Patient-derived Pancreatic Cancer Cells Research Articles

3D Chromatin Alteration by Disrupting β-Catenin/CBP Interaction Is Enriched with Insulin Signaling in Pancreatic Cancer.

The therapeutic potential of targeting the β-catenin/CBP interaction has been demonstrated in a variety of preclinical tumor models with a small molecule inhibitor, ICG-001, characterized as a β-catenin/CBP antagonist. Despite the high binding specificity of ICG-001 for the N-terminus of CBP, this β-catenin/CBP antagonist exhibits pleiotropic effects. Our recent studies found global changes in three-dimensional (3D) chromatin architecture in response to disruption of the β-catenin/CBP interaction in pancreatic cancer cells. However, an understanding of how the functional crosstalk between the antagonist and the β-catenin/CBP interaction affects changes in 3D chromatin architecture and, thereby, gene expression and downstream effects remains to be elucidated. Here, we perform Hi-C analyses on canonical and patient-derived pancreatic cancer cells before and after treatment with ICG-001. In addition to global alteration of 3D chromatin domains, we unexpectedly identify insulin signaling genes enriched in the altered chromatin domains. We further demonstrate that the chromatin loops associated with insulin signaling genes are significantly weakened after ICG-001 treatment. We finally elicit the deletion of a looping of IRS1-a key insulin signaling gene-significantly impeding pancreatic cancer cell growth, indicating that looping-mediated insulin signaling might act as an oncogenic pathway to promote pancreatic cancer progression. Our work shows that targeting aberrant insulin chromatin looping in pancreatic cancer might provide a therapeutic benefit.

Abstract 1494: Clonal dominance defines metastatic dissemination in pancreatic cancer

Abstract Tumors represent ecosystems where subclones compete during tumor growth. While extensively investigated, a comprehensive picture of the interplay of clonal lineages during dissemination is still lacking. Using patient-derived pancreatic cancer cells, we created orthotopically-implanted clonal replica tumors to trace clonal dynamics of unperturbed tumor expansion and dissemination. This model revealed the multifaceted nature of tumor growth, with rapid changes in clonal fitness leading to continuous reshuffling of tumor architecture and alternating clonal dominance as a distinct feature of cancer growth. Regarding dissemination, a large fraction of tumor lineages could be found at secondary sites each having distinctive organ growth patterns as well as numerous undescribed behaviors such as abortive colonization. Paired analysis of primary and secondary sites revealed fitness as major contributor to dissemination. From the analysis of pro- and non-metastatic isogenic subclones, we identified a transcriptomic signature able to identify metastatic cells in human tumors and predict patients’ survival. Citation Format: I-Lin Ho, Chieh-Yuan Li, Fuchenchu Wang, Li Zhao, Jingjing Liu, Er-Yen Yen, Charles A. Dyke, Rutvi Shah, Zhaoliang Liu, Ali Osman Çetin, Francesca Citron, Sergio Attanasio, Virgina Giuliani, Tim Heffernan, Kim-Anh Do, Gaetano Gargiulo, Giulio Draetta, Alessandro Carugo, Ruitao Lin, Andrea Viale. Clonal dominance defines metastatic dissemination in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1494.

Abstract 6940: The transcriptional and epigenetic regulation of epithelial-mesenchymal plasticity in patient-derived pancreatic cancer cell lines

Abstract Epithelial-mesenchymal transition (EMT) is a hallmark of pancreatic ductal adenocarcinoma (PDAC) invasion. In general, cellular plasticity is a major contributor to both tumor progression and therapy resistance. The major goals of our study are to 1) directly and quantitatively measure plasticity in PDAC3 cell lines, and 2) identify candidate genes that could alter the plasticity of cancer cells. By understanding the regulation of tumor cell plasticity, we may find ways to perturb cells and prevent the transition towards treatment resistant cell states. A strict definition of plasticity in cancer has yet to be established, however, and is imperative to our understanding of cell state regulation and for identifying vulnerabilities in plastic cells. We argue that in order to prove plasticity occurs within a population of cells, one must either 1) demonstrate that a new cell state has emerged that was previously not present, or 2) trace the lineage of a cell to show it can adopt multiple cell states. For our study, we profile 12 patient-derived PDAC cell lines to find convergent EMT programs and perform lineage tracing on these cell lines using a modified CROP-seq vector, called ClonMapper, to quantitatively measure plasticity. Towards this goal, we perform single-cell RNA-seq (scRNA) and single-cell multiomic measurements of 1000 uniquely barcoded cells expanded over a time course of 2, 3 and 4 weeks. We refer to cells that have the same lineage barcode - originating from the same initial cell - as families. We found that ~50% of families are composed of cells that are epithelial and mesenchymal, thus proving plasticity must exist, as it shows that a given parental cell of unknown state can give rise to progeny that are in multiple states. We then develop mathematical models to show that families have different levels of plasticity, defined by the rates of transition between distinct cell states. We next leverage our barcode-resolution time course data to identify factors at early time points that may influence plasticity at later time points. We show there is differential accessibility of several of these factors across families with different plasticity properties, thus proposing a role for epigenetic regulation of EMT. Importantly, we find that specific factors drive gene regulatory networks important for epithelial and mesenchymal programs, and therefore propose candidates for the modulation of EMT for therapeutic benefit. Citation Format: Deepika Yeramosu, Lynn Bi, Abigail Collins, Mike Bogaev, Martin Jankowiak, Aziz Al’Khafaji, Milan Parikh, Mehrtash Babadi, Alex Bloemendal, Surya Nagaraja, Ray Jones, Jay Shendure, David T. Ting, Andrew Aguirre, Nir Hacohen, Dana Pe’er, Eric S. Lander, Arnav Mehta. The transcriptional and epigenetic regulation of epithelial-mesenchymal plasticity in patient-derived pancreatic cancer cell lines [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 6940.

Clonal dominance defines metastatic dissemination in pancreatic cancer.

Tumors represent ecosystems where subclones compete during tumor growth. While extensively investigated, a comprehensive picture of the interplay of clonal lineages during dissemination is still lacking. Using patient-derived pancreatic cancer cells, we created orthotopically implanted clonal replica tumors to trace clonal dynamics of unperturbed tumor expansion and dissemination. This model revealed the multifaceted nature of tumor growth, with rapid changes in clonal fitness leading to continuous reshuffling of tumor architecture and alternating clonal dominance as a distinct feature of cancer growth. Regarding dissemination, a large fraction of tumor lineages could be found at secondary sites each having distinctive organ growth patterns as well as numerous undescribed behaviors such as abortive colonization. Paired analysis of primary and secondary sites revealed fitness as major contributor to dissemination. From the analysis of pro- and nonmetastatic isogenic subclones, we identified a transcriptomic signature able to identify metastatic cells in human tumors and predict patients' survival.

Fast-relaxing hydrogels with reversibly tunable mechanics for dynamic cancer cell culture

The mechanics of the tumor microenvironment (TME) significantly impact disease progression and the efficacy of anti-cancer therapeutics. While it is recognized that advanced in vitro cancer models will benefit cancer research, none of the current engineered extracellular matrices (ECM) adequately recapitulate the highly dynamic TME. Through integrating reversible boronate-ester bonding and dithiolane ring-opening polymerization, we fabricated synthetic polymer hydrogels with tumor-mimetic fast relaxation and reversibly tunable elastic moduli. Importantly, the crosslinking and dynamic stiffening of matrix mechanics were achieved in the absence of a photoinitiator, often the source of cytotoxicity. Central to this strategy was Poly(PEGA-co-LAA-co-AAPBA) (PELA), a highly defined polymer synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization. PELA contains dithiolane for initiator-free gel crosslinking, stiffening, and softening, as well as boronic acid for complexation with diol-containing polymers to give rise to tunable viscoelasticity. PELA hydrogels were highly cytocompatible for dynamic culture of patient-derived pancreatic cancer cells. It was found that the fast-relaxing matrix induced mesenchymal phenotype of cancer cells, and dynamic matrix stiffening restricted tumor spheroid growth. Moreover, this new dynamic viscoelastic hydrogel system permitted sequential stiffening and softening to mimic the physical changes of TME.

Integrated single-dose kinome profiling data is predictive of cancer cell line sensitivity to kinase inhibitors.

Protein kinase activity forms the backbone of cellular information transfer, acting both individually and as part of a broader network, the kinome. Their central role in signaling leads to kinome dysfunction being a common driver of disease, and in particular cancer, where numerous kinases have been identified as having a causal or modulating role in tumor development and progression. As a result, the development of therapies targeting kinases has rapidly grown, with over 70 kinase inhibitors approved for use in the clinic and over double this number currently in clinical trials. Understanding the relationship between kinase inhibitor treatment and their effects on downstream cellular phenotype is thus of clear importance for understanding treatment mechanisms and streamlining compound screening in therapy development. In this work, we combine two large-scale kinome profiling data sets and use them to link inhibitor-kinome interactions with cell line treatment responses (AUC/IC50). We then built computational models on this data set that achieve a high degree of prediction accuracy (R2 of 0.7 and RMSE of 0.9) and were able to identify a set of well-characterized and understudied kinases that significantly affect cell responses. We further validated these models experimentally by testing predicted effects in breast cancer cell lines and extended the model scope by performing additional validation in patient-derived pancreatic cancer cell lines. Overall, these results demonstrate that broad quantification of kinome inhibition state is highly predictive of downstream cellular phenotypes.

Priming therapy by targeting enhancer-initiated pathways in patient-derived pancreatic cancer cells.

Systems biology leveraging multi-OMICs technologies, is rapidly advancing development of precision therapies and matching patients to targeted therapies, leading to improved responses. A new pillar of precision oncology lies in the power of chemogenomics to discover drugs that sensitizes malignant cells to other therapies. Here, we test a chemogenomic approach using epigenomic inhibitors (epidrugs) to reset patterns of gene expression driving the malignant behavior of pancreatic tumors. We tested a targeted library of ten epidrugs targeting regulators of enhancers and super-enhancers on reprogramming gene expression networks in seventeen patient-derived primary pancreatic cancer cell cultures (PDPCCs), of both basal and classical subtypes. We subsequently evaluated the ability of these epidrugs to sensitize pancreatic cancer cells to five chemotherapeutic drugs that are clinically used for this malignancy. To comprehend the impact of epidrug priming at the molecular level, we evaluated the effect of each epidrugs at the transcriptomic level of PDPCCs. The activating epidrugs showed a higher number of upregulated genes than the repressive epidrugs (χ2 test p-value <0.01). Furthermore, we developed a classifier using the baseline transcriptome of epidrug-primed-chemosensitized PDPCCs to predict the best epidrug-priming regime to a given chemotherapy. Six signatures with a significant association with the chemosensitization centroid (R≤-0.80; p-value < 0.01) were identified and validated in a subset of PDPCCs. We conclude that targeting enhancer-initiated pathways in patient-derived primary cells, represents a promising approach for developing new therapies for human pancreatic cancer. This work was supported by INCa (Grants number 2018-078 to ND and 2018- 079 to JI), Canceropole PACA (ND), Amidex Foundation (ND), and INSERM (JI).

Biological evaluation of novel gemcitabine analog in patient-derived xenograft models of pancreatic cancer

Gemcitabine (Gem) has been a standard first-line drug for pancreatic cancer (PCa) treatment; however, Gem's rapid metabolism and systemic instability (short half-life) limit its clinical outcome. The objective of this study was to modify Gem into a more stable form called 4-(N)-stearoyl-gemcitabine (4NSG) and evaluate its therapeutic efficacy in patient-derived xenograft (PDX) models from PCa of Black and White patients.Methods 4NSG was synthesized and characterized using nuclear magnetic resonance (NMR), elemental analysis, and high-performance liquid chromatography (HPLC). 4NSG-loaded solid lipid nanoparticles (4NSG-SLN) were developed using the cold homogenization technique and characterized. Patient-derived pancreatic cancer cell lines labeled Black (PPCL-192, PPCL-135) and White (PPCL-46, PPCL-68) were used to assess the in vitro anticancer activity of 4NSG-SLN. Pharmacokinetics (PK) and tumor efficacy studies were conducted using PDX mouse models bearing tumors from Black and White PCa patients.Results 4NSG was significantly stable in liver microsomal solution. The effective mean particle size (hydrodynamic diameter) of 4NSG-SLN was 82 ± 6.7 nm, and the half maximal inhibitory concentration (IC50) values of 4NSG-SLN treated PPCL-192 cells (9 ± 1.1 µM); PPCL-135 (11 ± 1.3 µM); PPCL-46 (12 ± 2.1) and PPCL-68 equaled to 22 ± 2.6 were found to be significantly lower compared to Gem treated PPCL-192 (57 ± 1.5 µM); PPCL-135 (56 ± 1.5 µM); PPCL-46 (56 ± 1.8 µM) and PPCL-68 (57 ± 2.4 µM) cells. The area under the curve (AUC), half-life, and pharmacokinetic clearance parameters for 4NSG-SLN were 3–fourfold higher than that of GemHCl. For in-vivo studies, 4NSG-SLN exhibited a two-fold decrease in tumor growth compared with GemHCl in PDX mice bearing Black and White PCa tumors.Conclusion 4NSG-SLN significantly improved the Gem's pharmacokinetic profile, enhanced Gem's systemic stability increased its antitumor efficacy in PCa PDX mice bearing Black and White patient tumors.

Abstract 816: Biological evaluation of novel stearoyl gemcitabine nanoparticles in primary pancreatic cancer cells and PDX mice tumor models

Abstract Purpose: This study aimed to improve the systemic stability and enhance the anticancer activity of gemcitabine (Gem) by modifying Gem with stearic acid to form 4-N-stearoylGem (4NSG). The 4NSG compound was formulated into solid lipid nanoparticles and tested against patient-derived pancreatic cancer (PCa) cell lines and patient-derived xenograft (PDX) mice bearing subcutaneous tumors. Methods: Gem was modified by linking the 4-amino group of Gem and stearoyl acyl derivative to form 4-(N)-stearoyl-gemcitabine (4NSG). 4NSG was characterized using high-performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), and elemental analysis. 4NSG was developed into solid lipid nanoparticles (4NSG-SLN) and characterized using a particle size analyzer. Patient-derived primary PCa cells (PPCL-46 and PPCL-68) were treated with GemHCl and 4NSG-SLN. Cytotoxicity, cell migration, and cell-cycle studies were performed to determine the effectiveness of 4NSG-SLN against primary PCa cell lines. We performed antitumor efficacy testing using GemHCl and 4NSG-SLN in PDX mice bearing subcutaneous pancreatic tumors. Results: Analysis of the H-NMR spectra displayed an amide bond at 10.88 ppm, confirming the conjugated bond between the 4-amino group of Gem and stearic acid. The purity of 4NSG was 99.8%. The hydrodynamic diameter (particle size) of 4NSG-SLN was 82±2.3nm, while the blank nanoparticle (SLN) was found to be 35 ± 4.3 nm. The half-maximal inhibitory concentration of 4NSG-SLN-treated PPCL-46 (IC50 =12 ± 2.1µM) and PPCL-68 cultures (IC50 =22 ± 2.6µM) showed higher cytotoxic activity compared with GemHCl-treated PPCL-46 and PPCL-68 cultures respectively (IC50 = 56 ± 2.4 µM, IC50 = 57 ± 1.5 µM, p-value: p&amp;lt;0.001). Our migration study showed 4NSG-SLN treated PPCL-46 cells at 5μΜ concentrations significantly reduced cell mobility towards the wound area with (28 ± 3.5) number of cells migrated compared to GemHCl treatment which showed (120 ± 3.8). The cell-cycle analysis showed that 4NSG-SLN treated PPCL-46 cells at 5μΜ concentration had a higher G1 population (78.25%) than GemHCl (72.75%). 4NSG-SLN may have triggered apoptosis in PPCL-46 cells (2.45%) at 20μΜ in the S and G2/M phases compared to GemHCl treatments which showed a higher cell population of (9.54%).4NSG-SLN treated PDX mice exhibited a two-fold decrease in tumor growth compared to GemHCl-treated PDX mice bearing tumors. Conclusion: This study reveals that 4NSG-SLNs may potentially prolong Gem's systemic circulation, increase its bioavailability, and, most importantly, enhance the therapeutic efficacy of Gem in the treatment of PCa. Citation Format: Esther K. Frimpong, Andriana Inkoom, Nkafu B. Ndemazie, Raviteja Bulusu, Joy C. Okoro, Bo Han, Jose Trevino, Edward Agyare. Biological evaluation of novel stearoyl gemcitabine nanoparticles in primary pancreatic cancer cells and PDX mice tumor models [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 816.

A Cyclodiaryliodonium NOX Inhibitor for the Treatment of Pancreatic Cancer via Enzyme-Activatable Targeted Delivery by Sulfated Glycosaminoglycan Derivatives.

Pancreatic cancer renders a principal cause of cancer mortalities with a dismal prognosis, lacking sufficiently safe and effective therapeutics. Here, diversified cyclodiaryliodonium (CDAI)NADPH oxidase (NOX) inhibitors are rationally designed with tens of nanomolar optimal growth inhibition, and CD44-targeted delivery is implemented using synthesized sulfated glycosaminoglycan derivatives. The self-assembled nanoparticle-drug conjugate (NDC) enables hyaluronidase-activatable controlled release and facilitates cellular trafficking. NOX inhibition reprograms the metabolic phenotype by simultaneously impairing mitochondrial respiration and glycolysis. Moreover, the NDC selectively diminishes non-mitochondrial reactive oxygen species (ROS) but significantly elevates cytotoxic ROS through mitochondrial membrane depolarization. Transcriptomic profiling reveals perturbed p53, NF-κB, and GnRH signaling pathways interconnected with NOX inhibition. After being validated in patient-derived pancreatic cancer cells, the anticancer efficacy is further verified in xenograft mice bearing heterotopic and orthotopic pancreatic tumors, with extended survival and ameliorated systemic toxicity. It is envisaged that the translation of cyclodiaryliodonium inhibitors with an optimized molecular design can be expedited by enzyme-activatable targeted delivery with improved pharmacokinetic profiles and preserved efficacy.

Abstract B034: CXCR4 targeting endogenous human peptides eliminate migrating cancer stem cells by disrupting tumor-stroma crosstalk in pancreatic ductal adenocarcinomas

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a lethal cancer characterized by late diagnosis, lack of early symptoms and extensive metastasis. One of the foremost reasons for such startling statistics is the presence of a subpopulation of highly plastic stem-like cells within the tumor called cancer stem cells (CSCs). We have previously identified a distinct subset of these CSCs within the invasive front of patient tumors. This subset, called migrating cancer stem cells (miCSCs), is characterized by CD133+CXCR4+ expression and determines the metastatic phenotype of pancreatic cancer. Therefore, targeting CXCR4 may represent a potential therapeutic approach to lower metastatic burden in PDAC. Here, we examined the effect of endogenous human peptides EPI-X4 and other derivatives thereof as CXCR4 antagonist on (i) patient-derived primary pancreatic cancer cells and (ii) tumor-stroma crosstalk by using a dual culture system with pancreatic stellate cells. We established these peptides as novel therapeutic strategy for combating the metastatic activity of pancreatic cancer using combinatorial therapeutic approaches and testing different in vivo delivery system such as peptide fatty-acid (FA) conjugates and silica nanoparticles (Si-NP). Our results show that EPI-X4 as well as its derivatives (e.g., JM#21) strongly inhibited migratory capacity of primary pancreatic cancer cells towards the CXCR4 ligand CXCL12 in vitro. Thereby, JM#21 was identified as the most potent EPI-X4 derivate. Mechanistical analysis by western blot, gene expression and immunofluorescence revealed that JM#21 increased Cadherin-1 expression by suppression of Snail1 via inactivation of SHH pathway. Moreover, JM#21 decreased CXCL12-induced phosphorylation of AKT and IKBa as well as NANOG expression, which further suppressed self-renewal capacity and EMT in the tumor cells. Strikingly, JM#21 sensitized selected cell lines towards gemcitabine and paclitaxel. Furthermore, FA conjugated and Si-NP encapsulated JM#21 restricted miCSCs maintenance which was predominantly regulated via stellate cell secreted CXCL12. In serum conditions, both FA conjugated, and Si-NP encapsulated JM#21 was found to be stable and active, proving as a valuable delivery system for in vivo studies. In conclusion, our study reveals that targeting CXCR4/CXCL12 signaling axis using human endogenous EPI-X4 derivates particularly JM#21 inhibits tumor-stroma crosstalk which is paramount for the propagation and maintenance of miCSC. Particularly, we demonstrate, in both mechanistic and preclinical set up, that these peptides abrogate the metastatic capacity of patient-derived pancreatic cancer cells by selective targeted elimination of miCSCs. Moreover, tumor cells show increased susceptibility towards conventional treatment strategies enforcing EPI-X4 derivate as a novel combinatory therapy to treat metastatic pancreatic cancer. Citation Format: Kanishka Tiwary, Mirja Harms, Bastian Beitzinger, Roman Schmid, Syeda Inaas, Karolin Walter, Alexander Kleger, Mika Lindén, Thomas Seufferlein, Jan Münch, Patrick Christian Hermann. CXCR4 targeting endogenous human peptides eliminate migrating cancer stem cells by disrupting tumor-stroma crosstalk in pancreatic ductal adenocarcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B034.

Abstract B035: CXCL12 / CXCR4 signaling enhances and sustains migrating cancer stem cells via BMI1 in pancreatic ductal adenocarcinomas

Abstract Pancreatic cancer is a fatal disease and is one of the most aggressive and metastatic malignancies worldwide. The dissemination of tumor cells is the prerequisite of metastases and correlates with a loss of epithelial differentiation and the acquisition of a migratory phenotype, a hallmark of malignant tumor progression. Migrating cancer stem cells (miCSCs) characterized by CD133+ and CXCR4+ expression play a pivotal role in malignant tumor formation, have been reported to form the invasive front of the metastasis and in silico analysis showed that both CSCs and miCSCs are significantly upregulated in PDAC. However, the regulatory pattern maintaining these CSCs and especially miCSCs in PDAC remains widely elusive. Thus, this study further helps to unravel pathways responsible for maintenance of CSC (CD133+) and miCSC (CD133+ CXCR4+) population. To identify key signaling pathways responsible for both CSCs and miCSCs maintenance, we first generated a protein-protein interaction network using STRING database. Afterwards, we validated these signaling pathway(s) involved in aiding CSC and miCSC population by performing shRNA mediated knockdown of key signaling proteins in different patient-derived pancreatic cancer cell lines. Moreover, we interrogated the involvement of the tumor-stroma crosstalk in the regulation of these pathways by co-culturing tumor cells with pancreatic stellate cells. Protein-protein interaction network incorporating relevant factors involved in EMT, stemness, as well as SHH, NF-kB and AKT signaling pathway identified a strong link between the CXCL12/CXCR4 signaling axis and BMI1. Migration assay, sphere formation assay and western blot upon shRNA mediated knockdown of either CXCR4 and/or BMI1 ascertained BMI1 as a key player downstream of the CXCL12/CXCR4 axis to mechanistically effect both EMT and stemness. Pathway focused gene expression analysis as well as ELISA and immunofluorescence for CXCL12 and actin filaments, respectively, revealed the indispensable nature of tumor-stroma crosstalk on promoting CSC and miCSC population through the chemokine CXCL12. In addition, co-culture systems revealed that particularly pancreatic stellate cells play a significant role in maintaining both CSCs and miCSCs population as well as their characteristic phenotype including but not limited to chemotherapy resistance. Taken together, our results obtained in this study established mechanistically that the CXCL12/CXCR4 signaling pathway driven by tumor-stroma crosstalk not only enhances but also maintains both CSCs and miCSCs in pancreatic ductal adenocarcinomas through BMI1 ultimately promoting metastases and therapeutic resistance. Citation Format: Kanishka Tiwary, Anton Lahusen, Syeda Inaas, Stefanie Hauff, Karolin Walter, Alexander Kleger, Thomas Seufferlein, Bruno Sainz Jr., Patrick Christian Hermann. CXCL12 / CXCR4 signaling enhances and sustains migrating cancer stem cells via BMI1 in pancreatic ductal adenocarcinomas [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B035.

Patient-Derived Pancreatic Cancer Cells Induce C2C12 Myotube Atrophy by Releasing Hsp70 and Hsp90.

Pancreatic cancer (PC) patients are highly prone to cachexia, a lethal wasting syndrome featuring muscle wasting with an undefined etiology. Recent data indicate that certain murine cancer cells induce muscle wasting by releasing Hsp70 and Hsp90 through extracellular vesicles (EVs) to activate p38β MAPK-mediated catabolic pathways primarily through Toll-like receptor 4 (TLR4). However, whether human PC induces cachexia through releasing Hsp70 and Hsp90 is undetermined. Here, we investigated whether patient-derived PC cells induce muscle cell atrophy directly through this mechanism. We compared cancer cells isolated from patient-derived xenografts (PDX) from three PC patients who had cachexia (PCC) with those of three early-stage lung cancer patients without cachexia (LCC) and two renal cancer patients who were not prone to cachexia (RCC). We observed small increases of Hsp70 and Hsp90 released by LCC and RCC in comparison to non-cancer control cells (NCC). However, PCC released markedly higher levels of Hsp70 and Hsp90 (~ 6-fold on average) than LCC and RCC. In addition, PCC released similarly increased levels of Hsp70/90-containing EVs. In contrast to RCC and LCC, PCC-conditioned media induced a potent catabolic response in C2C12 myotubes including the activation of p38 MAPK and transcription factor C/EBPβ, upregulation of E3 ligases UBR2 and MAFbx, and increase of autophagy marker LC3-II, resulting in the loss of the myosin heavy chain (MHC ~50%) and myotube diameter (~60%). Importantly, the catabolic response was attenuated by Hsp70- and Hsp90-neutralizing antibodies in a dose-dependent manner. These data suggest that human PC cells release high levels of Hsp70 and Hsp90 that induce muscle atrophy through a direct action on muscle cells.

Abstract 1746: Optimizing Gemcitabine efficacy through chemical modification for the treatment of pancreatic cancer using PDX mouse model

Abstract PURPOSE: Despite the remarkable advances in cancer treatment, the incidence rate of pancreatic cancer (PCa) for decades among African Americans (AA) has been 30% to 70% higher than other racial groups in the United States. Gemcitabine (Gem) remains the first-choice treatment for PCa. It is administered as monotherapy in patients with locally advanced and metastatic PCa; but, drug resistance develops over time. Gem treatment resistance coupled with poor pharmacokinetics (short half-life) has resulted in poor treatment outcomes. The purpose of this study is to enhance the anticancer efficacy and pharmacokinetic profile of Gem through chemical modification of Gem with stearic acid by using mice bearing tumors from AA and White pancreatic cancer patients. METHODS: The Gem was modified by conjugating the 4-amino group of Gem and stearoyl acyl derivative to form 4-(N)-stearoyl-gemcitabine (4NSG). 4NSG was developed into solid lipid nanoparticles (4NSG-SLN) and characterized using nuclear magnetic resonance (NMR), micro-elemental analysis, and high liquid chromatography (HPLC). Patient-derived primary pancreatic cancer cells from AA (PPCL-135 and PPCL-192) and Whites (PPCL-46 and PPCL-68) were used to evaluate the anticancer activity of Gem and 4NSG-SLN. We performed antitumor efficacy testing of Gem and 4NSG-SLN in a patient-derived xenograft (PDX) mouse model harboring pancreatic tumors from the same AA and White patients. RESULTS: The H-NMR spectra displayed an amide bond at 11ppm, confirming the conjugated bond between the 4-amino group of Gem and stearoyl derivative. The half-maximal inhibitory concentration (IC50 =12 ± 2.1µM) of 4NSG-SLN-treated PPCL-46 and (IC50 =22 ± 2.6µM) of 4NSG-SLN-treated PPCL-68 cultures were significantly higher than that of Gem treated PPCL-46 and PPCL-68 cultures respectively (IC50 = 56 ± 2.4 µM, IC50 = 57 ± 1.5 µM, p-value: p&amp;lt;0.001). We found a similar trend of higher growth inhibition of 4NSG-SLN treated PPCL-135 and PPCL-192 cultures (IC50 = 11± 1.3 µM; IC50 = 9 ± 1.1 µM) respectively, P&amp;lt; 0.001 (4NSG-SLN-PPCL-135 vs. Gem-PPCL-135) and P&amp;lt;0.001 (4NSG-SLN-PPCL-192-2 vs Gem-PPCL-192) compared with Gem treated PPCL-135 and PPCL-192 cultures (IC50 = 56 ± 1.8 µM; IC50 =57 ± 2.4 µM) respectively. The anticancer activity of 4NSG-SLN showed an enhanced efficacy in PPCL-46, PPCL-68, PPCL-135, and PPCL-192 treated cultures compared with their corresponding Gem treatments. In vivo studies, 4NSG-SLN treated mice bearing AA tumors showed significant tumor growth inhibition compared with GemHCl treated mice bearing AA tumors. A similar pattern was observed where 4NSG-SLN treated mice bearing White tumors showed significant tumor growth inhibition than GemHCl treated mice bearing White tumors. CONCLUSION: This study reveals that 4NSG-SLN may be a novel approach to significantly enhance the therapeutic efficacy of Gem in the treatment of pancreatic cancer. Citation Format: Andriana Inkoom, Edward Agyare, Nkafu Bechem Ndemazie, Taylor Smith, Bo Han, Jose Trevino Trevino. Optimizing Gemcitabine efficacy through chemical modification for the treatment of pancreatic cancer using PDX mouse model [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 1746.

Smart Nanoparticles as Advanced Anti-Akt Kinase Delivery Systems for Pancreatic Cancer Therapy.

Pancreatic cancer is one of the deadliest cancers partly due to late diagnosis, poor drug delivery to the target site, and acquired resistance to therapy. Therefore, more effective therapies are urgently needed to improve the outcome of patients. In this work, we have tested self-assembling genetically engineered polymeric nanoparticles formed by elastin-like recombinamers (ELRs), carrying a small peptide inhibitor of the protein kinase Akt, in both PANC-1 and patient-derived pancreatic cancer cells (PDX models). Nanoparticle cell uptake was measured by flow cytometry, and subcellular localization was determined by confocal microscopy, which showed a lysosomal localization of these nanoparticles. Furthermore, metabolic activity and cell viability were significantly reduced after incubation with nanoparticles carrying the Akt inhibitor in a time- and dose-dependent fashion. Self-assembling 73 ± 3.2 nm size nanoparticles inhibited phosphorylation and consequent activation of Akt protein, blocked the NF-κB signaling pathway, and triggered caspase 3-mediated apoptosis. Furthermore, in vivo assays showed that ELR-based nanoparticles were suitable devices for drug delivery purposes with long circulating time and minimum toxicity. Hence, the use of these smart nanoparticles could lead to the development of more effective treatment options for pancreatic cancer based on the inhibition of Akt.

Abstract PO-108: Evaluation of antitumor activity of modified-gemcitabine solid-lipid nanoparticle in pancreatic pdx models

Abstract Purpose: Pancreatic cancer (PCa) is one of the most aggressive and devastating malignancies in the United States, with a high mortality rate. Despite the remarkable advances in cancer therapy, there are limited treatment options for PCa patients. Gemcitabine (Gem), a nucleoside analog, remains the preferred choice, either alone or in combination with other chemotherapeutic agents, to treat PCa. While Gem has proven to be effective against PCa in the early stages of treatment, PCa cells resistance to Gem and rapid metabolism of Gem has resulted in poor treatment outcomes. The objective of this study was to address the issues associated with Gem via chemical modification of Gem to 4NSG. This significantly reduced the metabolism and highly sensitized PCa cells to Gem. Methods: Gem was modified by linking the 4-amino group of Gem and stearoyl acyl derivative to form 4NSG and characterized using nuclear magnetic resonance (NMR) for) to determine new bond formation, micro-elemental analysis micro-elemental analysis used to ascertain the presence of elemental composition and purity, high liquid chromatography (HPLC) used to determine percent purity of 4NSG. The 4NSG was developed into solid lipid nanoparticles (4NSG-SLN), and the size was determined using a particle size analyzer. Patient-derived primary pancreatic cancer cells (CMZ and G68Ca) and MiaPaCa-2 cells were treated with free Gem and 4NSG-SLN. Percent cell viability was determined using resazurin assay and antitumor efficacy testing of Gem, and 4NSG-SLN was performed in patient-derived xenograft (PDX) mouse models bearing G68Ca PCa. Results: Analysis of the H-NMR spectra displayed an amide bond at 11ppm, confirming the conjugated bond between the 4-amino group of Gem and stearoyl derivative. The half-maximal inhibitory concentration (IC50 =11±1.3µM) of 4NSG-SLN-treated CMZ culture was significantly higher than that of Gem treated CMZ culture (IC50 = 56±2 µM, p&amp;lt;0.001). We found a similar trend of higher growth inhibition of 4NSG-SLN treated G68Ca and MiaPaCa-2 cultures (IC50 (4NSG-SLN-G68Ca) = 12±2.1 µM; IC50(Gem-MiaPaCa-2) = 27±4 µM) respectively. Where p&amp;lt; 0.001 (4NSG-SLN-G68Ca vs. Gem-G68Ca) and p&amp;lt;0.001 (4NSG-SLN-MiaPaCa-2 vs Gem-MiaPaCa-2) compared with Gem treated G68Ca and Mia-PaCa-2 cultures (IC50 (G68Ca) = 68±26 µM; IC50 (Mia-PaCa-2) =54±5 µM) respectively. Put together, the anticancer activity of 4NSG-SLN demonstrated enhanced efficacy in CMZ, G68Ca, and MiaPaCa-2 treated cultures compared with their corresponding Gem treated cultures. For antitumor efficacy testing, 4NSG-SLN demonstrated significant tumor growth inhibition in PDX models harboring G68Ca pancreatic tumors compared with Gem treated with PDX models. Immunohistostaining studies of 4NSG-SLN treated pancreatic tumors significantly reduced vascular endothelial growth factor receptor (VEGFR) expression compared with Gem treated pancreatic tumors. Conclusion: This study demonstrated that 4NSG might be a novel approach to significantly enhance the therapeutic efficacy of Gem in the treatment of PCa. Citation Format: Edward Agyare, Taylor Smith, Andriana Inkoom, Bo Han, Jose Trevino, Nkafu Bechem Ndemazie. Evaluation of antitumor activity of modified-gemcitabine solid-lipid nanoparticle in pancreatic pdx models [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2021 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2021;81(22 Suppl):Abstract nr PO-108.

High-content single-cell combinatorial indexing.

Single-cell combinatorial indexing (sci) with transposase-based library construction increases the throughput of single-cell genomics assays but produces sparse coverage in terms of usable reads per cell. We develop symmetrical strand sci (‘s3’), a uracil-based adapter switching approach that improves the rate of conversion of source DNA into viable sequencing library fragments following tagmentation. We apply this chemistry to assay chromatin accessibility (s3-ATAC) in human cortical and mouse whole brain tissues, with mouse datasets demonstrating a 6-to-13-fold improvement in usable reads per cell compared with other available methods. Application of s3 to single-cell whole genome sequencing (s3-WGS) and to whole genome plus chromatin conformation (s3-GCC) yields 148 and 14.8 fold improvements, respectively, in usable reads per cell compared with sci-DNA-seq and sci-HiC. We show that s3-WGS and s3-GCC resolve subclonal genomic alterations in patient-derived pancreatic cancer cell lines. We expect that the s3 platform will be compatible with other transposase-based techniques, including sci-MET or CUT&Tag.

Telomerase and Pluripotency Factors Jointly Regulate Stemness in Pancreatic Cancer Stem Cells.

Simple SummaryPancreatic ductal adenocarcinoma (PDAC) is an extremely lethal cancer with very limited therapeutic options. Cancer stem cells (CSCs) are essential for propagation of PDAC, but also for its metastatic activity and chemoresistance. To date, it is still unclear how cancer stem cells (CSCs) regulate their ‘stemness’ and self-renewal properties, and to what extent they share common features with normal stem cells. Telomerase regulation is a key factor in stem cell maintenance. Here, we investigate how telomerase regulation affects CSC biology in PDAC, and delineate the mechanisms by which telomerase activity and CSC properties are linked.To assess the role of telomerase activity and telomere length in pancreatic CSCs we used different CSC enrichment methods (CD133, ALDH, sphere formation) in primary patient-derived pancreatic cancer cells. We show that CSCs have higher telomerase activity and longer telomeres than bulk tumor cells. Inhibition of telomerase activity, using genetic knockdown or pharmacological inhibitor (BIBR1532), resulted in CSC marker depletion, abrogation of sphere formation in vitro and reduced tumorigenicity in vivo. Furthermore, we identify a positive feedback loop between stemness factors (NANOG, OCT3/4, SOX2, KLF4) and telomerase, which is essential for the self-renewal of CSCs. Disruption of the balance between telomerase activity and stemness factors eliminates CSCs via induction of DNA damage and apoptosis in primary patient-derived pancreatic cancer samples, opening future perspectives to avoid CSC-driven tumor relapse. In the present study, we demonstrate that telomerase regulation is critical for the “stemness” maintenance in pancreatic CSCs and examine the effects of telomerase inhibition as a potential treatment option of pancreatic cancer. This may significantly promote our understanding of PDAC tumor biology and may result in improved treatment for pancreatic cancer patients.

Abstract 555: Mitochondrial fusion exerts KRAS-dependent therapeutic synergy with gemcitabine/nab-paclitaxel in preclinical models of pancreatic cancer

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is driven by activating Kras mutations which cause a fragmented mitochondrial phenotype. We recently demonstrated that leflunomide, an FDA-approved drug for rheumatoid arthritis, could be repurposed for pancreatic cancer therapy. The mode of action is through the activation of mitochondrial fusion, which inhibits PDAC growth in a KRAS-dependent fashion. Leflunomide also has the off-target effect of inhibiting dihydroorotate dehydrogenase (DHODH), which is critical for pyrimidine biosynthesis. Gemcitabine is part of the standard of care for pancreatic cancer and also inhibits nucleoside biosynthesis. Accordingly, we hypothesized that leflunomide might synergize with gemcitabine chemotherapy to further improve outcomes. In this pre-clinical study, we attempt to provide proof-of-concept for combining leflunomide with gemcitabine/nab-paclitaxel, the current gold standard for PDAC, in order to characterize a synergistic effect and to provide evidence for potentially repurposing the drug for clinical use. Methods: We heterotopically implanted patient-derived pancreatic cancer cell lines with an activating KRAS mutations (MDA-PATC148) or WT KRAS (MDA-PATC153) into the flanks of immunocompromised (NOD-SCID) mice. The tumors were grown to 5mm in size then assigned to one of four treatment groups: (1) vehicle control, (2) gemcitabine/nab-paclitaxel only, and (3) leflunomide + gemcitabine/nab-paclitaxel. Leflunomide was dosed at 20 mg/kg and given concurrently with gemcitabine and nab-paclitaxel. Gemcitabine was administered IP 2x/week at 100 mg/kg and nab-paclitaxel given once per week by tail vein at 10 mg/kg. Results: The combination of leflunomide with gemcitabine/nab-paclitaxel synergistically impeded tumor proliferation and reduced the median tumor volume by more than 50% when compared to vehicle (102.6 vs. 294.8 mm3, ****P-value &amp;lt; 0.0001 by Mann-Whitney U test) or gemcitabine/nab-paclitaxel only group (102.6 mm3 vs. 211.7 mm3, **P-value &amp;lt; 0.01 by Mann-Whitney U test) in KRAS mutant PATC148 mice. Interestingly, the addition of leflunomide to gemcitabine/nab-paclitaxel did not further improve tumor responses in KRAS wild-type PATC153 tumors, suggesting a dependence on mutant KRAS for therapeutic efficacy of the repurposed mitochondrial fusion actiator, leflunomide. Conclusions: Leflunomide exhibits therapeutic synergy with standard-of-care gemcitabine/nab-paclitaxel in patient-derived PDAC lines that harbor KRAS mutations, but are ineffective in KRAS WT lines, suggesting that this combination might be effective not only in PDAC, but in other cancer with mutant KRAS, such as cancers of colon, lung, or bile ducts. Citation Format: Nicholas D. Nguyen, Meifang Yu, Tara N. Fujimoto, Abagail Delahoussaye, Yanqing Huang, Manuel A. Estrada, Cullen M. Taniguchi. Mitochondrial fusion exerts KRAS-dependent therapeutic synergy with gemcitabine/nab-paclitaxel in preclinical models of pancreatic cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 555.

DNA methyltransferase mediates the hypermethylation of the microRNA 34a promoter and enhances the resistance of patient-derived pancreatic cancer cells to molecular targeting agents

DNA methyltransferase (DNMT) participates in the transformation or progression of human cancers by mediating the hypermethylation of cancer suppressors. However, the regulatory role of DNMT in pancreatic cancer cells remains poorly understood. In the present study, we demonstrated that DNMT1 repressed the expression of microRNA 34a (miR-34a) and enhanced the activation of the Notch pathway by mediating the hypermethylation of the miR-34a promoter. In patients with pancreatic cancer, the expression levels of DNMT1 were negatively related with those of miR-34a. Mechanistically, knockdown of DNMT1 decreased the methylation of the miR-34a promoter and enhanced the expression of miR-34a to inhibit the activation of the Notch pathway. Downregulation of the Notch pathway via the DNMT1/miR-34a axis significantly enhanced the sensitivity of pancreatic cells to molecular targeting agents. Therefore, the results of our study suggest that downregulation of DNMT enhances the expression of miR-34a and may be a potential therapeutic target for pancreatic cancer.