Joyce K. Thompson

Pleural fibrosis and malignant mesotheliomas (MM) occur after exposures to pathogenic fibers, yet the mechanisms initiating these diseases are unclear.We document priming and activation of the NLRP3 inflammasome in human mesothelial cells by asbestos and erionite that is causally related to release of IL-1β, IL-6, IL-8, and Vascular Endothelial Growth Factor (VEGF). Transcription and release of these proteins are inhibited in vitro using Anakinra, an IL-1 receptor antagonist that reduces these cytokines in a human peritoneal MM mouse xenograft model.These novel data show that asbestos-induced priming and activation of the NLRP3 inflammasome triggers an autocrine feedback loop modulated via the IL-1 receptor in mesothelial cell type targeted in pleural infection, fibrosis, and carcinogenesis.

Abstract Inflammation is a key mediator in the development of malignant mesothelioma, which has a dismal prognosis and poor therapeutic strategies. Curcumin, a naturally occurring polyphenol in turmeric, has been shown to possess anticarcinogenic properties through its anti-inflammatory effects. Inflammasomes, a component of inflammation, control the activation of caspase-1 leading to pyroptosis and processing of proinflammatory cytokines, interleukin (IL)-1β and IL-18. In the present study, we investigate the role of curcumin in pyroptotic cell death of malignant mesothelioma cells. Using in vitro models with mouse and human malignant mesothelioma cells, curcumin is shown to induce pyroptosis through activation of caspase-1 and increased release of high-mobility group box 1 (HMGB1) without processing of IL-1β and IL-18. Absence of IL-1β processing in response to curcumin-mediated caspase-1 activation is attributed to blockade of pro-IL-1β priming through inhibition of the NF-κB pathway. Furthermore, curcumin's cytotoxicity in malignant mesothelioma cells is demonstrated to be dependent on pyroptosis as inhibition of caspase-1 resulted in protection against curcumin-induced cell death. We also demonstrate that curcumin-mediated caspase-1 activation is oxidant dependent by using N-acetyl-L-cysteine (NAC) to inhibit pyroptosis. PCR array analysis using the human inflammasome template revealed that curcumin significantly downregulated levels of inflammasome-related gene expression involved in inflammation, e.g., NF-κB, toll-like receptors (TLR), and IL-1β. Our data indicate that curcumin has a double effect on malignant mesothelioma cells through induction of pyroptosis while subsequently protecting against inflammation. Cancer Prev Res; 7(3); 330–40. ©2014 AACR.

Pancreatic Cancer (PDA) is an aggressive malignancy characterized by early spread and a high mortality. Current studies suggest that a subpopulation of cells exist within tumors, cancer stem cell (CSC), which are capable of self-renewal and give rise to unique progeny which form the major neoplastic cellular component of tumors. While CSCs constitute a small cellular subpopulation within the tumor, their resistance to chemotherapy and radiation make them an important therapeutic target for eradication. Along with distinctive phenotypic properties, CSCs possess a unique metabolic plasticity allowing them to rapidly respond and adapt to environmental changes. These cells and their progeny also display a significantly altered epigenetic state with distinctive patterns of DNA methylation. Several mechanisms of cross-talk between epigenetic and metabolic pathways in PDA exist which ultimately contribute to the observed cellular plasticity and enhanced tumorigenesis. In this review we discuss various examples of this metabolic-epigenetic interplay and how it may constitute a new avenue for therapy specifically targeting CSCs in PDA.

Malignant mesothelioma (MM) is a fatal disease in dire need of therapy. The role of inflammasomes in cancer is not very well studied, however, literature supports both pro-and anti-tumorigenic effects of inflammasomes on cancer depending upon the type of cancer. Asbestos is a causative agent for MM and we have shown before that it causes inflammasome priming and activation in mesothelial cells. MM tumor cells/tissues showed decreased levels of inflammasome components like NLRP3 and caspase-1 as compared to human mesothelial cells or normal tissue counterpart of tumor. Based on our preliminary findings we hypothesized that treatment of MMs with chemotherapeutic drugs may elevate the levels of NLRP3 and caspase-1 resulting in increased cell death by pyroptosis while increasing the levels of IL-1β and other pro-inflammatory molecules. Therefore, a combined strategy of chemotherapeutic drug and IL-1R antagonist may play a beneficial role in MM therapy. To test our hypothesis we used two human MM tumor cell lines (Hmeso, H2373) and two chemotherapeutic drugs (doxorubicin, cisplatin). Through a series of experiments we showed that both chemotherapeutic drugs caused increases in NLRP3 levels, caspase-1 activation, pyroptosis and pro-inflammatory molecules released from MM cells. In vivo studies using SCID mice and Hmeso cells showed that tumors were smaller in combined treatment group of cisplatin and IL-1R antagonist (Anakinra) as compared to cisplatin alone or untreated control groups. Taken together our study suggests that chemotherapeutic drugs in combination with IL-1R antagonist may have a beneficial role in MM treatment.

Asbestos exposure is related to various diseases including asbestosis and malignant mesothelioma (MM). Among the pathogenic mechanisms proposed by which asbestos can cause diseases involving epithelial and mesothelial cells, the most widely accepted one is the generation of reactive oxygen species and/or depletion of antioxidants like glutathione. It has also been demonstrated that asbestos can induce inflammation, perhaps due to activation of inflammasomes. The oxidation state of thioredoxin was analyzed by redox Western blot analysis and ROS generation was assessed spectrophotometrically as a read-out of solubilized formazan produced by the reduction of nitrotetrazolium blue (NTB) by superoxide. Quantitative real time PCR was used to assess changes in gene transcription. Here we demonstrate that crocidolite asbestos fibers oxidize the pool of the antioxidant, Thioredoxin-1 (Trx1), which results in release of Thioredoxin Interacting Protein (TXNIP) and subsequent activation of inflammasomes in human mesothelial cells. Exposure to crocidolite asbestos resulted in the depletion of reduced Trx1 in human peritoneal mesothelial (LP9/hTERT) cells. Pretreatment with the antioxidant dehydroascorbic acid (a reactive oxygen species (ROS) scavenger) reduced the level of crocidolite asbestos-induced Trx1 oxidation as well as the depletion of reduced Trx1. Increasing Trx1 expression levels using a Trx1 over-expression vector, reduced the extent of Trx1 oxidation and generation of ROS by crocidolite asbestos, and increased cell survival. In addition, knockdown of TXNIP expression by siRNA attenuated crocidolite asbestos-induced activation of the inflammasome. Our novel findings suggest that extensive Trx1 oxidation and TXNIP dissociation may be one of the mechanisms by which crocidolite asbestos activates the inflammasome and helps in development of MM.

Despite the causal relationship established between malignant mesothelioma (MM) and asbestos exposure, the exact mechanism by which asbestos induces this neoplasm and other asbestos-related diseases is still not well understood. MM is characterized by chronic inflammation, which is believed to play an intrinsic role in the origin of this disease. We recently found that asbestos activates the nod-like receptor family member containing a pyrin domain 3 (NLRP3) inflammasome in a protracted manner, leading to an up-regulation of IL-1β and IL-18 production in human mesothelial cells. Combined with biopersistence of asbestos fibers, we hypothesize that this creates an environment of chronic IL-1β signaling in human mesothelial cells, which may promote mesothelial to fibroblastic transition (MFT) in an NLRP3-dependent manner. Using a series of experiments, we found that asbestos induces a fibroblastic transition of mesothelial cells with a gain of mesenchymal markers (vimentin and N-cadherin), whereas epithelial markers, such as E-cadherin, are down-regulated. Use of siRNA against NLRP3, recombinant IL-1β, and IL-1 receptor antagonist confirmed the role of NLRP3 inflammasome-dependent IL-1β in the process. In vivo studies using wild-type and various inflammasome component knockout mice also revealed the process of asbestos-induced mesothelial to fibroblastic transition and its amelioration in caspase-1 knockout mice. Taken together, our data are the first to suggest that asbestos induces mesothelial to fibroblastic transition in an inflammasome-dependent manner.

Journal of Cellular BiochemistryVolume 116, Issue 8 p. 1540-1552 Article Differential Susceptibility of Human Pleural and Peritoneal Mesothelial Cells to Asbestos Exposure Julie Dragon, Julie Dragon Department of Microbiology and Molecular Genetics, College of Medicine, University of Vermont, Burlington, Vermont, 05405Search for more papers by this authorJoyce Thompson, Joyce Thompson Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, Vermont, 05405Search for more papers by this authorMaximilian MacPherson, Maximilian MacPherson Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, Vermont, 05405Search for more papers by this authorArti Shukla, Corresponding Author Arti Shukla Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, Vermont, 05405 Correspondence to: Arti Shukla, Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, VT 05405. E-mail: Arti.Shukla@med.uvm.eduSearch for more papers by this author Julie Dragon, Julie Dragon Department of Microbiology and Molecular Genetics, College of Medicine, University of Vermont, Burlington, Vermont, 05405Search for more papers by this authorJoyce Thompson, Joyce Thompson Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, Vermont, 05405Search for more papers by this authorMaximilian MacPherson, Maximilian MacPherson Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, Vermont, 05405Search for more papers by this authorArti Shukla, Corresponding Author Arti Shukla Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, Vermont, 05405 Correspondence to: Arti Shukla, Department of Pathology and Laboratory Medicine, College of Medicine, University of Vermont, Burlington, VT 05405. E-mail: Arti.Shukla@med.uvm.eduSearch for more papers by this author First published: 10 March 2015 https://doi.org/10.1002/jcb.25095Citations: 18 Conflict of interest: The authors declare no competing interests. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat ABSTRACT Malignant mesothelioma (MM) is an aggressive cancer of mesothelial cells of pleural and peritoneal cavities. In 85% of cases both pleural and peritoneal MM is caused by asbestos exposure. Although both are asbestos-induced cancers, the incidence of pleural MM is significantly higher (85%) than peritoneal MM (15%). It has been proposed that carcinogenesis is a result of asbestos-induced inflammation but it is not clear what contributes to the differences observed between incidences of these two cancers. We hypothesize that the observed differences in incidences of pleural and peritoneal MM are the result of differences in the direct response of these cell types to asbestos rather than to differences mediated by the in vivo microenvironment. To test this hypothesis we characterized cellular responses to asbestos in a controlled environment. We found significantly greater changes in genome-wide expression in response to asbestos exposure in pleural mesothelial cells as compared to peritoneal mesothelial cells. In particular, a greater response in many common genes (IL-8, ATF3, CXCL2, CXCL3, IL-6, GOS2) was seen in pleural mesothelial cells as compared to peritoneal mesothelial cells. Unique genes expressed in pleural mesothelial cells were mainly pro-inflammatory (G-CSF, IL-1β, IL-1α, GREM1) and have previously been shown to be involved in development of MM. Our results are consistent with the hypothesis that differences in incidences of pleural and peritoneal MM upon exposure to asbestos are the result of differences in mesothelial cell physiology that lead to differences in the inflammatory response, which leads to cancer. J. Cell. Biochem. 116: 1540–1552, 2015. © 2015 Wiley Periodicals, Inc. Citing Literature Supporting Information Additional supporting information may be found in the online version of this article at the publisher's web-site Filename Description jcb25095-sup-0001-SuppData-S1.xls38.5 KB Supporting Information. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Volume116, Issue8August 2015Pages 1540-1552 RelatedInformation

Abstract Study of early pancreas neoplasia in humans had previously been limited by lack of available tissue. Recent work by our group on deceased donor pancreata identified pancreatic intraepithelial neoplasia (PanIN) lesions in non-diseased organs and defined a transcriptomic signature for the microenvironment of these pre-cancerous lesions. Prevalence of PanINs in healthy human tissue was higher than expected and established a novel model to expand understanding of the complex biology of these precursor lesions. Epigenetic changes in chromatin structure and the subsequent effects on gene expression are essential processes in neoplasia that have yet to be described at the single cell level in the human pancreas. We hypothesized that characterization of the epigenomic landscape in donor pancreata would identify differences in chromatin structure and gene expression between healthy human tissue and PanINs, thereby expanding prior knowledge and identifying novel targets of study in the biology of early neoplasia in the pancreas. Single nuclei from five donor pancreata (six total samples including one replicate sample from a single donor pancreas) were isolated for transposase-accessible chromatin preparation followed by high-throughput sequencing (ATAC-seq) using the 10x Genomics platform. Data analysis was conducted using CellRanger, Seurat, Signac, and AUCell to identify and label chromatin peaks in individual nuclei corresponding to acinar, ductal, and PanIN origin according to gene signatures identified by scRNA on matched pancreata (Carpenter et al 2023, Cancer Discovery). Using this method, cells corresponding to these gene signatures, including PanIN-like cells, were indeed identified in preliminary analysis. Differentially accessible regions of the genome were then analyzed for motif enrichment between these cell types. Motifs enriched in cells identified as PanINs compared to either ductal or acinar cells included several transcription factors implicated in tumorigenesis, including members of the AP-1/Fos/Jun family and multiple zinc-finger protein family members, such as the KLF. These results are similar to previously demonstrated essential factors in pancreatic neoplasia. Differentially expressed motifs between ductal and acinar cells included factors associated with differentiation, including Myod1, Ptf1a, and the TCF family of transcription factors. Together, this study demonstrates that PanINs can be identified in human pancreas tissue by ATAC-seq and that differentially expressed motifs can be identified this model, providing opportunity for further integration with transcriptomic information to elucidate detailed understanding of early neoplasia in the pancreas. Citation Format: Jamie N. Mills, Joyce Thompson, Jacqueline Morales, Padma Kadiyala, Ahmed M. Elhossiny, Howard Crawford, Eileen Carpenter, Marina Pasca di Magliano, Filip Bednar, Simone Benitz. Characterization of the epigenomic landscape of the human pancreas and early pancreatic neoplastic lesions [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 B113.

Abstract Dysregulation of epigenetic factors is a key component of tumorigenesis. BMI1, a member of the polycomb repressor complex 1 (PRC1), is an oncogenic factor studied in many types of cancer, including pancreatic ductal adenocarcinoma (PDAC). PDAC is the third most common cause of cancer-related death in the United States and investigation of the biology involved in transformation is critical for improving outcomes in this devastating disease. In PDAC, BMI1 is required for initiation of pancreatic cancer in mice and enhances in vitro growth of human and murine tumor cell lines. CRISPR-mediated knock out of BMI1 results in reduced subcutaneous and orthotopic tumor growth in mice and decreased expression of genes related to metabolism, specifically glycolysis, and cell proliferation. BMI1 has also been implicated in regulation of metabolism in other epithelial tumor types, including ovarian cancer. Given these preliminary findings, we hypothesized that loss of BMI1 in multiple human PDAC cell lines would result in altered metabolic activity, reducing the growth and tumorigenic properties of these cells. We demonstrated that BMI1 expression is higher in PDAC cell lines compared to normal pancreatic epithelial cells and normal human donor pancreatic tissue. Following CRISPR-mediated BMI1 knock out in human PDAC cell lines, we investigated the alterations to metabolism using metabolic flux analysis. Loss of BMI1 resulted in lower basal and compensatory rates of glycolysis and increased oxygen consumption rates compared to wild-type cells. Metabolomic analysis demonstrated reduction in all the enzymes involved in the glycolysis pathway with loss of BMI1. Cell growth was reduced by loss of BMI1 in vitro, and orthotopic injection into mice of human cells with BMI1 knock out resulted in decreased tumor size compared to wild-type, recapitulating these findings in vivo. Together, these data identify a role of BMI1 in promotion of tumor growth through regulation of metabolism in pancreatic cancer. Experiments are ongoing to determine the underlying mechanism of this regulation and implications on therapeutic targeting with BMI1/PRC1 complex inhibitors in conjunction with traditional chemotherapy. Citation Format: Jamie N. Mills, Dominik Awad, Heather K. Schofield, Joyce K. Thompson, Hannah Watkoske, Damien Sutton, Nicholas Nedzesky, Donovan Drouillard, Zeribe Nwosu, Carlos Espinoza, Yaqing Zhang, Annachiara Del Vecchio, Christopher J. Halbrook, Marina Pasca di Magliano, Costas A. Lyssiotis, Filip Bednar. The epigenetic factor BMI1 regulates metabolism and tumorigenesis in human pancreatic cancer cells [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 4441.

ABSTRACT Malignant mesothelioma (MM) is an aggressive cancer of the mesothelium caused by asbestos. Asbestos use has been reduced but not completely stopped. In addition, natural or man‐made disasters will continue to dislodge asbestos from old buildings into the atmosphere and as long as respirable asbestos is available, MM will continue to be a threat. Due to the long latency period of MM development, it would still take decades to eradicate this disease if asbestos was completely removed from our lives today. Therefore, there is a need for researchers and clinicians to work together to understand this deadly disease and find a solution for early diagnosis and treatment. This article focuses on developmental mechanisms as well as current therapies available for MM. J. Cell. Biochem. 115: 1–7, 2014. © 2013 Wiley Periodicals, Inc.

Malignant mesothelioma is an aggressive cancer in desperate need of treatment. We have previously shown that extracellular signaling regulated kinase 5 (ERK5) plays an important role in mesothelioma pathogenesis using ERK5 silenced human mesothelioma cells exhibiting significantly reduced tumor growth in immunocompromised mice. Here, we used a specific ERK 5 inhibitor, XMD8-92 in various in vitro and in vivo models to demonstrate that inhibition of ERK5 can slow down mesothelioma tumorigenesis. First, we show a dose dependent toxicity of XMD8-92 to 2 human mesothelioma cell lines growing as a monolayer. We also demonstrate the inhibition of ERK5 phosphorylation in various human mesothelioma cell lines by XMD8-92. We further confirmed the toxicity of XMD8-92 towards mesothelioma cell lines grown as spheroids in a 3-D model as well as in intraperitoneal (immune-competent) and intrapleural (immune-deficient) mouse models with and without chemotherapeutic drugs. To ascertain the mechanism, we explored the role of the nod-like receptor family member containing a pyrin domain 3 (NLRP3) inflammasome in the process. We found XMD8-92 attenuated naïve and chemotherapeutic-induced inflammasome priming and activation in mesothelioma cells. It can thus be concluded that ERK5 inhibition attenuates mesothelioma tumor growth and this phenomenon in part is regulated by the inflammasome.

Malignant mesothelioma (MM), lung cancers, and asbestosis are hyperproliferative diseases associated with exposures to asbestos.All have a poor prognosis; thus, the need to develop novel and effective therapies is urgent.Vandetanib (Van) (ZD6474, ZACTIMA) is a tyrosine kinase inhibitor that has shown equivocal results in clinical trials for advanced non-small cell lung cancer.However, tyrosine kinase inhibitors alone have shown no significant clinical activity in phase II trials of patients with unresectable MM.Using epithelioid (HMESO) and sarcomatoid (H2373) human MM lines, the efficacy of tumor cell killing and signaling pathways modulated by Van with and without doxorubicin (Dox) was examined.Van alone reduced total cell numbers in HMESO MM and synergistically increased the toxicity of Dox in HMESO and H2373 cells.Most importantly, we identified two novel cell survival/resistance pathways, ERK5 and cyclic AMP response element binding protein (CREB), that were inhibited by Van and Dox.After silencing of either ERK5 or CREB, significant decreases in cell numbers in the Dox-resistant sarcomatoid H2373 line were observed.Results suggest that a plethora of cell signaling pathways associated with cell survival are induced by Dox but inhibited by the addition of Van in MM.Data from our study support the combined efficacy of Van and Dox as a novel approach in the treatment of MM that is further enhanced by blocking ERK5 or CREB signaling cascades.

A 62-year-old woman with known pyruvate kinase deficiency and previous splenectomy in early adulthood was investigated for breathlessness. A chest radiograph demonstrated a right retro-cardiac mass and computed tomography/magnetic resonance imaging scans confirmed a 56 × 37 × 60 mm heterogeneous soft tissue mass in the right paravertebral region at the T9/T10 level (top and bottom). The provisional diagnosis was of a neurogenic tumour and surgical excision was planned. Her full blood count at the time was as follows: haemoglobin concentration 101 g/l, white cell count 10·76 × 109/l, platelet count 660 × 109/l and mean cell volume 105·9 fl. Bilirubin was 28 μmol/l. She had been transfusion independent. At thoracotomy the mass appeared very vascular and adherent to the ribs and vertebra without bony erosion and with no involvement of nerve roots. The mass was completely excised at thoracotomy and microscopically was composed of varying proportions of mature adipocytes mixed with haemopoietic tissue with normal maturation. Extramedullary haemopoiesis is typically (>90% of cases) found in the liver and spleen in the context of myelofibrosis or thalassaemia but can occur rarely in the paravertebral area, central nervous system, eye, lymph nodes, myocardium, lung and pleura. There have been four previous reports of extramedullary haemopoiesis affecting the paravertebral area associated with pyruvate kinase deficiency with one case leading to spinal cord compression.

Pancreatic cancer is a molecularly heterogeneous disease. Epigenetic changes and epigenetic regulatory mechanisms underlie at least some of this heterogeneity and contribute to the evolution of aggressive tumor biology in patients and the tumor's intrinsic resistance to therapy. Here we review our current understanding of epigenetic dysregulation in pancreatic cancer and how it is contributing to our efforts in early diagnosis, predictive and prognostic biomarker development and new therapeutic approaches in this deadly cancer.

<div>Abstract<p>Inflammation is a key mediator in the development of malignant mesothelioma, which has a dismal prognosis and poor therapeutic strategies. Curcumin, a naturally occurring polyphenol in turmeric, has been shown to possess anticarcinogenic properties through its anti-inflammatory effects. Inflammasomes, a component of inflammation, control the activation of caspase-1 leading to pyroptosis and processing of proinflammatory cytokines, interleukin (IL)-1β and IL-18. In the present study, we investigate the role of curcumin in pyroptotic cell death of malignant mesothelioma cells. Using <i>in vitro</i> models with mouse and human malignant mesothelioma cells, curcumin is shown to induce pyroptosis through activation of caspase-1 and increased release of high-mobility group box 1 (HMGB1) without processing of IL-1β and IL-18. Absence of IL-1β processing in response to curcumin-mediated caspase-1 activation is attributed to blockade of pro-IL-1β priming through inhibition of the NF-κB pathway. Furthermore, curcumin's cytotoxicity in malignant mesothelioma cells is demonstrated to be dependent on pyroptosis as inhibition of caspase-1 resulted in protection against curcumin-induced cell death. We also demonstrate that curcumin-mediated caspase-1 activation is oxidant dependent by using N-acetyl-L-cysteine (NAC) to inhibit pyroptosis. PCR array analysis using the human inflammasome template revealed that curcumin significantly downregulated levels of inflammasome-related gene expression involved in inflammation, e.g., NF-κB, toll-like receptors (TLR), and IL-1β. Our data indicate that curcumin has a double effect on malignant mesothelioma cells through induction of pyroptosis while subsequently protecting against inflammation. <i>Cancer Prev Res; 7(3); 330–40. ©2014 AACR</i>.</p></div>

<p>PDF file - 62K, Supplementary Figure 1. Curcumin in combination with asbestos resulted in a synergistic effect on NLRP3 and pro-IL-1beta priming in human mesothelial cells. LP9 cells pretreated with curcumin (Cur 10 microM for 24-48 h) and exposed to asbestos (5 microg/cm2 for 24-48 h) showed increased NLRP3 (A) and pro-IL-1beta (B) mRNA levels with no significant change in caspase-1 activation (C) compared to control and asbestos exposed LP9 cells.</p>

<p>PDF file - 61K, Supplementary Figure 2. Curcumin treatment did not reduce MM tumor burden. (A) Allograft model comparing gavage administered curcumin to control group. (B) Allograft model comparing IP administered curcumin to control, cisplatin only and cisplatin plus curcumin groups. (C) Xenograft model comparing gavage and IP administered curcumin to control groups.</p>

Abstract The fundamental biology of pancreatic ductal adenocarcinoma has been greatly impacted by the characterization of genetically modified mouse models that allow temporal and spatial activation of oncogenic KRAS (KRAS G12D ). The most commonly used model involves targeted insertion of a cre recombinase into the Ptf1a gene. However, this approach disrupts the Ptf1a gene, resulting in haploinsufficiency that likely affects sensitivity to oncogenic KRAS ( KRAS G12D ). The goal of this study was to determine if Ptf1a haploinsufficiency affected the acinar cell response to KRAS G12D before and after induction of pancreatic injury. We performed morphological and molecular analysis of three mouse lines that express a tamoxifen-inducible cre recombinase to activate KRAS G12D in acinar cells of the pancreas. The cre-recombinase was targeted to the acinar-specific transcription factor genes, Ptf1a and Mist1/Bhlha15 , or expressed within a BAC-derived Elastase transgene. Up to two months after tamoxifen induction of KRAS G12D , morphological changes were negligible. However, induction of pancreatic injury by cerulein resulted in stark differences in tissue morphology between lines within seven days, which were maintained for at least five weeks after injury. Ptf1a creERT pancreata showed widespread PanIN lesions and fibrosis, while the Mist1 creERT and Ela-creERT models showed reduced amounts of pre-neoplastic lesions. RNA-seq analysis prior to inducing injury suggested Ptf1a creERT and Mist1 creERT lines have unique profiles of gene expression that predict a differential response to injury. Multiplex analysis of pancreatic tissue confirmed different inflammatory responses between the lines. These findings suggest understanding the mechanisms underlying the differential response to KRAS G12D will help in further defining the intrinsic KRAS-driven mechanisms of neoplasia initiation.

<div>Abstract<p>Inflammation is a key mediator in the development of malignant mesothelioma, which has a dismal prognosis and poor therapeutic strategies. Curcumin, a naturally occurring polyphenol in turmeric, has been shown to possess anticarcinogenic properties through its anti-inflammatory effects. Inflammasomes, a component of inflammation, control the activation of caspase-1 leading to pyroptosis and processing of proinflammatory cytokines, interleukin (IL)-1β and IL-18. In the present study, we investigate the role of curcumin in pyroptotic cell death of malignant mesothelioma cells. Using <i>in vitro</i> models with mouse and human malignant mesothelioma cells, curcumin is shown to induce pyroptosis through activation of caspase-1 and increased release of high-mobility group box 1 (HMGB1) without processing of IL-1β and IL-18. Absence of IL-1β processing in response to curcumin-mediated caspase-1 activation is attributed to blockade of pro-IL-1β priming through inhibition of the NF-κB pathway. Furthermore, curcumin's cytotoxicity in malignant mesothelioma cells is demonstrated to be dependent on pyroptosis as inhibition of caspase-1 resulted in protection against curcumin-induced cell death. We also demonstrate that curcumin-mediated caspase-1 activation is oxidant dependent by using N-acetyl-L-cysteine (NAC) to inhibit pyroptosis. PCR array analysis using the human inflammasome template revealed that curcumin significantly downregulated levels of inflammasome-related gene expression involved in inflammation, e.g., NF-κB, toll-like receptors (TLR), and IL-1β. Our data indicate that curcumin has a double effect on malignant mesothelioma cells through induction of pyroptosis while subsequently protecting against inflammation. <i>Cancer Prev Res; 7(3); 330–40. ©2014 AACR</i>.</p></div>

<p>PDF file - 62K, Supplementary Figure 1. Curcumin in combination with asbestos resulted in a synergistic effect on NLRP3 and pro-IL-1beta priming in human mesothelial cells. LP9 cells pretreated with curcumin (Cur 10 microM for 24-48 h) and exposed to asbestos (5 microg/cm2 for 24-48 h) showed increased NLRP3 (A) and pro-IL-1beta (B) mRNA levels with no significant change in caspase-1 activation (C) compared to control and asbestos exposed LP9 cells.</p>

<p>PDF file - 61K, Supplementary Figure 2. Curcumin treatment did not reduce MM tumor burden. (A) Allograft model comparing gavage administered curcumin to control group. (B) Allograft model comparing IP administered curcumin to control, cisplatin only and cisplatin plus curcumin groups. (C) Xenograft model comparing gavage and IP administered curcumin to control groups.</p>

ABSTRACT H3K27M diffuse intrinsic pontine gliomas (DIPG) exhibit cellular heterogeneity comprising less-differentiated, stem-like glioma cells that resemble oligodendrocyte precursors (OPC) and more differentiated astrocyte (AC)-like cells. H3K27M DIPG stem-like cells exhibit tumor-seeding capabilities in vivo , a feature lost or greatly diminished in the more differentiated AC-like cells. In this study, we established isogenic in vitro models of DIPG that closely recapitulated the OPC-like and AC-like phenotypes of DIPG cells. Using these tools, we performed transcriptomics, metabolomics, and bioenergetic profiling to identify metabolic programs operative in the different cellular states. From this, we defined new strategies to selectively target metabolic vulnerabilities within the specific tumor populations. Namely, we showed that the AC-like cells exhibited a more mesenchymal phenotype and were thus sensitized to ferroptotic cell death. In contrast, OPC-like cells upregulated cholesterol metabolism and mitochondrial oxidative phosphorylation (OXPHOS) and were accordingly more sensitive to statins and OXPHOS inhibitors. Additionally, statins and OXPHOS inhibitors showed efficacy and extended survival in preclinical orthotopic models established with stem-like H3K27M DIPG cells. Together, this study demonstrates that cellular subtypes within DIPGs harbor distinct metabolic vulnerabilities that can be uniquely and selectively targeted for therapeutic gain.

Abstract Malignant mesothelioma (MM) is an aggressive cancer in dire need of successful treatment. We are first to demonstrate that Extracellular signal regulated kinase 5 (ERK5) plays an important role in MM pathogenesis (Clinical Cancer Research, 19, 2013). In this study we used genetically manipulated (ERK5 inhibited) MM cell lines to show that MM tumorigenesis is influenced by ERK5. In the present study we used recently discovered specific ERK5 inhibitor (XMD8-92) to show that it regulates MM tumor growth both in vitro and in vivo. First, we show that XMD8-92 inhibits ERK5 phosphorylation in 3 out of 4 MM cell lines showing constitutive activation of ERK5. Next, time and dose dependent study demonstrated significant cytotoxicity of XMD8-92 on 3 different MM cell lines. We also showed that soft agar colony forming efficiency of H2373 MM cells was significantly reduced in response to XMD8-92 treatment. Furthermore, in vivo studies using syngeneic mouse model of intraperitoneal mesothelioma showed that XMD8-92 treatment (50 mg/kg, ip, 1x daily for 3 weeks) resulted in significant decreases in tumor weights as compared to vehicle treated mice. Total and differential cell counts in peritoneal lavage fluid (PLF) were also inhibited by XMD8-92 suggesting the role of inflammation in MM tumor growth and their inhibition by ERK5 inhibitor. Cytokine profile assessment in PLF demonstrated the reduced levels of VEGF and IL-6 in XMD8-92 treated animals as compared to control animals receiving saline. FGF2, MCP-1, G-CSF, KC and RAGE levels were, however, not significantly impacted by the ERK5 inhibition. Taken together these data suggest that ERK5 inhibitor/ERK5 inhibition could be used for inhibiting the growth of MM tumors. Currently we are exploring the role of XMD8-92 alone as well as in combination with chemotherapeutic drugs for the treatment of MM using various mouse models. We are also exploring the role(s) of ERK5 inhibitor in inflammasome regulation by chemotherapeutics. This work is supported by NIEHS RO1 ES021110 and VCC/LCCRO grants. Citation Format: Arti Shukla, Joyce Thompson, Anurag Shukla, Alan Leggett, Maximilian MacPherson, Stacie Beuschel. ERK5 inhibitor XMD8-92 for malignant mesothelioma treatment: A preclinical study. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 38. doi:10.1158/1538-7445.AM2015-38

Abstract Malignant mesothelioma (MM) is a deadly cancer and inflammation plays an important role in this carcinogenesis. In about 80% of cases, asbestos is the cause of MM development. We have previously shown that asbestos-induced NLRP3 priming and activation plays a significant role in MM development. We have also demonstrated that MM cells and tumor tissues show decreased levels of NLRP3 and caspase-1 activity as compared to normal mesothelial cells or tissue. Here using various human MM lines and two chemotherapeutic agents, doxorubicin and cisplatin, we show that chemotherapeutic agents can prime and activate NLRP3 in various MM cell lines, resulting in caspase-1 activation which may help in reducing drug resistance and increase tumor toxicity. The process of NLRP3 priming/activation was in part based on reactive oxygen species generated by these chemotherapeutic agents, as N-acetyl cysteine (NAC) was able to block these responses partly but significantly. The process of NLRP3 activation by these drugs also resulted in the release of pro-inflammatory cytokine IL-1β and IL-18. Our current studies are focused on blocking IL-1 receptor by an antagonist to find out if this can attenuate the release of IL-1-related cytokines like IL-6, IL-8 and VEGF. If successful, the combination of chemotherapeutic drugs and IL-1 receptor antagonists may have potential in MM treatment. This work is supported by NIH RO1 grant and a fellowship from the Department of Pathology, UVM. Citation Format: Arti Shukla, Catherine Westbom, Joyce Thompson, Maximilian MacPherson, Stacie Beuschel. Modulation of NLRP3 by chemotherapeutics: Potential application in mesothelioma treatment. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3183. doi:10.1158/1538-7445.AM2014-3183

Abstract Malignant mesothelioma (MM) is a cancer of mesothelial cells of the pleura or peritoneum caused exclusively by asbestos exposure. It is a deadly cancer which is only diagnosed at terminal stages, possibly due to the long latency period of development and lack of biomarkers for early diagnosis. Both pleural and peritoneal mesothelial cells can develop MM in response to asbestos, however, pleural MM (85%) is more common than peritoneal MM (15%). To get an insight of this difference we first isolated exosomes from pleural (Hmeso, H2373) and peritoneal (ORT) MM cells and performed proteomic analysis. We found that 45 proteins are common and specific to MM cells derived from pleural cavity (Hmeso/H2373) while 86 proteins are specific to ORT cells lines derived from peritoneal cavity. On the other hand, the non-transformed mesothelial cell line, LP9, showed a completely different profile with 269 proteins secreted in exosomes not shared with MM models. This led us to hypothesize that mesothelial cells of two different locations may have different susceptibility to asbestos, accounting for differences observed in pleural and peritoneal MM. To test this hypothesis we exposed human primary pleural and peritoneal mesothelial cells to asbestos for 8 h, and performed massively parallel sequencing on the RNA. Gene expression analysis performed on the RNA-Seq data showed a higher magnitude of responses from pleural mesothelial cells as compared to peritoneal mesothelial cells for the same genes. In addition, at a p<0.05 and 2-fold threshold, unique genes expressed in pleural mesothelium were pro-inflammatory genes known to be involved in MM tumorigenesis. Taken together, results from our study suggest that exosomal cargo from pleural and peritoneal MM cells bear unique signatures which may be due to the different responses of pleural and peritoneal mesothelial cells to asbestos. Future directions involve assessing miRNA cargo from exosomes of pleural and peritoneal MM cells and identifying potential biomarker candidates. This work is supported by grants from NIH (RO1 ES021110), DoD (W81XWH-14-1-0199) and the University of Vermont Cancer Center/Lake Champlain Cancer Research Organization. Citation Format: Arti Shukla, Phillip Munson, Joyce K. Thompson, Julie Dragon, Maximilian B. MacPherson, Hector Peinado. Unique pleural vs peritoneal mesothelioma exosomal signature: does mesothelial cell susceptibility to asbestos matter. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3892.

Abstract Mechanisms involved in the tumorigenesis of the devastating cancer, malignant mesothelioma (MM) are poorly understood. We have recently shown that interleukin-1β (IL-1β), an inflammatory cytokine is upregulated by asbestos via the activation of the inflammasome (a molecular platform that assembles for the activation of caspase-1) in mesothelial cells. Furthermore we have demonstrated that IL-1β secretion may lead to the activation of downstream signaling cascades involved in malignant transformation of mesothelial cells. Preliminary data from our lab indicate that in addition to IL-1β, asbestos exposure upregulated the secretion of basic fibroblast growth factor (bFGF/FGF2) and tissue factor pathway inhibitor 2 ((TFPI2) a kunitz type protease inhibitor). These factors were also regulated by the inflammasome and have never before been implicated in asbestos-induced mesothelial to fibroblastic transition (MFT). Based on our preliminary data, we hypothesized that upregulation of IL-1β by asbestos-induced inflammasome activation increases FGF2 secretion and signaling. Furthermore, we hypothesize that FGF2 together with increased TFPI2 secretion induces transition of mesothelial cells to a fibroblastic phenotype that facilitates MM carcinogenesis. In the proposed study we will delineate the role of TFPI2 (siRNA) and FGF2 (pan FGFR inhibitor, BGJ398) in the process of asbestos-induced MFT. Data from this study will provide added insight into the mechanisms involved in the initiation of MM and indicate whether TFPI2 and FGF2 can serve as drugable targets for combination therapy against MM. This work is supported by NIH grant, RO1 ES021110. Citation Format: Joyce K. Thompson, Jill Miller, Maximilian B. MacPherson, Arti Shukla. The role of TFPI2 and FGF2 in asbestos-induced mesothelial to fibroblastic transition. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4046.

Abstract Malignant mesothelioma (MM) is an aggressive and devastating cancer of the pleural/peritoneal mesothelium related to asbestos exposure. MM has a low survival rate (average: less than 12 months). Despite the causal relationship between asbestos and MM development, the exact mechanism by which asbestos causes MM is still poorly understood. There is an urgent need for the identification of mechanism(s) that may help in early detection and finding new treatment targets for prevention and treatment of MM. We have recently shown that asbestos exposure of human mesothelial cells (HMCs) leads to the activation of the NLRP3 inflammasome and a concomitant increase in the secretion of the inflammasome products, IL-1β and IL-18. Other studies have shown that IL-1β promotes the stemness of colon cancer cells and an epithelial to mesenchymal transition (EMT) of corneal endothelial cells. The activation of the inflammasome by asbestos is protracted in mesothelial cells and led us to hypothesize that the inflammasome and its products play a crucial role in the tumorigenesis of MM by promoting a mesothelial to fibroblastic transition (MFT) of HMCs. Our studies using EMT PCR array revealed that asbestos exposure results in the down regulation of E-cadherin and KRT19 among others in HMCs. Western blot analysis revealed decreases in expression of the epithelial markers, E-cadherin, Claudin-1 and β-catenin in response to asbestos exposure, while levels of the transcription factors ZEB-1, Snail and Slug (drivers of EMT) were increased in response to asbestos exposure in primary human pleural mesothelial cells. The secretion of MFT cytokines/mediators, IL-1β, IL-18, IL-6, IL-8, FGF2 and TFPI2 were also upregulated after asbestos exposure. Inhibition of NLRP3 by siRNA attenuated various parameters, suggesting a role for inflammasomes in the process. In vivo studies using an intra-peritoneal model of asbestos exposure in wild type and knockout mice (NLRP3-/-, ASC-/- and caspase-1-/-) also showed that asbestos exposure causes a thickening of the parietal peritoneal mesothelium over an 8 week period and an early increase in IL-1β and IL-18 levels in the peritoneal lavage fluid. An increase in collagen deposition and expression of vimentin and alpha smooth muscle actin was also observed, indicating a shift towards a mesenchymal phenotype in the mesothelium. Our results demonstrated that caspase-1 plays a significant role in asbestos-induced peritoneal wall thickening. Taken together, our results indicate that asbestos exposure activates the NLRP3 inflammasome thereby increasing secretion of the pro-inflammatory cytokines, IL-6 and IL-8 in an IL-1β dependent manner while compromising the fibrinolytic capacity of the mesothelium through the increased expression of TFPI2 and other protease inhibitors involved in fibrin clearance (e.g. SERPINE1). This altogether leads to MFT and may eventually result in MM development. This work is supported by funding from NIEHS grant RO1 ES021110 Citation Format: Joyce K. Thompson, Maximilian B. MacPherson, Stacie L. Beuschel, Arti Shukla. Inflammasomes: fanning the flames of malignant mesothelioma initiation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3164. doi:10.1158/1538-7445.AM2015-3164

Asbestos exposure can lead to many lung and mesothelial cell diseases, including fibrosis and malignant mesothelioma. These are devastating diseases that are difficult to treat due to the long latency period and lack of predictive markers. Available literature shows that there is consensus among researchers that inflammation plays a significant role in the development of these diseases. Furthermore, there is a potential that early inflammatory signatures could be exploited as biomarkers for diagnosis and targets for treatment. This chapter reviews recent information, ranging from experimental disease models to asbestos-exposed individuals, that suggests a critical role for asbestos-induced inflammation in disease causation; this information has implications for the identification of novel predictive biomarkers and therapeutic targets to aid in early diagnosis and treatment of asbestos-associated diseases.

Abstract The five-year survival rate of pancreatic ductal adenocarcinoma (PDAC) is currently 11% [CP1] and by 2030 PDAC is expected to become the second leading cause of cancer related death. Understanding PDAC initiation and maintenance is important for the advancement of treatment. Mouse models allow us to study the mechanisms of PDAC initiation and progression. The most common genetically engineered mouse model (GEMM) of PDAC utilizes a knock-in of Cre recombinase [FB2] into the pancreatic transcription factor 1a (Ptf1a)locus to drive pancreas specific oncogenic KRAS expression. Alternative GEMMs express Cre recombinase from the Mist1/Bhlha15 locus (Mist1-CreER) or from elastase enzyme promoter-driven constructs (Ela-CreER). How these models differ in their ability to promote neoplasia is unknown. We hypothesized that differences in GEMMs may provide insight into mechanisms of PDAC initiation and progression. Here, we compare the tamoxifen-inducible Ptf1a-CreER, [FB3] Mist1-CreER and Ela-CreER models in their response to oncogenic KRAS expression. KRASG12D was induced with tamoxifen gavage 6-8 weeks postpartum and caerulein was used to induce pancreatitis. Histological analyses (H&E and IHC for amylase, cytokeratin 19, Fos and Jun) were performed on fixed tissue to assess associated phenotypic changes. Tissues from Ptf1a-CreER, Mist1-CreER and Ela-CreER mice were harvested 3 weeks after tamoxifen administration and processed for RNA sequencing. Phenotypically, in the presence of oncogenic KRAS and after pancreatitis induction, Ptf1a-CreER pancreata exhibited extensive tissue remodeling - acinar to ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia (PanIN) - with a dense fibroinflammatory response in contrast to both Mist1-CreER and Ela-CreER pancreata. Mist1-CreER and Ela-CreER pancreata had much more variable penetrance with tissues often showing little to no ADM and PanINs despite oncogenic KRAS expression and prior pancreatitis. At the gene expression level, Mist1-CreER pancreata showed fewer differentially expressed genes when compared to wild type tissue than Ptf1a-CreER under the same conditions. In total, less than 70 differentially expressed genes were common between the two models. Members of the AP-1 transcriptional complex (fos and c-jun) previously implicated in neoplastic progression were specifically increased only in Ptf1a-CreER mice.[FB4] Taken together, our histological, and transcriptional data indicates mice lacking one copy of Ptf1a (Ptf1a-CreER) undergo more dramatic histological changes after pancreatitis injury and oncogenic KRAS induction when compared to the GEMMs with both copies of Ptf1a intact. Additionally, mice in the Ptf1a-CreER group show a more marked molecular response to oncogenic KRAS induction, suggesting the absence of a single copy of the acinar fate transcription factor Ptf1a disrupted pancreatic homeostasis and poised it for transformation. Further comparison of PDAC GEMMs will yield clearer insight into mechanisms of PDAC initiation and progression. Citation Format: Joyce K. Thompson, Fatemeh Mousavi, Justine Lau, Emily Wu, Christopher Pin, Filip Bednar. Comparison of pancreatic cancer mouse models identifies potential drivers of tumor initiation and progression [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 A070.

Abstract The initial stages of pancreatic ductal adenocarcinoma (PDA) involve an oncogenic Kras-driven metaplastic transition of acinar cells to a more duct-like phenotype referred to as acinar to ductal metaplasia (ADM). The exact mechanism by which Kras reprograms acinar cells to a more duct-like cell at the genomic level is not completely understood. Developmental transcription factor (TF) networks regulate and maintain the fate of cells in other contexts and rely on appropriate chromatin context for their activity. Deletion of Bmi1, a component of the Polycomb Repressor Complex 1, completely represses Kras-driven ADM and neoplasia in genetically engineered mouse models of PDA. Thus, we hypothesized that Kras reprograms the acinar fate TF network to promote PDA in a Bmi1-dependent epigenetic context. We used a genetically engineered mouse model of early pancreatic neoplasia bearing an acinar cell-specific tamoxifen inducible Cre-recombinase under the elastase promoter (Ela-CreER) combined with the Kras G12D oncogenic allele (Kras−LSL-G12D/+). We added the conditional Bmi1 knockout allele (Bmi1−fl) and the Rosa26 tdTomato reporter to generate Ela-CreER, Kras−LSL-G12D/+, Bmi1−fl/fl, R26 tdTomato mice and their litter mate controls with and without KrasG12D and Bmi1. We activated the Cre-recombinase in 6- to 8-week-old mice with 5 daily tamoxifen gavages (4mg/day) prior to induction of acute pancreatitis by intraperitoneal caerulein administration (eight doses per day for two days) one week after the first tamoxifen dose. We used FACS to sort tdTomato+ cells one week after pancreatitis induction and analyzed RNA expression levels of 26 TFs, previously reported to play a role in pancreatic development, homeostasis and neoplasia. Expression levels of three lineage markers (amylase, elastase and cytokeratin 19) were also measured by quantitative RT-PCR. Tamoxifen gavage induced high tdTomato expression in the acinar compartment of Ela-CreER+ mice. RT-qPCR of tdTomato+ cells indicated a loss of acinar specific TFs in mice expressing mutant Kras one week after induction of pancreatitis. This is consistent with cells undergoing ADM. Genetic ablation of Bmi1 in the presence of mutant Kras restored elastase and amylase expression at the mRNA level. At the morphologic level, loss of Bmi1 also led to recovery of acinar cells from ADM and correlated with an increase in expression of three key acinar-specific fate TFs, Mist1, Hnf1a and Nr5a2. The mRNA expression levels of other acinar TFs like Pdx1 and Ptf1a/p48 did not recover with loss of Bmi1 and mutant Kras expression. Together, our data suggest that oncogenic Kras-driven ADM is controlled by changes in master TF gene regulatory networks. Bmi1 deletion leads to partial reprogramming of these networks to allow acinar cells to resist Kras-driven oncogenesis. Citation Format: Joyce K. Thompson, Jack Blumberg, Osama Alkahili, Howard C. Crawford, Marina Pasca di Magliano, Filip Bednar. Kras drives changes in acinar-specific gene regulatory networks in early pancreatic neoplasia in conjunction with Bmi1 [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr A22.

Abstract Rare acinar cells expressing Bmi1, a component of the Polycomb Repressor Complex 1 (PRC1), represent a reservoir of cells contributing to pancreatic repair during injury and stress. This is marked by acinar-ductal metaplasia (ADM), a transition of acinar cells to a more duct-like phenotype, which is seen in pancreatitis and early stages of Kras-driven pancreatic neoplasia. The mechanism behind Kras-driven ADM at the genomic level is not completely understood. Developmental transcription factor (TF) networks regulate and maintain the fate of cells and rely on appropriate chromatin context for their activity. Deletion of Bmi1 represses Kras-driven ADM and neoplasia in genetically engineered mouse models (GEMMs) of PDA. Thus, we hypothesized that Kras reprograms the acinar fate TF network to promote PDA in a Bmi1-dependent epigenetic context. We used GEMMs to study the expression of Bmi1 in the acinar compartment and its role in early pancreatic neoplasia. We combined an acinar cell-specific tamoxifen-inducible Cre-recombinase under the elastase promoter (Ela-CreER) with the Kras G12D oncogenic allele and the conditional Bmi1 knockout allele and the Rosa26 tdTomato reporter to generate Ela-CreER, KrasLSL-G12D/+, Bmi1, R26 tdTomato mice and their littermate controls. We also generated Ela-CreER, Bmi1GFP, R26tdTomato and Bmi1-CreER, R26 tdTomato mice to quantify the fraction of acinar cells expressing Bmi1. We activated the Cre-recombinase in 6- to 8-week-old Ela-CreER mice with 5 tamoxifen gavages (4mg/day) prior to induction of acute pancreatitis by intraperitoneal caerulein administration (8 hourly injections x 2 days) one week after the first tamoxifen dose. We FACS sorted tdTomato+ cells from untreated mice (0 hr) and one week (168 hr) after pancreatitis induction and analyzed RNA levels of 8 TFs, reported to play a role in pancreatic development, homeostasis, and neoplasia. Expression levels of two lineage markers (amylase and elastase) were also measured by RT-qPCR. Lineage tracing in Bmi1-CreER and Ela-CreER, Bmi1GFP mice revealed expression of Bmi1 in vast majority of acinar cells. RT-qPCR of tdTomato+ cells in the Ela-CreER models indicated a loss of acinar-specific TFs in mice expressing mutant Kras one week after induction of pancreatitis consistent with cells undergoing ADM. Genetic ablation of Bmi1 in the presence of mutant Kras restored elastase and amylase mRNA levels. At the morphologic level, loss of Bmi1 also led to recovery of acinar cells from ADM and correlated with an increase in RNA expression of three key acinar-specific fate TFs—Mist1, Hnf1a, and Nr5a2. The mRNA levels of other key acinar TFs—Pdx1 and Ptf1a/p48—did not recover with the loss of Bmi1 in the presence of KrasG12D. Together, our data suggest that Bmi1 is expressed in the majority of acinar cells and regulates oncogenic Kras-driven ADM by altering master TF gene regulatory networks. Its deletion leads to partial reprogramming of these networks to allow acinar cells to resist Kras-driven oncogenesis. Citation Format: Joyce K. Thompson, Emily Wu, Osama Alkhalili, Howard C. Crawford, Marina Pasca di Magliano, Filip Bednar. Bmi1 is widely expressed in acini and regulates Kras-driven transcription factor networks in early pancreatic neoplasia [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer: Advances in Science and Clinical Care; 2019 Sept 6-9; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2019;79(24 Suppl):Abstract nr C56.

Abstract Pancreatic cancer is the third leading cause of cancer-related death in the United States, and deeper understanding into factors that control its progression will be key to developing future treatments. Previously, we showed that Bmi1 is required for the initiation of pancreatic cancer in mice, and enhances growth of pancreatic cancer cells in vitro. However, the role of Bmi1 in established tumors and the mechanisms underlying its requirement have so far been elusive. To investigate this, we used a CRISPR/Cas9 strategy to delete BMI1 in primary cell lines derived from human pancreatic tumors as well as murine pancreatic cancer cell lines, and created clonal lines lacking BMI1 expression. We then used subcutaneous tumor growth models as well as transcriptomic and metabolomic profiling to define the role of BMI1 expression in tumor growth. BMI1 knockdown in human and mouse pancreatic cancer cells resulted in slower growth in vitro and in vivo compared to controls. Histologically, control tumors were moderately differentiated, while BMI1-/- tumors were small or histologically undetectable. BMI1-/- tumors with sufficient tumor tissue displayed prominent extracellular mucin pools, with few cancer cell clusters interspersed within mucin and dead cell debris. Interestingly, the remaining cancer cells in the BMI1-/- tumors, identified by CK19 immunostaining, still expressed phosphorylated ERK, indicating MAPK pathway activation, as expected in Kras mutant cells. Thus, BMI1 expression was required for the growth of both human and murine pancreatic cancer cells. Given this, we sought to determine the functional processes regulated by BMI1 to facilitate pancreatic cancer growth by using both transcriptomic and metabolomic comparison of WT or BMI1-/- human pancreatic cancer cells. Further analysis revealed changes in pathways with known importance in pancreatic cancer, including many related to metabolism and cell proliferation. Strikingly, the expression of every enzyme in the glycolytic pathway was downregulated in BMI1-/- cells. Loss of BMI1 also resulted in changes in pathways that control cell proliferation, and specifically the G2/M checkpoint, possibly explaining the growth defect observed in vitro and in vivo. Together, our data show that BMI1 is a key regulator of essential cellular processes for human and mouse pancreatic cancer growth, including metabolism and cell cycle progression. Further study will be required to determine the exact mechanism of BMI1 regulation of these pathways, as well as its potential as a therapeutic target in pancreatic cancer. Citation Format: Heather K. Schofield, Christopher J. Halbrook, Annachiara Del Vecchio, Donovan Drouillard, Zeribe C Nwosu, Joyce K. Thompson, Carlos Espinoza, Yaqing Zhang, Costas A Lyssiotis, Marina Pasca di Magliano, Filip Bednar. BMI1 is required for human and murine pancreatic cancer progression and controls metabolism and cell proliferation [abstract]. In: Proceedings of the AACR Virtual Special Conference on Pancreatic Cancer; 2020 Sep 29-30. Philadelphia (PA): AACR; Cancer Res 2020;80(22 Suppl):Abstract nr PO-025.