug-anchored CRISPR/Cas9 genetic screens to identify compensatory pathways to DHODH inhibition (DHODHi) and targets for combination strategies. We demonstrate that DHODHi alters the apoptotic regulatory proteome thereby enhancing sensitivity to inhibitors of the anti-apoptotic BCL2L1 (BCL-XL) protein. Combinatorial regimens with DHODH and BCL-XL inhibition synergistically induce apoptosis in PDAC cell lines and patient-derived PDAC organoids. In vivo DHODH inhibition with Brequinar and BCL-XL degradation with DT2216, a proteolysis targeting chimera (PROTAC), significantly inhibits the growth of PDAC tumors. Our data defines mechanisms of adaptation to DHODH inhibition and identifies a combination therapy strategy in PDAC. Citation Format: huan zhang, Qijia yu, Naiara Santana-Codina, Clara Poupault, Claudia Campos, Xingping Qin, Aparna Padhye, Nicole Sindoni, Miljan Kuljanin, Junning Wang, Matthew J. Dorman, Andrew J. Aguirre, Stephanie K. Dougan, Kristopher A. Sarosiek, Joseph D. Mancias. De novo pyrimidine biosynthesis inhibition synergizes with BCL-XL targeting in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pancreatic Cancer Research; 2024 Sep 15-18; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl_2):Abstract nr C032.

Allysine-Targeted Molecular MRI Enables Early Prediction of Chemotherapy Response in Pancreatic Cancer.

Neoadjuvant therapy is routinely used in pancreatic ductal adenocarcinoma (PDAC), but not all tumors respond to this treatment. Current clinical imaging techniques are not able to precisely evaluate and predict the response to neoadjuvant therapies over several weeks. A strong fibrotic reaction is a hallmark of a positive response, and during fibrogenesis, allysine residues are formed on collagen proteins by the action of lysyl oxidases. Here, we report the application of an allysine-targeted molecular MRI probe, MnL3, to provide an early, noninvasive assessment of treatment response in PDAC. Allysine increased 2- to 3-fold after one dose of neoadjuvant therapy with FOLFIRINOX in sensitive human PDAC xenografts in mice. Molecular MRI with MnL3 could specifically detect and quantify fibrogenesis in PDAC xenografts. Comparing the MnL3 signal before and 3 days after one dose of FOLFIRINOX predicted subsequent treatment response. The MnL3 tumor signal increased by 70% from day 0 to day 3 in mice that responded to subsequent doses of FOLFIRINOX, whereas no signal increase was observed in FOLFIRINOX-resistant tumors. This study indicates the promise of allysine-targeted molecular MRI as a noninvasive tool to predict chemotherapy outcomes. Significance: Allysine-targeted molecular MRI can quantify fibrogenesis in pancreatic tumors and predict response to chemotherapy, which could guide rapid clinical management decisions by differentiating responders from nonresponders after treatment initiation.

The role of DPYD and the effects of DPYD suppressor luteolin combined with 5-FU in pancreatic cancer.

Despite advances in the treatment of cancer, pancreatic ductal adenocarcinoma (PDAC) remains highly lethal due to the lack of effective therapies. Our previous study showed that Luteolin (Lut), a flavonoid, suppressed pancreatocarcinogenesis and reduced the expression of dihydropyrimidine dehydrogenase (DPYD), an enzyme that degrades pyrimidines such as 5-fluorouracil (5-FU), in PDACs. In this study, we investigated the role of DPYD and evaluated the therapeutic potential of combining 5-FU with Lut in PDACs. PDAC cells overexpressing DPYD showed increased proliferation, and invasiveness, adding to the resistance to 5-FU. The xenograft tumors of DPYD-overexpressing PDAC cells also exhibit enhanced growth and invasion compared to the control xenograft tumors. RNA-seq analysis of the DPYD-overexpressing PDAC xenograft tumors revealed an upregulation of genes associated with metallopeptidase activity-MMP9 and MEP1A. Furthermore, the overexpression of MEP1A in PDAC was associated with invasion. Next, we investigated the combined effects of Lut, a DPYD suppressor, and 5-FU on DPYD-overexpressing xenograft tumors and PDAC of Pdx1-Cre; LSL-KrasG12D/+; Trp53flox/flox(KPPC) mice. Neither single administration of 5-FU nor Lut showed significant inhibitory effects; however, the combined administration of 5-FU and Lut exhibited a significant tumor-suppressive effect in both the xenograft tumors and KPPC models. We have elucidated that DPYD expression contributes to proliferation, invasiveness, and 5-FU resistance, in PDACs. The combination therapy of Lut and 5-FU holds the potential for enhanced efficacy against PDACs.

Development of novel MUC1-C targeting antibody-drug conjugate for pancreatic cancer treatment.

e15025 Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the most aggressive forms of cancers, which is usually diagnosed in advanced stages and presents an abysmal 5-year survival rate of 3-10%. With late detection of metastasized/advanced disease, curative surgery is not an option for most patients. Accordingly, there is an urgent unmet need for development of therapeutics targeting the pancreatic cancer cells. MUC1 is one such cell surface antigen that is aberrantly overexpressed in most solid tumors including pancreatic cancer. In this study we report preclinical efficacy of pharmacological inhibition of MUC1 signaling by GO-203 and targeted delivery of toxin conjugated to novel MUC1-C antibodies (7B8 and 3D1) binding to the extra cellular domain (ECD) to the PDAC xenografts. Methods: Subjects from TCGA-PAAD were stratified into high (75th percentile) and low (25th percentile) MUC1-expressing cohorts. Differential expression was performed using DESeq2, followed by GSEA, and signaling network analysis using partial correlation networks, ARACNE, and BC3NET. Anti-MUC1 antibody-drug conjugates (ADCs) were generated by coupling monomethyl auristatin E (3D1-MMAE, 7B8-MMAE) via V-C-PAB linker and characterized for anti-tumor activity against pancreatic cancer cells including MIA PaCa-1, CAPAN2 and HAPF-II. Paired-end RNA-seq (50bp) was aligned and quantified to the Human Ensembl GRC38 reference using STAR aligner. Subcutaneous xenografts were obtained in nude mice by injecting 5X106 HPAF-II tumor cells. Results: Differential expression, pathway, and network analyses revealed consistent dysregulation of cancer survival-related processes, and P53-related pathways in the cohort with increased MUC1 expression. The ADCs (DAR of ~3.5) exhibited efficient internalization in cancer cells and associated apoptotic cell death (Kds = 2 - 5nM). Pharmacological inhibition of MUC1 signaling by GO-203 demonstrated reduction in canonical pathways including P53 and EMT. Treatment with 3D1-MMAE compared to both vehicle and GO-203 showed dysregulation of cellular metabolism pathways (cholesterol homeostasis, glycolysis) and exhibited several strong indicators of cellular stress including activation of unfolded protein response processes and mTorc1 signaling. Excellent tumor-suppressive effects of ADCs were observed in HPAF-II xenografts in nude mice. The 7.5 mg/kg (i.v., q7dx3), treatment showed complete tumor regression from 14th day (end of treatment) till the date of termination (Day 50). Doses up to 40 mg/kg were well tolerated by the mice and this anti-tumor efficacy was observed without any appreciable loss in body weight or adverse effects on tissues. Conclusions: Targeted delivery of MMAE toxin to the pancreatic cancer cells presents an exciting opportunity for a novel therapeutic intervention for the treatment of pancreatic cancer patients with MUC1 over expression in tumors.

Nanotechnology-Based Strategy for Enhancing Therapeutic Efficacy in Pancreatic Cancer: Receptor-Targeted Drug Delivery by Somatostatin Analog

A novel nanotechnology-based drug delivery system (DDS) targeted at pancreatic cancer cells was developed, characterized, and tested. The system consisted of liposomes as carriers, an anticancer drug (paclitaxel) as a chemotherapeutic agent, and a modified synthetic somatostatin analog, 5-pentacarbonyl-octreotide, a ligand for somatostatin receptor 2 (SSTR2), as a targeting moiety for pancreatic cancer. The cellular internalization, cytotoxicity, and antitumor activity of the DDS were tested in vitro using human pancreatic ductal adenocarcinoma (PDAC) cells with different expressions of the targeted SSTR2 receptors, and in vivo on immunodeficient mice bearing human PDAC xenografts. The targeted drug delivery system containing paclitaxel exhibited significantly enhanced cytotoxicity compared to non-targeted DDS, and this efficacy was directly related to the levels of SSTR2 expression. It was found that octreotide-targeted DDS proved exceptionally effective in suppressing the growth of PDAC tumors. This study underscores the potential of octreotide-targeted liposomal delivery systems to enhance the therapeutic outcomes for PDAC compared with non-targeted liposomal DDS and Paclitaxel-Cremophor® EL, suggesting a promising avenue for future cancer therapy innovations.

Cannabidiol’s cytotoxicity in pancreatic cancer is induced via an upregulation of ceramide synthase 1 and ER stress

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most aggressive malignancies with a median 5 year-survival rate of 12%. Cannabidiol (CBD) has been found to exhibit antineoplastic potential and may potentiate the anticancer effects of cytotoxic’s such as gemcitabine. CBD therapy has been linked to de novo synthesis of ceramide. The sphingolipid ceramide is a potent tumour suppressor lipid with roles in apoptosis and autophagy. One of the key players involved is ceramide synthase, an enzyme with six isoforms (CerS1-CerS6), reported to have disease prognostic value. Quantitative real time PCR was used to determine mRNA expression levels of ceramide synthase isoforms, GRP78, ATF4 and CHOP. Western blotting was used to analyze protein expression of these markers and knockdown of CerS1 and GRP78 were applied via an siRNA and confirmed by the two mentioned methods. Mice with PDAC xenografts were injected via intraperitoneal method with drugs and tumours were analysed with flow cytometry and processed using H&E and IHC staining. siRNA knockdown of ceramide synthase 1 (CerS1) and analysis point to evidence of a putative CerS1 dependent pathway driven by CBD in activating endoplasmic reticulum (ER) stress target; GRP78. Upon CBD treatment, CerS1 was upregulated and downstream this led to the GRP78/ATF4/CHOP arm of the unfolded protein response (UPR) pathway being activated. In an in vivo model of PDAC in which CerS1 was not upregulated on IHC, there was no observed improvement in survival of animals, however a reduction in tumour growth was observed in combination chemotherapy and CBD group, indicating further investigations in vivo. These findings provide evidence of a potential ceramide induced cytotoxic mechanism of action of CBD in pancreatic ductal adenocarcinoma.

CCK Receptor Inhibition Reduces Pancreatic Tumor Fibrosis and Promotes Nanoparticle Delivery.

The poor prognosis for pancreatic ductal adenocarcinoma (PDAC) patients is due in part to the highly fibrotic nature of the tumors that impedes delivery of therapeutics, including nanoparticles (NPs). Our prior studies demonstrated that proglumide, a cholecystokinin receptor (CCKR) antagonist, reduced fibrosis pervading PanIN lesions in mice. Here, we further detail how the reduced fibrosis elicited by proglumide achieves the normalization of the desmoplastic tumor microenvironment (TME) and improves nanoparticle uptake. One week following the orthotopic injection of PDAC cells, mice were randomized to normal or proglumide-treated water for 3-6 weeks. Tumors were analyzed ex vivo for fibrosis, vascularity, stellate cell activation, vascular patency, and nanoparticle distribution. The histological staining and three-dimensional imaging of tumors each indicated a reduction in stromal collagen in proglumide-treated mice. Proglumide treatment increased tumor vascularity and decreased the activation of cancer-associated fibroblasts (CAFs). Additionally, PANC-1 cells with the shRNA-mediated knockdown of the CCK2 receptor showed an even greater reduction in collagen, indicating the CCK2 receptors on tumor cells contribute to the desmoplastic TME. Proglumide-mediated reduction in fibrosis also led to functional changes in the TME as evidenced by the enhanced intra-tumoral distribution of small (<12 nm) Rhodamine-loaded nanoparticles. The documented in vivo, tumor cell-intrinsic anti-fibrotic effects of CCK2R blockade in both an immunocompetent syngeneic murine PDAC model as well as a human PDAC xenograft model demonstrates that CCK2R antagonists, such as proglumide, can improve the delivery of nano-encapsulated therapeutics or imaging agents to pancreatic tumors.

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.

Abstract 6513: Discovery and characterization of a KRASG12C/D/V degrader with potent anti-tumor activity in KRASmut-driven preclinical models

Abstract KRAS mutations are one of the most common oncogenic drivers for multiple cancers. KRASG12X mutations are identified in approximately 25% of lung cancer, 24% of colorectal (CRC), and 79% of pancreatic ductal adenocarcinoma (PDAC). Although the approval of Sotorasib in 2021 by the FDA partially addressed the needs of patients with KRASG12C mutant, other mutations, such as G12D and G12V, still lack effective target therapies. Here, we report RD0255359, a proteolysis targeting chimera (PROTAC) KRASG12C/D/V degrader with highly potent cell viability and KRASG12C/D/V degradation ability. In vitro, RD0255359 exhibited a remarkable KRAS degradation property in NCI-H358 (G12C), AGS (G12D), and SW620 (G12V) cell lines at concentrations below 10 nM. RD0255359 suppressed the proliferation of these cells with nanomolar IC50 without affecting the viability of KRASWT or KRAS-independent cell lines. Meanwhile, using protease inhibitor MG132 or corresponding ligand pretreatment and VHLKD-AGS cell experiment confirmed that RD0255359-induced KRAS degradation was VHL and UPS dependent. In vivo, weekly IV injection of 1-10 mg/kg RD0255359 led to tumor growth suppression or tumor regression in PK-59 PDAC xenograft model. The in vivo PK/PD demonstrated that durable KRAS degradation could be achieved with once- weekly IV injection. Notably, after injection, the drug can distribute into tumor tissue rapidly and abundantly, and maintaining a high concentration till the next administration. Taken together, RD0255359 is a highly potent and selective KRASG12C/D/V degrader with favorable PK properties. It exhibits a promising therapeutic index in preclinical studies. Citation Format: Zhengqing Li, Renqi Xu, Xiaofeng Yang, Zhengyao Zou, Xiaoqiang Kong, Haibo Chen, Yongqing Sun, Ying Zhao, Jing Guo, Cheng Yang, Han Han, Xiaoyun Liu, Hong Lan, Lieming Ding, Quan Zhou, Hao Wu, Jiabing Wang. Discovery and characterization of a KRASG12C/D/V degrader with potent anti-tumor activity in KRASmut-driven preclinical models [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 6513.

Abstract 1392: Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling

Abstract Neutrophil extracellular traps (NETs) are an important contributor to the immunosuppressive tumor microenvironment and facilitate metastasis of solid cancers. NETs have been shown to stimulate breast and colon cancer cell metastasis through a mechanism potentially involving an interaction between integrin-linked kinase (ILK) and the cell surface receptor coiled-coil domain containing protein 25 (CCDC25). Here, we have interrogated whether ILK is required for NET-induced metastasis in pancreatic ductal adenocarcinoma (PDAC), an inflammation-driven tumor that is difficult to treat. Evaluation of the circulating neutrophil to lymphocyte ratio (NLR) in the blood samples of patients presenting with metastatic PDAC showed that a higher baseline NLR is associated with significantly shorter overall patient survival. Western blot and immunofluorescence analyses demonstrated that human PDAC cell lines, patient-derived xenograft cells, cells derived from the KPCY genetic model of PDAC and patient-derived organoids co-express CCDC25 and ILK. Co-immunoprecipitation analyses showed that CCDC25 and ILK associate in both mouse and human PDAC cells, and that exposure of the cells to NETs results in an increased amount of ILK associated with CCDC25. Exposure of human and KPCY mouse PDAC cells to NETs increases phosphorylation of GSK3-β and ERK, increases activation of Rac1 and Cdc42 and upregulates markers of epithelial-to-mesenchymal transition (EMT), including ZEB1 and Snail, in a time and concentration-dependent manner. Furthermore, time-lapse imaging using Incucyte-based wounding assays showed that exposure to NETs induces pseudopodia-like protrusions, migration and invasion by these cells. RNAi-mediated suppression of ILK expression or inhibition of ILK activity using a highly selective inhibitor of ILK, QLT-0267, significantly inhibits the NET-induced increase in GSK3-β phosphorylation, Rac1/Cdc42 activation, migration and invasion of PDAC cells. In vivo, co-localization of CCDC25 and ILK is observed in tumors and metastases from KPCY mouse and MIA PaCa-2 human PDAC xenograft models stained by multispectral immunofluorescence and analyzed by confocal microscopy. Tumors and metastases show the presence of infiltrating neutrophils and NETs as indicated by staining for myeloperoxidase and citrullinated histone H3. Importantly, doxycycline-inducible shRNA-mediated suppression of ILK expression by luciferase-positive PDAC cells following injection of these cells through the tail vein of mice administered LPS intranasally results in a significant reduction in metastasis as measured by bioluminescence-based imaging, compared to control animals. Collectively, these data identify NET-induced ILK signaling as an important contributor to PDAC progression and suggest the potential for targeting this axis to prevent PDAC progression and metastasis. Citation Format: Paul C. McDonald, Shannon J. Awrey, Hossein Tavakoli, Rebekah Carroll, Zachary J. Gerbec, Joanna M. Karasinska, David F. Schaeffer, Daniel J. Renouf, Shoukat Dedhar. Neutrophil extracellular trap (NET)-induced pancreatic tumor metastasis requires integrin-linked kinase (ILK) signaling [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 1392.

Abstract 2618: BCG017, a bispecific ADC targeting PTK7 and EGFR exhibits anti-tumor efficacy in PDX models

Abstract EGFR is a favored target in various cancer therapies. However, EGFR-based treatments have been challenged by drug resistance and cytotoxicity. Targeting EGFR with an antibody-drug conjugate (ADC) is a promising new therapeutic strategy to address the drug resistance, due to the potent killing effects of the payload. Furthermore, we hypothesized that development of a bispecific ADC (bsADC) targeting EGFR and another tumor-associated antigen (TAA) might improve the tumor selectivity, thereby limiting the on-target off-tumor toxicity. One such TAA candidate is PTK7, which belongs to the same receptor tyrosine kinase family as EGFR, and is co-expressed with EGFR in multiple types of solid tumors, including lung, esophageal, head and neck, and colon cancers. Herein, we developed a fully human anti-human PTK7 EGFR bsAb using our proprietary common light chain RenLite® mouse platform and knobs-into-holes technology. The anti-PTK7 EGFR bsAb demonstrates good purity by SEC-HPLC, and can cross-react with human and cynomolgus monkey antigens with favorable affinity. The PTK7 x EGFR bsAb demonstrated higher binding to various types of cancer cell lines, as well as enhanced internalization in vitro compared with PTK7 parental antibody. Next, the bsAb was conjugated with monomethyl auristatin E (MMAE) to generate an anti-PTK7 x EGFR bsADC, BCG017, with a drug-to-antibody ratio of approximately 4. BCG017 demonstrated superior anti-tumor activity compared to benchmarks and its PTK7 parental ADC in cell-derived non-small-cell lung cancer (NSCLC) xenografts, and showed enhanced anti-tumoractivity when compared to benchmark ADCs and parental ADCs in patient-derived breast and pancreatic ductal adenocarcinoma xenograft models. These results suggest that BCG017 have the potential to be a novel therapeutic alternative for PTK7 and EGFR co-expressing tumors. Citation Format: Sufei Yao, Chengzhang Shang, Zhuolin Li, Gao An, Chaoshe Guo, W. Frank An, Yi Yang. BCG017, a bispecific ADC targeting PTK7 and EGFR exhibits anti-tumor efficacy in PDX models [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 2618.

Skeletal Muscle-Derived Irisin Enhances Gemcitabine Sensitivity and Suppresses Migration Ability in Pancreatic Ductal Adenocarcinoma.

High skeletal muscle mass might be a prognostic factor for patients with pancreatic ductal adenocarcinoma (PDAC); however, the underlying reason is unclear. We hypothesized that myokines, which are cytokines secreted by the skeletal muscle, function as suppressors of PDAC. We specifically examined irisin, a myokine, which plays a critical role in the modulation of metabolism, to clarify the anticancer mechanisms. First, the effect of the conditioned medium (CM) from skeletal muscle cells and from irisin-knockdown skeletal muscle cells on PDAC cell lines was evaluated. We then investigated the effects and anticancer mechanism of irisin in PDAC cells, and evaluated the anticancer effect of recombinant irisin in a PDAC xenograft mouse model. Finally, patients undergoing pancreatic resection for PDAC were divided into two groups based on their serum irisin level, and the long-term outcomes were evaluated. The CM enhanced gemcitabine sensitivity by inducing apoptosis and decreasing cell migration by inhibiting epithelial-mesenchymal transition (EMT) in PDAC cell lines. The CM derived from irisin-knockdown skeletal muscle cells did not affect the PDAC cell lines. The addition of recombinant irisin to PDAC cell lines facilitated sensitivity to gemcitabine by inhibiting the mitogen-activated protein kinase (MAPK) pathway, and decreased migration by inhibiting EMT via the transforming growth factor-β/SMAD pathway. Xenografts injected with gemcitabine and recombinant irisin grew slower than the xenografts injected with gemcitabine alone. The overall survival was prolonged in the high-irisin group compared with that in the low-irisin group. Skeletal muscle-derived irisin may affect PDAC by enhancing its sensitivity to gemcitabine and suppressing EMT.

Anti-proliferative and apoptotic effect of cannabinoids on human pancreatic ductal adenocarcinoma xenograft in BALB/c nude mice model

Human pancreatic ductal adenocarcinoma (PDAC) is a highly malignant and lethal tumor of the exocrine pancreas. Cannabinoids extracted from the hemp plant Cannabis sativa have been suggested as a potential therapeutic agent in several human tumors. However, the anti–tumor effect of cannabinoids on human PDAC is not entirely clarified. In this study, the anti–proliferative and apoptotic effect of cannabinoid solution (THC:CBD at 1:6) at a dose of 1, 5, and 10 mg/kg body weight compared to the negative control (sesame oil) and positive control (5-fluorouracil) was investigated in human PDAC xenograft nude mice model. The findings showed that cannabinoids significantly decreased the mitotic cells and mitotic/apoptotic ratio, meanwhile dramatically increased the apoptotic cells. Parallelly, cannabinoids significantly downregulated Ki-67 and PCNA expression levels. Interestingly, cannabinoids upregulated BAX, BAX/BCL-2 ratio, and Caspase-3, meanwhile, downregulated BCL-2 expression level and could not change Caspase-8 expression level. These findings suggest that cannabinoid solution (THC:CBD at 1:6) could inhibit proliferation and induce apoptosis in human PDAC xenograft models. Cannabinoids, including THC:CBD, should be further studied for use as the potent PDCA therapeutic agent in humans.

Bispecific, Exosome‐Mimetic Lipid Nanoparticles Facilitate Dual siRNAs for Synergistic Therapy against Pancreatic Cancer

AbstractWhile RNA therapeutics hold great promise for combating pancreatic ductal adenocarcinoma (PDAC), the limited therapy outcome due to the insufficient delivery efficiency of vehicles and inadequate innate therapeutic potency for single siRNA hinders the broader application. Herein, report bispecific, exosome‐mimetic lipid nanoparticles (EM‐LNPs) carrying high loads of siKras and siTP53, which can efficiently overcome layers of barriers to accumulate cytoplasm in vitro and in vivo and achieve synergistic treatment for PDAC tumors. EM‐LNPs inherit features of synthetic LNPs and the protein characteristics of exosomes with outer CD47 proteins, membrane‐inserted integrin α6β4, and connexin 43. The siRNAs‐loaded EM‐LNPs can efficiently evade the host immune clearance in the circulation, considerably accumulate and penetrate dense tumors, and simultaneously target EGFR/VEGF of PDAC cells, following leakage into the cytoplasm. The intracellular siKras and siTP53 achieved synergistic therapeutic effects for PDAC cells with as high as 20 times enhancement in vitro and 3.5 times increase in vivo, significantly suppressed xenograft PDAC tumors, considerably reduced lung metastasis, and vastly extended mice survival at a low injection dose. These results explore an approach for delivering multiple gene cargoes to dense PDAC tumors for synergistic therapeutics.

Physico-chemical characterization of the tumour microenvironment of pancreatic ductal adenocarcinoma

Pancreatic ductal adenocarcinoma (PDAC) is a highly aggressive lethal malignancy that accounts for more than 90% of pancreatic cancer diagnoses. Our research is focused on the physico-chemical properties of the tumour microenvironment (TME), including its tumoural extracellular matrix (tECM), as they may have an important impact on the success of cancer therapies. PDAC xenografts and their decellularized tECM offer a great material source for research in terms of biomimicry with the original human tumour. Our aim was to evaluate and quantify the physico-chemical properties of the PDAC TME. Both cellularized (native TME) and decellularized (tECM) patient-derived PDAC xenografts were analyzed. A factorial design of experiments identified an optimal combination of factors for effective xenograft decellularization. Our results provide a complete advance in our understanding of the PDAC TME and its corresponding stroma, showing that it presents an interconnected porous architecture with very low permeability and small pores due to the contractility of the cellular components. This fact provides a potential therapeutic strategy based on the therapeutic agent size.

Abstract B081: SOAT1 as a targetable KRAS dependency in PDAC

Abstract Metabolic rewiring is an emerging hallmark of neoplasia. In pancreatic ductal adenocarcinoma (PDAC), KRAS alters myriad metabolic programs to promote cellular transformation. Among them, the cholesterol synthesis pathway has emerged as a vulnerability of cancer cells due to the importance of cholesterol in membrane fluidity and the production of isoprenoid intermediates that enable KRAS membrane localization and activation in pancreatic cancer. We recently reported that the non-essential gene SOAT1 (Sterol-O-Acyl transferase 1), encoding an enzyme that esterifies free cholesterol into an insoluble form for storage and transport, is markedly upregulated in metastatic PDAC cells. SOAT1 promotes the hyperactivation of the mevalonate synthesis pathway due to loss of negative feedback by free cholesterol, thereby leading to increased production of isoprenoids such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), which are required for RAS and RHO membrane localization and activity, respectively. Genetic ablation of Soat1 reduced RAS and RHO membrane localization and inhibited cellular proliferation and mitochondrial function in vitro, and these effects were largely rescued by exogenous FPP and GGPP. Therefore, our study nominated SOAT1 as a new candidate gene for therapeutic targeting. Based on SuFEx Click Chemistry (ASCC) technology, we have designed and synthesized a panel of drug-like candidates. The first series of Soat1 probes selectively impair the proliferation of PDAC cells that express SOAT1, reduce SOAT1 protein content in cells, and covalently bind to SOAT1. In mice, the most active SOAT1 probes were tolerated in a 28-day daily dosing study up to 120 mg/kg. Marked tumor growth inhibition was noted in mice bearing PDAC xenografts and GEMM mice treated as monotherapy with 15 mg/kg/d of the most active SOAT1 probe, and tissue analysis demonstrated tumor necrosis, decreased proliferation, and increased apoptosis. Mechanistically, SOAT1 loss or inhibition targets multiple important components of cellular transformation in PDAC including KRAS, RHO, and mitochondrial biology, and our work suggests that it is an attractive target to pursue for further drug development in this cancer. Citation Format: Wenjun Lan, Shoujun Sun, Jeremy Nigri, Josh Homer, Young Park, Astrid Deschenes, Paolo Cifani, Justin Cross, Victoria Gaeth, Sunny Kim, Shawn Ting, Rachel Rubino, John Moses, David Tuveson. SOAT1 as a targetable KRAS dependency in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr B081.

A Cancer-Specific Monoclonal Antibody against Podocalyxin Exerted Antitumor Activities in Pancreatic Cancer Xenografts.

Podocalyxin (PODXL) overexpression is associated with poor clinical outcomes in various tumors. PODXL is involved in tumor malignant progression through the promotion of invasiveness and metastasis. Therefore, PODXL is considered a promising target of monoclonal antibody (mAb)-based therapy. However, PODXL also plays an essential role in normal cells, such as vascular and lymphatic endothelial cells. Therefore, cancer specificity or selectivity is required to reduce adverse effects on normal cells. Here, we developed an anti-PODXL cancer-specific mAb (CasMab), PcMab-6 (IgG1, kappa), by immunizing mice with a soluble PODXL ectodomain derived from a glioblastoma LN229 cell. PcMab-6 reacted with the PODXL-positive LN229 cells but not with PODXL-knockout LN229 cells in flow cytometry. Importantly, PcMab-6 recognized pancreatic ductal adenocarcinoma (PDAC) cell lines (MIA PaCa-2, Capan-2, and PK-45H) but did not react with normal lymphatic endothelial cells (LECs). In contrast, one of the non-CasMabs, PcMab-47, showed high reactivity to both the PDAC cell lines and LECs. Next, we engineered PcMab-6 into a mouse IgG2a-type (PcMab-6-mG2a) and a humanized IgG1-type (humPcMab-6) mAb and further produced the core fucose-deficient types (PcMab-6-mG2a-f and humPcMab-6-f, respectively) to potentiate the antibody-dependent cellular cytotoxicity (ADCC). Both PcMab-6-mG2a-f and humPcMab-6-f exerted ADCC and complement-dependent cellular cytotoxicity in the presence of effector cells and complements, respectively. In the PDAC xenograft model, both PcMab-6-mG2a-f and humPcMab-6-f exhibited potent antitumor effects. These results indicated that humPcMab-6-f could apply to antibody-based therapy against PODXL-expressing pancreatic cancers.