Primary Pancreatic Ductal Adenocarcinoma Tumors Research Articles

Abstract C074: Influence of Cell Death and Stress on the Tumor Microenvironment and EMT in Pancreatic Ductal Adenocarcinoma Through Single Cell and Spatial Transcriptomics

Abstract Pancreatic cancer remains one of the most deadly cancers, with over 450,000 fatalities in 2022 alone. The most lethal variation of this cancer is Pancreatic Ductal Adenocarcinoma (PDAC), with a 5-year survival rate of under 10%. Combination chemotherapy has provided major advances in this disease but it remains challenging for many reasons including the immunosuppressive nature of the tumor microenvironment (TME). PDAC has a highly desmoplastic microenvironment that suppresses antitumor immunity and promotes tumor cell invasion, metastasis, and resistance through epithelial to mesenchymal transition (EMT) of cancer cells. Cancer cells can undergo various forms of cell death, notably apoptosis, necrosis, autophagy, pyroptosis, and ferroptosis. These mechanisms are triggered by different cellular stressors and shape the microenvironment. Through the use of 15 Visium spatial transcriptomics samples from publicly available primary PDAC tumors and paired single-cell RNA (scRNA) data, we aim to understand how cell stress and cell death shape the tumor microenvironment (TME) and epithelial-mesenchymal transition (EMT). By leveraging information from known pathways in literature, we will identify cellular programs related to stress, death, and EMT. We will explore intracellular correlations within single-cell data and extracellular interactions within the TME using spatial data to uncover how different forms of cell death and stress signatures influence the TME and EMT spectrum. The initial phase involves identifying the types of stress that lead to various forms of cell death intracellularly and validating these gene signatures. Subsequently, we will apply these validated signatures to the spatial data to correlate each score and search for significant overlapping programs. This will extend beyond stress and death, incorporating TME signatures corresponding to different cell types and the EMT spectrum. By understanding the signals that promote the death of various cell populations, we aim to identify combination treatments that target both ends of the EMT spectrum. Additionally, this research will provide insights into the inflammatory impact of cell death on the TME. Our approach aims to inform the development of therapies that disrupt resistance mechanisms, ultimately improving treatment outcomes for patients with PDAC. This research can potentially reveal new therapeutic targets and strategies, enhancing our ability to combat this deadly disease. Citation Format: Rahul Bansal, Izabella Zamora, Samuel Wright, Nir Hacohen, Arnav Mehta. Influence of Cell Death and Stress on the Tumor Microenvironment and EMT in Pancreatic Ductal Adenocarcinoma Through Single Cell and Spatial Transcriptomics [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 C074.

Abstract B081: In situ multi-modal characterization of pancreatic ductal adenocarcinoma reveals tumor cell identity as a defining factor of the surrounding microenvironment

Abstract Pancreatic Ductal Adenocarcinoma (PDAC) is heterogeneous with low tumor purity, prominent microenvironment and complex architecture, which preclude identification of shared tumor intrinsic biology within and across patients. We overcame these challenges by applying complementary spatial omics approaches – providing necessary resolution and context – to primary untreated PDAC tumors from 39 patients capturing 341,949 low-bulk and 531,718 single-cell spatial transcriptomes. Of these, 59,403 low-bulk profiles and 205,665 single-cells represented tumor cells. We leveraged this data to build on prior reports of tumor cell subtypes in PDAC. We identify 5 distinct malignant subtypes, excluding ductal-like and intraepithelial neoplasia cells, that span the classical-to-basal spectrum. One tumor subtype is highly proliferative and enriched for the DNA synthesis-condensation-mitosis transcriptional network suggesting this subset disproportionately contribute to tumor growth. Strikingly, the spectrum of tumor cell subtypes was present within all patients, suggesting that subtype-based treatment stratification may need refinement. We find that each subtype has its own distinct regulators and histology. The classical subtype has extensive cell intrinsic regulation, higher cell density and lower extracellular matrix content. In contrast, the basal subtype phenotype results from a combination of cell intrinsic and tumor microenvironment (TME) interactions, wherein the basal subtype has a lower cell density, is surrounded by activated stroma and dense collagen matrix, and is poised for hypoxic adaptation. We derive the composition of cellular neighborhoods providing an explanation for the complex architecture seen in PDAC. Tumor cell neighborhoods stratified based on which side of the classical-to-basal spectrum they fell. For example, cells towards the classical-side of the spectrum had more homogenous tumor neighborhoods that generally lacked intermediate and basal tumor cells. In contrast, cells towards the basal-side of the spectrum were likely to contain classical neighbors. More broadly, neighborhoods along this continuum also contained different proportions of immune and stromal cells, cell densities, collagen deposition, and TME adaptation. These observations support a model where tumor subtypes exist within disparate niches. Lastly, our atlas unifies the catalog of previously reported stromal cells (fibroblasts and stellate cells versus myofibroblastic and inflammatory cancer associated fibroblasts (CAF)) and describes a novel interferon stimulated gene (ISG) resistance signature (ISG.RS) fibroblast. We map the location of these various stromal cells relative to tumor subtypes. For example, myofibroblast CAFs and ISG.RS fibroblasts are enriched near the basal tumor subtype whereas inflammatory CAF abundance increases with distance away from tumor cells. In sum, our atlas provides a lens for stratifying tumor complexity in a cancer cell centric manner and provides a path for testing therapeutic hypothesis in the right cell subtype and context. Citation Format: Brian Rabe, Anna Lyubetskaya, Andrew Kavran, Yulong Bai, Andrew Fisher, Hannah Pliner, Alba Font-Tello, Anne Lewin, Ruifeng Hu, Alexandre P Alloy, Yelena Cheng, Chao Dai, Yunfan Fan, Constance Brett, Todd Brett, Lauren Giampapa, Soeren Stahlschmidt, Fayaz Seifuddin, Steven Vasquez Grinnell, Daniel Carrera, Carlos Rios, Tom Lila, Pradeep Kar, Abhishek Shukla, Rachael Bashford Rogers, Lara Heij, Mike Mason, Ryan Golhar, Isaac Neuhaus, Enas Abu Shah, Shivan Sivakumar, Jimena Trillo Tinoco, Benjamin J Chen, Konstantinos J Mavrakis, Eugene Drokhlyansky. In situ multi-modal characterization of pancreatic ductal adenocarcinoma reveals tumor cell identity as a defining factor of the surrounding microenvironment [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 B081.

Abstract A074: Integrated clinical and single-cell profiling analysis reveals cellular states associated with metastatic organotropism and survival in patients with pancreatic ductal adenocarcinoma

Abstract Some patients with localized pancreatic ductal adenocarcinoma (PDAC) undergo resection with curative intent, but most experience disease recurrence and succumb to their disease. Through examination of 743 PDAC patients in a single center we found that the site of first recurrence is a major predictor for survival. Patients with liver-metastatic recurrence (LIV-MR) had significantly shorter disease free and overall survival compared to those with recurrence to the lung (LUN-MR). We performed single-nucleus RNA-seq (snRNA-seq) and whole-genome sequencing (WGS) of primary PDAC with either LIV-MR or LUN-MR and found that malignant cells adopt transcriptional programs that mimicked that of their ultimate metastatic target organ. We validated this observation in independent data sets of human adult and fetal liver and lung tissues, and two additional patient cohorts who underwent molecular profiling of their actual PDAC liver and lung metastatic lesions. We reconstructed two different trajectories of malignant transformation in PDAC and found that the LIV-MR and LUN-MR are associated with these, suggesting that subsequent metastatic organotropism may be imprinted early in tumorigenesis. Furthermore, primary PDAC tumors recurring with LIV-MR compared to LUN-MR were depleted of infiltrating T cells and enriched with M2-like macrophages, highlighting potential contributions to subsequent outcomes. Taken together, this work provides evidence for pre-existing cellular adaptations that may determine metastatic organotropism in the context of cancer recurrence. These insights may be useful for prognostication and development of therapeutic strategies at the time of diagnosis of resectable PDAC. Citation Format: Morteza Chalabi Hajkarim, Michael May, Amit Dipak Amin, Jacob Jamison, Somnath Tagore, Lindsay Caprio, Zachary Walsh, Parin Shah, Sinan Abuzaid, Alissa Michel, Sun Dajiang, Winston Wong, Neha Shaikh, Priyanka Ramaradj, Basil Bakir, Michael D Kluger, John Chabot, Hanina Hibshoosh, Anil K Rustgi, Alexander Raufi, Gulam A Manji, Benjamin Izar. Integrated clinical and single-cell profiling analysis reveals cellular states associated with metastatic organotropism and survival in patients with pancreatic ductal adenocarcinoma [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 A074.

Abstract C077: Functional Dissection of the Highly Plastic Basal Cell State in Pancreatic Cancer

Abstract Pancreatic ductal adenocarcinoma (PDAC) is a treatment-refractory malignancy with a dismal prognosis. Targeting cancer cell plasticity—the capacity to adapt to cell-extrinsic pressure via non-genetic mechanisms—is a promising therapeutic strategy, yet cell states within PDAC harboring high plasticity remain poorly understood. Using genetically engineered mouse models driven by oncogenic KrasG12D and loss of p53 (“KPC” model), we profiled primary PDAC tumors using single-cell RNA sequencing and single cell ATAC sequencing. We identified a cancer cell differentiation state that co-expresses both epithelial and mesenchymal programs and has a distinct epigenetic landscape with accessible chromatin across gene sets that mark cancer cell states with divergent phenotypes, suggesting that this cell state is primed for cell state switching. Notably, this high-plasticity cell state (HPCS) signature aligns with a "basal" signature in human PDAC, which associates with poor prognosis and chemoresistance. To functionally interrogate the HPCS in PDAC progression, we developed a novel lineage-tracing and ablation system. Lineage-tracing experiments revealed that the HPCS has a robust capacity for differentiation into epithelial and mesenchymal states, indicating that the HPCS is functionally plastic and acts as a source of fixed malignant states. Strikingly, targeted elimination of the HPCS over short time periods resulted in tumor collapse, underscoring a critical role in tumor maintenance. Further, we found that the HPCS is highly dependent on KRAS activity, with acute KRAS inhibitor treatment leading to a selective depletion of the HPCS. Taken together, our data indicates that PDAC harbors a striking dependency on the HPCS and encourages therapeutic approaches aimed at eradicating this critical subset of cancer cells. Citation Format: Anupriya Singhal, Hannah C. Styers, Jonathan Rub, Zhuxuan Li, Zeynep Tarcan, Jill Hallin, Olca Basturk, Rona Yaeger, James G. Christensen, Doron Betel, Yan Yan, Elisa de Stanchina, Tuomas Tammela. Functional Dissection of the Highly Plastic Basal Cell State 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 C077.

Optimization of laser dosimetry based on patient-specific anatomical models for the ablation of pancreatic ductal adenocarcinoma tumor

Laser-induced thermotherapy has shown promising potential for the treatment of unresectable primary pancreatic ductal adenocarcinoma tumors. Nevertheless, heterogeneous tumor environment and complex thermal interaction phenomena that are established under hyperthermic conditions can lead to under/over estimation of laser thermotherapy efficacy. Using numerical modeling, this paper presents an optimized laser setting for Nd:YAG laser delivered by a bare optical fiber (300 µm in diameter) at 1064 nm working in continuous mode within a power range of 2–10 W. For the thermal analysis, patient-specific 3D models were used, consisting of tumors in different portions of the pancreas. The optimized laser power and time for ablating the tumor completely and producing thermal toxic effects on the possible residual tumor cells beyond the tumor margins were found to be 5 W for 550 s, 7 W for 550 s, and 8 W for 550 s for the pancreatic tail, body, and head tumors, respectively. Based on the results, during the laser irradiation at the optimized doses, thermal injury was not evident either in the 15 mm lateral distances from the optical fiber or in the nearby healthy organs. The present computational-based predictions are also in line with the previous ex vivo and in vivo studies, hence, they can assist in the estimation of the therapeutic outcome of laser ablation for pancreatic neoplasms prior to clinical trials.

PSGL-1 deficiency eradicates orthotopic pancreatic ductal adenocarcinoma tumors in combination with anti-PD-1 therapy and enhances T cell anti-tumor immunity

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) is highly refractory to all current immune checkpoint blockade (ICB) therapies and display limited CD8 +T cell infiltration. We previously showed that deficiency of P-selectin glycoprotein 1 (PSGL-1) promotes CD8 +T cell responses that clear chronic lymphocytic choriomeningitis virus (LCMV) Cl13 virus infection and dramatically inhibits the growth of anti-PD-1 blockade resistant melanoma. Here we evaluated if PSGL-1 regulates CD8 +T cell responses to PDAC tumors. Results: PSGL-1 −/−mice reproducibly showed T cell dependent, significant reduction of KPC.4662 primary PDAC tumors and reduced metastasis to lungs compared to control mice. Furthermore, anti-PD-1 treatment of PSGL-1 −/−mice completely obliterated tumor growth as opposed to control mice. Histology of primary tumors revealed marked increase of immune infiltration (CD45 +cells), including CD3 +T cells in PSGL-1 −/−mice. Flow cytometry and immunofluorescence staining confirmed a significant increase in tumor-infiltrating CD8 +T cells in PDAC tumors from PSGL-1 −/−mice. Importantly, PSGL-1 −/−CD8 +T cells were phenotypically and functionally less exhausted. Conclusion: PSGL-1 deficiency significantly reduced growth of primary orthotopic PDAC tumors and decreased metastases and this effect was enhanced in combination with anti-PD-1 ICB treatment. This protective effect was associated with increased immune cell infiltration in the tumors and notably, increased CD8 +T cell effector responses. PSGL-1 therefore represents a novel target for promoting immune responses to PDAC tumors and responses to PD-1 blockade. NCI Cancer Center Support Grant P30 CA030199

Abstract 3504: Evaluating the molecular determinants of PDAC racial health disparities

Abstract Pancreatic cancer is the 3rd leading cause of cancer-related deaths with a 5-year survival rate of just 11.5%. Pancreatic ductal adenocarcinoma (PDAC), accounting for over 90% of pancreatic malignancies, has the highest incidence rate, mortality rate, and shortest survival times in non-Hispanic black (defined here as African and/or African American ancestry) patients compared to other races. Due to the racial health disparities of PDAC, this study seeks to quantify the proteomic signatures associated with tumor racial origin to identify the underlying molecular pathways that could contribute to these disparities. Using mass spectrometry on primary PDAC tumor samples collected from 30 Caucasian and 12 African American patients, 183 proteins were differentially expressed between these groups. The most over-expressed protein in African American tumors was Ring Finger Protein 2 (RNF2) with a log2 fold-change greater than six. RNF2 had previously been found to regulate GATA Binding Protein 6 (GATA6) in embryonic stem cells. Additionally, GATA6 is also an important determinant in PDAC subtyping with higher GATA6 expression leading to greater chemosensitivity. To expand upon these findings, chromatin immunoprecipitation and sequencing (ChIP-seq) was performed on MIA-PaCa2 to identify binding sites for RNF2 in PDAC cells. Peaks were called with MACS2 (q <0.01) and a final peak list with an irreproducible discovery rate (IDR) of less than 0.05 across two replicates was generated. From the 1,142 replicated peaks identified, RNF2 peaks were localized over the transcription start site of 644 unique transcripts, including GATA6. Modulating RNF2 levels by exogenous expression or short hairpin RNA (shRNA) knockdown confirms that GATA6 is a target of RNF2 repression in PDAC. To further identify its downstream targets in PDAC, RNF2 knock down in MIA-PaCa2 was analyzed by RNA-seq. Differential gene expression analysis revealed RNF2 knockdown decreased expression of genes related to innate immunity, which we have previously identified as a marker of the aggressive Inflammatory PDAC subtype using proteomics. Of the 4,763 differentially expressed genes (P-adj < 0.05), 226 had RNF2 binding sites, demonstrating the scope of RNF2 regulation in MIA-PaCa2. Our work identifies differences in the underlying proteome of PDAC tumors as a function of racial origin, with overexpression of RNF2 in African American tumors potentially contributing to inflammation and subtype delineation. Because PDAC subtypes are predictive of therapeutic response, additional research is necessary to determine whether targeting RNF2 can mitigate oncogenic phenotypes that contribute to racial health disparities. Citation Format: Anthony E. Johansen-Sallee, Alexa M. Barber, Henry Law, Jose Trevino, Nicholas T. Woods. Evaluating the molecular determinants of PDAC racial health disparities [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 3504.

Modulation of myeloid and T cells in vivo by Bruton’s tyrosine kinase inhibitor ibrutinib in patients with metastatic pancreatic ductal adenocarcinoma

BackgroundIn preclinical studies of pancreatic ductal adenocarcinoma (PDAC), ibrutinib improved the antitumor efficacy of the standard of care chemotherapy. This led to a phase 1b clinical trial to determine the...

Abstract B036: The role of whole genome duplication in progression of pancreatic ductal adenocarcinoma

Abstract Introduction: Pancreatic ductal adenocarcinoma (PDA) is a lethal disease that tends to present at an advanced stage, but the evolutionary mechanisms responsible for rapid progression in PDA are poorly understood. Whole genome duplication (WGD) is a hallmark of cancer observed more frequently in late-stage tumors that predicts poorer overall patient survival across cancer types, including PDA. The genome-wide instability that occurs during WGD events can lead to gene dosage imbalances that promote tumor progression and may explain the rapid progression of aneuploidy. Although WGD has been studied extensively in advanced cancer, there is limited data on WGD in primary tumors. We hypothesize that WGD occurs frequently in primary PDA and promotes the rapid progression and high rate of relapse in early stage PDA. We leveraged scWGS to infer copy number profiles and WGD events from tumor cells to determine the rate of subclonal WGD in primary PDA tumors and understand how aneuploidy and genome instability evolve in the earliest stages of tumor progression. Methods: Single cell barcoded libraries from primary treatment naive tumors were prepared with the 10X Genomics Single Cell CNV platform and sequenced to a depth of 20-60x per tumor. Reads were processed using CellRanger DNA pipeline, demultiplexed, and analyzed with Gingko CNV. A total of 10,323 cells from 8 primary PDA tumors (range, 192 – 3146; median = 1286) were recovered, resulting in an average coverage of 0.06x per single cell (range, 0.02x – 0.14x). Results: 5/8 tumors analyzed had evidence of at least one WGD event. The WGD event was identified as a minor subclone in 3 of these 5. Copy number profiles revealed 1-6 distinct subclones in each tumor, demonstrating copy number evolution is ongoing. WGD+ tumors trend towards having higher diversity and higher percentage of genome altered. Conclusion: WGD occurs more frequently in primary PDA than previously understood, and may confer high levels of genetic instability. Citation Format: Lauren Hummel, Irene Xie, Karen Ng, Amy Zhang, Stephanie Ramotar, Anna Dodd, Grainne O'Kane, Jennifer Knox, Julie Wilson, Steven Gallinger, Faiyaz Notta. The role of whole genome duplication in progression of pancreatic ductal adenocarcinoma [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr B036.

Cancer Associated Fibroblast (CAF) Regulation of PDAC Parenchymal (CPC) and CSC Phenotypes Is Modulated by ECM Composition.

Simple SummaryHere, we demonstrate for the first time that ECM composition cooperates with CAFs to jointly regulate/modulate the highly dynamic interactions between the CPC and CSC cell lines and establish a continuum between tumor initiation and progression in primary PDAC tumors. Altogether, these findings propose a scenario in which the ECM composition and the cellular secretome of the CAFs cooperate to jointly regulate both growth and morphology of the CPC and CSC cell lines and, by modulating the highly dynamic interactions between them, establishes a continuum between tumor initiation and progression in primary PDAC tumors.Background: Pancreatic ductal adenocarcinoma (PDAC) is one of the deadliest of all cancers, having one of the lowest five-year survival rates. One of its hallmarks is a dense desmoplastic stroma consisting in the abnormal accumulation of extracellular matrix (ECM) components, especially Collagen I. This highly fibrotic stroma embeds the bulk cancer (parenchymal) cells (CPCs), cancer stem cells (CSCs) and the main producers of the stromal reaction, the Cancer Associated Fibroblasts (CAFs). Little is known about the role of the acellular ECM in the interplay of the CAFs with the different tumor cell types in determining their phenotypic plasticity and eventual cell fate. Methods: Here, we analyzed the role of ECM collagen I in modulating the effect of CAF-derived signals by incubating PDAC CPCs and CSCs grown on ECM mimicking early (low collagen I levels) and late (high collagen I levels) stage PDAC stroma with conditioned medium from primary cultured CAFs derived from patients with PDAC in a previously described three-dimensional (3D) organotypic model of PDAC. Results: We found that CAFs (1) reduced CPC growth while favoring CSC growth independently of the ECM; (2) increased the invasive capacity of only CPCs on the ECM mimicking the early tumor; and (3) favored vasculogenic mimicry (VM) especially of the CSCs on the ECM mimicking an early tumor. Conclusions: We conclude that the CAFs and acellular stromal components interact to modulate the tumor behaviors of the PDAC CPC and CSC cell types and drive metastatic progression by stimulating the phenotypic characteristics of each tumor cell type that contribute to metastasis.

Single-nucleus and spatial transcriptome profiling of pancreatic cancer identifies multicellular dynamics associated with neoadjuvant treatment.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal and treatment-refractory cancer. Molecular stratification in pancreatic cancer remains rudimentary and does not yet inform clinical management or therapeutic development. Here, we construct a high-resolution molecular landscape of the cellular subtypes and spatial communities that compose PDAC using single-nucleus RNA sequencing and whole-transcriptome digital spatial profiling (DSP) of 43 primary PDAC tumor specimens that either received neoadjuvant therapy or were treatment naive. We uncovered recurrent expression programs across malignant cells and fibroblasts, including a newly identified neural-like progenitor malignant cell program that was enriched after chemotherapy and radiotherapy and associated with poor prognosis in independent cohorts. Integrating spatial and cellular profiles revealed three multicellular communities with distinct contributions from malignant, fibroblast and immune subtypes: classical, squamoid-basaloid and treatment enriched. Our refined molecular and cellular taxonomy can provide a framework for stratification in clinical trials and serve as a roadmap for therapeutic targeting of specific cellular phenotypes and multicellular interactions.

Abstract 4069: The potent quadruplex-binding compound SOP1812 shows anti-tumor activity in patient-derived in vivo models of pancreatic cancer

Abstract The compound SOP1812, a tetra-substituted naphthalene diimide derivative has been previously disclosed to have single-digit nM anti-proliferative activity in a panel of human pancreatic ductal adenocarcinoma (PDAC) cell lines (Ahmed et al., ACS Med Chem Lett, 2020, 11, 1634-1644). It also has significant anti-tumor activity in the MIA-PACA2 xenograft model for PDAC as well as in the more demanding KPC model. We now report that this compound also shows significant anti-tumor activity in three patient-derived xenograft (PDX) models for PDAC. One is from a primary PDAC tumor at stage I and the two others from patients with stage IV PDAC (PDX tumor studies undertaken under contract by Champions Oncology). Transcriptome studies on cellular responses to SOP1812 have previously demonstrated that expression of a number of oncogenes and related signaling pathways are down-regulated by this agent. The promoter regions of these genes have an over-representation of quadruplex-containing sequences and several have also been shown by us to be also down-regulated in treated tumor xenografts. Our working hypothesis that the mechanism of action of SOP1812 in vivo involves targeting of quadruplex-containing genes, is also supported by the PDX data since these tumors have elevated levels of several quadruplex-containing genes previously identified by us in cells and in vivo. SOP1812 is bio-available at therapeutic doses and is well tolerated at these levels in these animal models. It is currently being evaluated as a candidate for clinical development by Qualigen Therapeutics Inc. Citation Format: Stephen Neidle, Ahmed Ahmed, Richard Angell, Sally Oxenford. The potent quadruplex-binding compound SOP1812 shows anti-tumor activity in patient-derived in vivo models of pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 4069.

Repurposing HDAC and mTOR inhibitors for pancreatic cancer.

e16234 Background: Pancreatic ductal adenocarcinoma (PDAC) will be the second leading cause of cancer death by 2030 through early onset dedifferentiation and metastasis that results in limited treatments. FDA-approved targeted therapies–including proteasome, mitogen-activated protein kinase (MEK), histone deacetylase (HDAC), and mammalian target of rapamycin (mTOR) inhibitors–have shown preclinical benefit but failed in clinic. However, combination targeted therapy has been largely unexplored in clinical trials. We hypothesized based on prior drug screening efforts in the lab on aggressive subtypes of pancreatic cancer that combinations of these targeted therapies could be synergistically active in preclinical PDAC models. Methods: We optimized combinations of proteasome, MEK, mTOR, and HDAC inhibitors by performing cell viability experiments in human PDAC lines in 2D and 3D cell culture conditions in vitro. Optimized drug combinations from these experiments were then trialed in two different in vivo animal models–human FG xenografts in immunocompromised mice (NSG) and spontaneous primary PDAC tumors from transgenic pancreatic-specific Kras-overexpressing, P53-deficient mice (KPC). RNA-sequencing and Seahorse metabolic assays were utilized to analyze molecular mechanisms of drug synergy. Dedifferentiation was assessed by histology of KPC tumors. Results: The HDAC inhibitor Panobinostat and mTOR inhibitor Everolimus synergistically killed human pancreatic cancer cell lines grown in 2D and 3D cell culture in vitro. Synergy was highest at low concentrations of Panobinostat. In vivo, low-dose Panobinostat and Everolimus synergistically blocked growth of human FG xenografts in NSG mice. In vivo synergy of low-dose Panobinostat and Everolimus was highest in the KPC mouse model, where only combination therapy could significantly reduce tumor growth, stall dedifferentiation, and improve survival. Mechanistically, RNA-sequencing of Panobinostat/Everolimus-treated FG cells revealed that Panobinostat increased tumor suppressor genes opposing dedifferentiation such as p21, EGR1, and CCN2. Everolimus complemented Panobinostat by decreasing expression of enzymes metabolizing acetyl groups such as fatty acid synthase, stearoyl-CoA desaturase, and sterol response element binding factors. Functionally, Everolimus’ activity to reduce oxidative consumption increased histone acetylation specifically in the presence of Panobinostat. Conclusions: A combination of FDA-approved, targeted oral therapies (low-dose Panobinostat/Everolimus) is synergistically active in preclinical models of pancreatic cancer in vitro and in vivo. mTOR inhibitors create drug synergy by specifically increasing histone acetylation in HDAC inhibitor-treated cells by reversing HDAC inhibitor de-repression of acetyl/oxidative metabolism, illustrating a novel connection between cancer epigenetics and metabolism.

HDAC4 Mediates Smoking-Induced Pancreatic Cancer Metastasis.

Cigarette smoking is an established risk factor for pancreatic ductal adenocarcinoma (PDAC). In this project, we investigated the effect of smoking and the role of histone deacetylase 4 (HDAC4) in PDAC invasion and metastasis. Cells were treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and cigarette smoke extract and the mRNA levels of HDACs were measured by real-time polymerase chain reaction. Invasion was measured using the Matrigel Invasion Assay. Syngeneic PDAC mice were treated with 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone and metastasis measured. Human PDAC primary and metastatic tissues were analyzed by immunohistochemistry. Levels of HDAC4 mRNA were increased by smoking. Smoking compounds significantly promoted invasion of cancer cells and promoted metastasis of PDAC cells to different organs, including the liver and the lung, whereas inhibition of HDAC4 prevented this effect. The effect of HDAC4 inhibition on preventing smoking-induced metastasis was greater in the liver compared with the lung. We found that HDAC4 is highly expressed in primary and metastatic PDAC tumors. We found that HDAC4 is the only HDAC induced by smoking among all HDACs analyzed. We found that smoking promotes invasion and metastasis of PDAC cells through a mechanism that involves HDAC4 and that HDAC4 is a promising target for preventing PDAC metastasis.

Spatially defined enrichment of a neuronal-like malignant phenotype in pancreatic cancer after neoadjuvant treatment.

610 Background: Pancreatic ductal adenocarcinoma (PDAC) is highly lethal and resistance to chemotherapy and radiotherapy is a major obstacle to improving clinical outcomes. Hence, there is an urgent need to elucidate the gene expression programs, spatial context, and interactions among different cell types in residual disease after neoadjuvant treatment. Methods: We optimized and applied single-nucleus RNA-seq (snRNA-seq) to 43 frozen primary PDAC tumors. Eighteen were treatment-naïve, 14 received FOLFIRINOX followed by radiotherapy with 5-FU or capecitabine (CRT), and 5 were subjected to CRT combined with losartan on protocol (CRTL). We performed unsupervised clustering of single nucleus profiles and then annotated and quantified cell subsets. Malignant and fibroblast gene expression programs were identified by consensus non-negative matrix factorization (cNMF). We mapped our cell type signatures and expression programs onto the tumor architecture using whole-transcriptome digital spatial profiling (DSP) to uncover distinct multicellular spatial neighborhoods and intercellular interactions that compose PDAC and are remodeled by neoadjuvant treatment. Results: Consistent with treatment effect, the proportion of malignant cells was significantly lower in tumors treated with neoadjuvant therapy. Within the immune compartment, CRTL was associated with a higher fraction of CD8+ T cells and Tregs compared to untreated and CRT tumors. Differential expression analysis of CD8+ T cells revealed greater effector function (e.g., IL2, CCL4, CCL5) and reduced quiescence/dysfunction markers (e.g., TIGIT, TCF7, KLF2, LEF1) associated with CRTL. We discovered expression programs across malignant and fibroblast profiles that formed a refined molecular taxonomy, including a novel neuronal-like malignant program enriched in the neoadjuvant groups and associated with the worst prognosis in independent cohorts. Ex vivo treatment of organoids derived from an untreated PDAC with FOLFIRINOX chemotherapy and radiotherapy recapitulated enrichment of the neuronal-like program. Whole-transcriptome DSP revealed three distinct multicellular neighborhoods: classical, squamoid-basaloid, and treatment-enriched. The observed enrichment in post-treatment residual disease of multiple spatially-defined receptor-ligand interactions and a neighborhood featuring colocalization of the neuronal-like malignant program, neurotropic CAF program, and CD8+ T cells may open new opportunities for therapeutic targeting in PDAC. Conclusions: Our work provides a high-resolution molecular framework for understanding the inter- and intra-tumoral heterogeneity of pancreatic cancer, spatial organization into discrete multicellular communities, and treatment-associated reprogramming as a blueprint for exploring novel therapeutic strategies tailored to residual disease.

Magnetic resonance molecular imaging of extradomain B fibronectin enables detection of pancreatic ductal adenocarcinoma metastasis

Extradomain-B Fibronectin (EDB-FN) is an oncomarker that can be visualized with magnetic resonance molecular imaging (MRMI) to detect pancreatic ductal adenocarcinoma (PDAC) metastasis. In this study, we sought to assess the expression of EDB-FN in clinical samples of PDAC and to evaluate MRMI of PDAC metastasis with an EDB-FN-specific gadolinium-based contrast agent (MT218) in an orthotopic KPC-GFP-Luc mouse model. EDB-FN expression was evaluated in PDAC tissue samples through immunohistochemistry. RNA-Seq data obtained from the GEPIA2 project was evaluated to demonstrate EDB-FN expression in large patient cohorts. FLASH-3D MRI at 3 T of the KPC-GFP-Luc metastasis model was performed following injection of MT218. Tumor enhancement in MR images was correlated to postmortem distribution of KPC-GFP-Luc tumors using fluorescent and bright-field cryo-imaging and anatomical landmarks. EDB-FN immunohistochemical staining scores of human metastatic tumor stroma, (2.17 ± 0.271), metastatic tumor parenchyma (2.08 ± 0.229), primary tumor stroma (1.61 ± 0.26), and primary tumor parenchyma (1.61 ± 0.12) were significantly (p < 0.0001) higher than normal pancreas stroma (0.14 ± 0.10) and normal pancreas parenchyma (0.14 ± 0.14). EDB-FN mRNA expression in tumors is 4.98 log2(TPM + 1) and 0.18 log2(TPM + 1) in normal tissue (p < 0.01). A mouse model of EDB-FN rich PDAC metastasis exhibited T1-weighted contrast to noise (CNR) changes of 21.80 ± 4.34 in perimetastatic regions and 8.38 ± 0.79 in metastatic regions identified through cryo-imaging, significantly higher (p < 0.05) than CNR changes found in normal liver (−6.43 ± 0.92), mesentery (2.24 ± 0.92), spleen (−3.06 ± 2.38) and intestine (1.08 ± 2.15). We conclude that EDB-FN is overexpressed in metastatic and primary PDAC tumors and MRMI with MT218 enables the detection of metastatic and perimetastatic tissues.

Phospho-Ser784-VCP Drives Resistance of Pancreatic Ductal Adenocarcinoma to Genotoxic Chemotherapies and Predicts the Chemo-Sensitizing Effect of VCP Inhibitor.

Simple SummaryDefining the mechanisms underlying the heterogeneous cancer cell response to DNA damage is important for our ability to tailor individualized strategies to improve chemotherapy efficacy. In this study, we focus on a central DNA repair regulator named VCP whose loss-of-function has been shown by prior studies to increase chemotherapy effects. However, since VCP participates in numerous cellular processes, its total expression level cannot differentiate between VCP-dependent vs. -independent tumors with specific regard to chemotherapy-induced DNA repair. Here, by examining cell lines and patient samples of pancreatic ductal adenocarcinoma (PDAC), we made two major observations. First, DNA damage-induced Ser784 phosphorylation is essential for DNA repair by VCP and drives chemo-resistance. Second, pSer784-VCP instead of total VCP protein predicts cellular reliance on VCP function during DNA repair and consequently chemo-sensitizing effects of VCP inhibition. Thus, pSer784-VCP may be a potential predictive biomarker and sensitizing target for cancer chemotherapy treatments. Pancreatic ductal adenocarcinoma (PDAC) patients have a dismal prognosis due in large part to chemotherapy resistance. However, a small subset containing defects in the DNA damage response (DDR) pathways are chemotherapy-sensitive. Identifying intrinsic and therapeutically inducible DDR defects can improve precision and efficacy of chemotherapies for PDAC. DNA repair requires dynamic reorganization of chromatin-associated proteins, which is orchestrated by the AAA+ ATPase VCP. We recently discovered that the DDR function of VCP is selectively activated by Ser784 phosphorylation. In this paper, we show that pSer784-VCP but not total VCP levels in primary PDAC tumors negatively correlate with patient survival. In PDAC cell lines, different pSer784-VCP levels are induced by genotoxic chemotherapy agents and positively correlate with genome stability and cell survival. Causal effects of pSer784-VCP on DNA repair and cell survival were confirmed using VCP knockdown and functional rescue. Importantly, DNA damage-induced pSer784-VCP rather than total VCP levels in PDAC cell lines predict their chemotherapy response and chemo-sensitizing ability of selective VCP inhibitor NMS-873. Therefore, pSer784-VCP drives genotoxic chemotherapy resistance of PDAC, and can potentially be used as a predictive biomarker as well as a sensitizing target to enhance the chemotherapy response of PDAC.

Neoadjuvant FOLFIRINOX Therapy Is Associated with Increased Effector T Cells and Reduced Suppressor Cells in Patients with Pancreatic Cancer.

FOLFIRINOX has demonstrated promising results for patients with pancreatic ductal adenocarcinoma (PDAC). Chemotherapy-induced immunogenic cell death can prime antitumor immune responses. We therefore performed high-dimensional profiling of immune cell subsets in peripheral blood to evaluate the impact of FOLFIRINOX on the immune system. Peripheral blood mononuclear cells (PBMC) were obtained from treatment-naïve (n = 20) and FOLFIRINOX-treated patients (n = 19) with primary PDAC tumors at the time of resection. PBMCs were characterized by 36 markers using mass cytometry by time of flight (CyTOF). Compared with treatment-naïve patients, FOLFIRINOX-treated patients showed distinct immune profiles, including significantly decreased inflammatory monocytes and regulatory T cells (Treg), increased Th1 cells, and decreased Th2 cells. Notably, both monocytes and Treg expressed high levels of immune suppression-associated CD39, and the total CD39+ cell population was significantly lower in FOLFIRINOX-treated patients compared with untreated patients. Cellular alterations observed in responders to FOLFIRINOX included a significantly decreased frequency of Treg, an increased frequency of total CD8 T cells, and an increased frequency of CD27-Tbet+ effector/effector memory subsets of CD4 and CD8 T cells. Our study reveals that neoadjuvant chemotherapy with FOLFIRINOX enhances effector T cells and downregulates suppressor cells. These data indicate that FOLFIRINOX neoadjuvant therapy may improve immune therapy and clinical outcome in patients with PDAC.

Genetic and immune divergence in pancreatic cancer lesions at the time of metastatic relapse compared to primary tumor.

4140 Background: Relapse of pancreatic ductal adenocarcinoma (PDA) is common even after complete resection and adjuvant therapy. Compared to the resected tumor, the biological characteristics of metastatic tumors at the time of first relapse are poorly understood. Methods: Whole-exome sequencing (WES) (250x) and bulk RNA sequencing were performed on samples from 30 patients with PDA. Paired primary tumor samples were obtained after R0 or R1 resection, and metastatic tumor samples were obtained by biopsy at the time of first relapse. 74.1% of patients had received adjuvant chemotherapy and radiation therapy, 7.4% had received adjuvant chemotherapy only, and 3.7% had received adjuvant radiation therapy only. Most common metastatic sites were liver and lung. The cohort was 60% male with a median age at diagnosis of 64 years. The vast majority of patients had stage IIA or IIB disease at diagnosis. Median disease-free survival was 481 days. Analysis used Freebayes (somatic variant calling), Kallisto (transcript quantification), Danaher et al. (cell type deconvolution), and antigen.garnish (neoantigen prediction). Results: High-quality WES and/or RNA sequencing were available for 27/30 patients. Among these were 16 pairs of primary and metastatic samples for WES and 15 paired samples for RNA sequencing. Median tumor purity was 32% (primary) and 42% (metastatic). KRAS mutations were present in 43/48 evaluable samples, with conserved KRAS mutations in 14/16 primary-metastatic pairs. Tumors were otherwise highly variable, with 13/16 patients developing oncogenic mutations in metastatic tumors that were undetected in primary tumors (BRCA1 [3/16], AKT3 [3/16], TP53 [2/16], ROS1 [2/16]). Overall, primary and metastatic tumors had similar tumor mutation burden and neoantigen production rate. However, neoantigens were highly variable at the peptide and gene level, with conservation rates of 2.73% and 11.57%, respectively, across primary-metastatic pairs. PDA transcriptomic subtype also differed across primary-metastatic pairs in all cases. Furthermore, metastatic tumors contained lower immune suppressive signal by transcripts and deconvolution (CTLA4: p = 0.0012, FOXP3: p = 0.0026, PDCD11: p = 0.012, regulatory T cells: p = 0.012), while myeloid cells were higher (CD33: p = 0.0067). Conclusions: With the exception of KRAS, metastatic PDA tumors at relapse contain new oncogenes, distinct neoantigens, and lower immune-suppressive signal compared to primary PDA tumors. These data suggest a potential clinical utility for tumor biopsies at the time of first metastatic relapse and caution against clinical decisions for relapsed, metastatic patients based solely on sequencing of the originally resected tumor.

Polycation fluorination improves intraperitoneal siRNA delivery in metastatic pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) is a growing medical problem associated with extensive metastasis and high mortality. Intraperitoneal (IP) administration of therapeutics promises to help the treatment of cancers originated from organs in the peritoneal cavity. In this study, we evaluated how physicochemical properties of self-assembled polycation/siRNA nanoparticles affect their IP delivery efficacy in an orthotopic PDAC model. We have examined the effect of covalent polycation modification with lipophobic and hydrophobic tetrafluoro-p-toluic acid (TFTA), hydrophobic cholesterol, and hydrophilic poly(ethylene glycol) respectively. The surface charge of the three different nanoparticles was also modulated by coating the surface with serum albumin. We found that positively charged fluorine-containing particles with lipophobic properties based on a mixture of positively charged polymeric AMD3100 CXCR4 antagonist (PAMD) and PAMD modified with TFTA (mPAMD-TFTA)/siRNA displayed the best cell uptake and transfection efficacy in vitro. Biodistribution evaluation of the nanoparticles in a syngeneic orthotopic PDAC model revealed that the fluorine-containing formulation also achieved the highest PDAC tumor accumulation after IP administration. With a combination of CXCR4 inhibition by PAMD and PLK1 downregulation by siRNA, the treatment with mPAMD-TFTA/siPLK1 showed significant inhibition of both primary and metastatic PDAC tumors. Overall, our study provides insights into and guides the design of the nanoparticles for improved IP delivery of siRNA in PDAC.