bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract C067: Mechanistic study of the superior anti-cancer properties of a first-in-class small molecule targeting PCNA
Authors: Gu Long , Li Min , Lingeman Robert , Hickey Robert J , Liu Yilun , Malkas Linda H
Published: March 2020

Proliferating cell nuclear antigen (PCNA), through its interaction with various proteins involved in DNA synthesis, cell cycle regulation, and DNA repair, plays a central role in maintaining genome stability. We previously reported a novel cancer associated PCNA isoform (dubbed caPCNA), which was predominantly expressed in a broad range of cancer cells and tumor tissues, but not significantly in non-malignant cells. We found that the caPCNA-specific antigenic site lies between L126 and Y133, a region within the interdomain connector loop of PCNA that is known to be a major binding site for many of PCNA’s interacting proteins. A cell permeable peptide harboring the L126-Y133 sequence inhibited PCNA function in cancer cells and selectively kills cancer cells and xenograft tumors. Based on these observations, we sought small molecules targeting this peptide region of PCNA as potential broad-spectrum anti-cancer agents. Our effort led to a drug candidate, AOH1996, which selectively kills a broad range of cancer cells at high nanomolar concentrations, but is not associated with significant toxicity to non-malignant cells. It also works synergistically with DNA damaging chemotherapeutic drugs, such as cisplatin and irinotecan, to selectively kill cancer cells. This compound is orally available to animals and suppresses tumor growth in a dosage form compatible to clinical applications. Importantly, it doesn’t cause significant toxicity at 2.5 times its effective dose. Mechanistically, AOH1996 competes with T3, a known PCNA ligand, for binding to PCNA. However, the mechanism by which AOH1996 exerts its effect on cancer cells may not be identical to what have been reported for the T3 analogs. In particular, we found that AOH1996 interferes with the association of PCNA and its binding proteins, leading to DNA replication stress, blockade of homologous recombination-mediated DNA repair, and induction of apoptosis in cancer cells. These findings demonstrated the potential of this compound as a novel therapeutic agent warranting clinical investigation for cancer treatment. We have started planning a phase 1 clinical study for this compound.

Citation Format: Long Gu, Min Li, Robert Lingeman, Robert J Hickey, Yilun Liu, Linda H Malkas. Mechanistic study of the superior anti-cancer properties of a first-in-class small molecule targeting PCNA [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C067. doi:10.1158/1535-7163.TARG-19-C067
Abstract C084: Crizotinib resistance mechanisms and potential therapeutic strategies to overcome the resistance in ROS1-rearranged non-small cell lung cancer
Authors: Katayama Ryohei , Gong Bo , Nishio Makoto , Fujita Naoya
Published: March 2020

ROS1 rearrangement was observed in about 1 % of NSCLC patients and in several other cancers such as cholangiocarcinoma, glioblastoma, or colorectal cancer. To date, crizotinib, an ALK/ROS1 inhibitor was approved and widely used for the treatment of ROS1 rearranged NSCLC. However, the tumor relapses within a few years due to the emergence of crizotinib resistance by the secondary mutations in ROS1 kinase domain (e.g. G2032R) or other unidentified mechanisms. In this study, we explored crizotinib resistance mechanisms using ENU mutagenesis screening and the analysis of crizotinib resistant patient’s pleural effusion. We found several secondary mutations in ROS1 kinase domain including previously reported mutations. From a crizotinib resistant patient’s specimen, no secondary mutation in ROS1 was observed. By establishing a cell line from the specimen, we performed kinase inhibitor screening combined with crizotinib, and found that a combination of crizotinib with dasatinib or a few other tyrosine kinase inhibitors can suppress the growth. These results suggested that a bypass pathway mediated resistance through Src family kinase conferred resistance in this crizotinib resistant case. For overcoming the secondary ROS1 mutations mediated crizotinib resistance, we characterized several ROS1 inhibitors including a new selective ROS1/NTRK inhibitor DS-6051b in preclinical models of ROS1-rarranged cancers by newly establishing cell lines from TKI naïve ROS1 fusion positive NSCLC patients, or developing resistant mutation harboring HCC78 (SCL34A2-ROS1) or Ba/F3-CD74-ROS1 cell line models. Using these preclinical models, we demonstrated that DS-6051b induced dramatic growth inhibition of both wild type and G2032R mutant ROS1–rearranged cancers in vitro and in vivo, suggesting that DS-6051b might be effective in treating wildtype and crizotinib resistant mutant ROS1-rearranged lung cancer. This study revealed that potential resistance mechanisms in ROS1-rearranged NSCLC are similar to those identified in ALK rearranged NSCLC, and next-generation ROS1 inhibitor provides future promising therapeutic strategies to overcome the secondary mutation mediated resistance.

Citation Format: Ryohei Katayama, Bo Gong, Makoto Nishio, Naoya Fujita. Crizotinib resistance mechanisms and potential therapeutic strategies to overcome the resistance in ROS1-rearranged non-small cell lung cancer [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C084. doi:10.1158/1535-7163.TARG-19-C084
Abstract C126: A new therapeutic strategy for cancers with transcription elongation defects
Authors: Muhammad Belal
Published: March 2020

Increasing evidence suggest that the tumorigenic process is driven by various forms of transcriptional and epigenetic defects beyond somatic mutations. We recently described a novel non-genomic phenomenon associated with tumorigenesis that we call Transcription Elongation Defects (TEdeff), which is characterized by high levels of truncated transcripts, intron retention, and loss of gene body exon expression. The TEdeff phenotype exists in more than 20% of all human tumors and is linked to immunotherapy resistance (Modur et al, 2018). The aim of this study was to find a new therapeutic strategy for these types of tumors by targeting cellular pathways that are essential for TEdeff tumor survival. Computational analyses of RNA-sequencing data indicate that TEdeff tumors have extensive overexpression of the cytoplasmic protein homeostasis machinery, including several components of autophagy and proteasome degradation pathways. This strongly suggests that these cells may have increased dependency on cellular protein quality control due to elevated proteotoxic stress. Furthermore, we experimentally show that TEdeff cancer cell lines have a profound accumulation of protein aggregates (aggresomes) that appear to be associated with autophagy for clearance. Importantly, TEdeff cells, but not control cells with proficient transcription elongation (TEprof), are highly sensitive to autophagy inhibitors like chloroquine (CQ), but not proteasome inhibitors, which indicates their increased dependency on autophagy for survival. We also show evidence that the proteasomal degradation system may be defective in TEdeff cells, further supporting the hypothesis that these cells are dependent on alternative protein quality control mechanisms. Mechanistically, we propose that blocking the autophagy pathway maximizes proteotoxic stress, due to the accumulation of toxic aggresomes and proteins generated from defective transcripts, leading to a strong synthetic lethality in TEdeff cells. Therefore, autophagy inhibition may be a potent treatment strategy for TEdeff tumors, which are inherently resistant to current immunotherapies.

Citation Format: Belal Muhammad. A new therapeutic strategy for cancers with transcription elongation defects [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr C126. doi:10.1158/1535-7163.TARG-19-C126
Abstract LB-A02: AMPK activation by metformin promotes survival of dormant ER+ breast cancer cells
Authors: Hampsch Riley , Traphagen Nicole , Wells Jason , McCleery Charlotte , Fields Jennifer , Shee Kevin , Dillon Lloye , Pooler Darcy , Lewis Lionel , Demidenko Eugene , Huang Yina , Marotti Jonathan , Kinlaw William , Miller Todd Wayne
Published: March 2020

Despite adjuvant anti-estrogen therapy for patients with ER+ breast cancer, clinically dormant residual disease can persist for years and eventually cause tumor recurrence. Mimicking the aromatase inhibitor-induced depletion of estrogen levels used to treat patients, we developed clinically relevant xenograft models of dormancy induced by estrogen withdrawal. Dormant breast cancer cells exhibited upregulated AMPK levels and activity. Metformin is an AMPK-activating drug approved to treat diabetes that is being tested in many ongoing clinical trials for treatment of cancer. We found that metformin promoted survival of dormant ER+ breast cancer cells through increased mitochondrial respiration driven by fatty acid oxidation. Pharmacologic or genetic inhibition of AMPK or fatty acid oxidation promoted clearance of dormant residual disease, while dietary fat increased tumor cell survival. These findings indicate that AMPK has context-dependent effects in cancer, caution against the widespread use of an AMPK activator across disease settings, and warrant the development of cancer therapeutics targeting fat metabolism.

Citation Format: Riley Hampsch, Nicole Traphagen, Jason Wells, Charlotte McCleery, Jennifer Fields, Kevin Shee, Lloye Dillon, Darcy Pooler, Lionel Lewis, Eugene Demidenko, Yina Huang, Jonathan Marotti, William Kinlaw, Todd Wayne Miller. AMPK activation by metformin promotes survival of dormant ER+ breast cancer cells [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A02. doi:10.1158/1535-7163.TARG-19-LB-A02
Abstract LB-A17: Discovery and characterization of a novel small molecule antagonist of PD-L1
Authors: Ying Haiyan , Yang Fei , Zhang Mingming , Zhang Yongxian , Zhao Yuan , Chen Xin , Zhang Lei , Yu Hongping , Chen Zhui , Xu Yao-chang
Published: March 2020

Introduction: Immunotherapy has revolutionized cancer treatment in the last decade. Several monoclonal PD-1 and PD-L1 antibodies have been approved for various cancers. Unlike antibodies, small molecule inhibitors may offer potential advantages in oral dosing, adjustable control of drug exposure, improved accessibility to tumor and many other aspects. Therefore, small molecule antagonist which can efficiently block interaction of PD-1 and PD-L1 may render a novel alternative treatment approach with potentially improved clinical benefits. Using advanced computation-aided structural analysis and medicinal chemistry design, we have discovered a novel and orally available small molecule PD-L1 antagonist ABSK-041, demonstrating robust T-cell activating ability and strong anti-tumor activity in various preclinical models. Method: ABSK-041’s ability to block PD-1-PD-L1 interaction was evaluated by HTRF assay and cellular luciferase reporter assay. Its biological activity was evaluated in vitro in MLR and in vivo in syngeneic tumor models. Results: ABSK-041 strongly inhibits PD-1-PD-L1 interaction with IC50<1 nM in vitro and very potently rescues PD-L1-induced suppression of T cell activation signaling in cells. It also efficiently rescues cytokine production of PD-L1 suppressed CD8+ T cell to a level comparable with PD-L1 antibody. In in vivo studies, oral administration of ABSK-041 strongly inhibits tumor growth to a extent similar to therapeutic anti-PD-L1 antibody. DMPK and safety profiling demonstrate excellent drug-like properties of ABSK-041. Conclusion: ABSK-041, presented here by Abbisko Therapeutics, is a highly potent and orally available small molecule PD-L1 antagonist. Its superior profile supports fast-track preclinical development.

Citation Format: Haiyan Ying, Fei Yang, Mingming Zhang, Yongxian Zhang, Yuan Zhao, Xin Chen, Lei Zhang, Hongping Yu, Zhui Chen, Yao-chang Xu. Discovery and characterization of a novel small molecule antagonist of PD-L1 [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-A17. doi:10.1158/1535-7163.TARG-19-LB-A17
Abstract LB-B01: Investigation of immuno-oncology drugs with humanized mouse models: a panel of technologies to evaluate different pharmacological approaches
Authors: Mignard Caroline , Duchamp Olivier , Caradec Josselin , Bichat Francis , France Damien , Viviani Fabrice , Mirjolet Jean François
Published: March 2020

Immunotherapy is one of the most exciting recent breakthroughs in the field of cancer treatment. Different approaches are developed such as cancer vaccines, adoptive cellular immunotherapy or immune checkpoint blockade, and a number have been regulatory approved or are currently investigated in clinic. Effective immunity against cancer involves complex interaction between the tumor, the host and the environment. The assessment of cancer immunotherapy approaches in preclinical settings requires the use of appropriate animal models that sufficiently reflect the physiological situation in humans and that must be chosen carefully to address specific mechanisms of action. In light of evaluating the therapeutic potential of different immunomodulatory agents in mice xenografted with cancer cell lines or patient-derived xenografts, we developed multiple humanization strategies on different immuno-deficient mouse strains. Either human PBMCs, Hematopoietic Stem Cells (HSCs), or specific human immune cells such as Dendritic Cells (DCs), T cells, subset of T cells (e.g. gamma9 delta2 T cells) and NK cells were used, but also the combinations of immune subpopulations such as the co-transfer of autologous T cells and DCs. We will highlight some results of our therapeutic efficacy investigation and evaluation of novel immuno-oncology therapies eg. adoptive cell therapy, bispecific T-cell engager antibody, and vaccine immunomodulatory agents in such humanized mouse models, assessing survival, tumor growth and effects on tumors and immune cells by flow cytometry and immunohistochemistry analyses. All these different humanized models can permit to evidence significant in vivo antitumor efficacy of immunomodulatory agents and might help to increase the success rate in clinical trials.

Citation Format: Caroline Mignard, Olivier Duchamp, Josselin Caradec, Francis Bichat, Damien France, Fabrice Viviani, Jean François Mirjolet. Investigation of immuno-oncology drugs with humanized mouse models: a panel of technologies to evaluate different pharmacological approaches [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B01. doi:10.1158/1535-7163.TARG-19-LB-B01
Abstract LB-B15: New causal drivers of estrogen signaling revealed by dense time series of nascent RNA transcription
Authors: Langendorf Ryan , Basken Joel , Lai Maria , Azofeifa Joseph
Published: March 2020

Mechanistic understanding of Estrogen Receptor (ER) signaling will lead to new therapeutic targets for ER positive breast cancer. To this end, Arpeggio performed nascent RNA sequencing following estradiol (E2) treatment in MCF7 breast cancer cells every 15 minutes for 6 hours. To date, our study represents the densest time series of estrogen signaling ever reported. Arpeggio noted three transcriptional waves following E2 treatment: peaks at 30 minutes, 2.5 hours and 5 hours respectively. We noted that the early response was enriched for canonical ER signaling, however mid and late response were enriched for metabolic processes. Because our study was statistically powered with 24 time points, we could use delay-coordinate embedding to look for causal gene interactions. We implicated NME2 as the causal driver of the 5-hour transcriptional wave. Indeed, NME2 and Estrogen Receptor (ER) have been shown to co-localize where NME2 blocks ER effect. Our study represents a new methodology for target discovery in hormone driven cancers.

Citation Format: Ryan Langendorf, Joel Basken, Maria Lai, Joseph Azofeifa. New causal drivers of estrogen signaling revealed by dense time series of nascent RNA transcription [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B15. doi:10.1158/1535-7163.TARG-19-LB-B15
Abstract PL02-03: The zinc-finger degrome
Authors: Sievers Quinlan L. , Petzold Georg , Bunker Richard D. , Renneville Aline , Liddicoat Brian , Abdulrahman Wassim , Mikkelsen Tarjei , Ebert Benjamin L. , Thoma Nicolas H.
Published: March 2020

The small molecule drugs thalidomide, lenalidomide, and pomalidomide induce the ubiquitination and proteasomal degradation of Ikaros (IKZF1) and Aiolos (IKZF3) by mediating their interaction with Cereblon (CRBN), the substrate receptor of the CRL4CRBN ubiquitin ligase. Here we screened the human Cys2-His2 (C2H2) zinc finger (ZF) proteome for degradation by CRL4CRBN in the presence of thalidomide analogues, identifying 11 ZF targets. Structural and functional characterization of the C2H2 zinc finger degron demonstrates how diverse ZF domains bind the drug-CRBN interface. Computational ZF docking, in conjunction with biochemical analysis, predicts that at least 50 zinc-fingers bind the drug-CRBN complex in vitro, a larger number than previously anticipated. These results provide strategies to degrade other zinc finger transcription factors.

Citation Format: Quinlan L. Sievers, Georg Petzold, Richard D. Bunker, Aline Renneville, Brian Liddicoat, Wassim Abdulrahman, Tarjei Mikkelsen, Benjamin L. Ebert, Nicolas H. Thoma. The zinc-finger degrome [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr PL02-03. doi:10.1158/1535-7163.TARG-19-PL02-03
Abstract A03: All the roads bring to Rome: How acetyl-CoA metabolism supports multistep pancreatic carcinogenesis
Authors: Carrer Alessandro , Trefely Sophie , Zhao Steven , Campbell Sydney , Sela Yogev , Sidoli Simone , Garcia Benjamin A. , Snyder Nathaniel W. , Stanger Ben Z. , Wellen Kathryn E.
Published: March 2020

Mutant KRAS is thought to initiate pancreatic tumorigenesis, orchestrating a program that leads to cell de-differentiation, proliferation, and symbiotic cooperation with neighboring cells, enabling the cancer cells to thrive in a particularly harsh microenvironment. Recent studies have highlighted the role of metabolites in regulating the epigenome. Although oncogenic KRAS is known to reprogram cellular metabolism, the role of metabolic control of the epigenome in pancreatic tumorigenesis is poorly understood. We showed that expression of KRASG12D in mouse pancreas promotes elevated histone acetylation levels in pancreatic acinar cells, and that this precedes tumor development. We hypothesized that augmented acetyl-CoA metabolism may play a role in facilitating pancreatic tumorigenesis. To test this, we generated mice deficient for Acly (acetyl-CoA producing enzyme) in pancreas (Pdx1-Cre; Aclyf/f mice). In the context of KRASG12D expression, ACLY deficiency reduces histone acetylation levels in pancreatic acinar cells and impairs formation of neoplastic lesions. ACLY deficiency also impairs pancreatitis-induced tumor development. In testing roles for acetyl-CoA-dependent processes in ADM, we found that targeting either histone acetylation by BET inhibition or cholesterol synthesis with statins suppressed tumor onset. In vivo, response to BET and cholesterol synthesis blockade is associated with recruitment of CD8+ T-cells. The findings indicate that ACLY-dependent metabolic and epigenetic remodeling promote tumor development and point to the potential to target acetyl-CoA metabolism for pancreatic cancer. Potential role of tumor-infiltrating leukocytic cells in modulating acetyl-CoA metabolism in vivo, cancer cell-autonomous mechanisms for acetyl-CoA levels regulation, as well as consequences for therapeutic targeting and dietary interventions, will be discussed.

Citation Format: Alessandro Carrer, Sophie Trefely, Steven Zhao, Sydney Campbell, Yogev Sela, Simone Sidoli, Benjamin A. Garcia, Nathaniel W. Snyder, Ben Z. Stanger, Kathryn E. Wellen. All the roads bring to Rome: How acetyl-CoA metabolism supports multistep pancreatic carcinogenesis [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 A03.
Abstract A06: Elucidation of the extracellular adenosine pathway in metastatic pancreatic cancer
Authors: Chiarella Anna M. , Rustgi Rustgi K.
Published: March 2020

Pancreatic cancer is a deadly disease that is expected to kill over 45,000 people in the United States this year alone. More than 80% of pancreatic cancer patients begin treatment after the disease has already metastasized. We recently published on an autochthonous mouse model of pancreatic cancer (Pdx1-cre; LSL-KRASG12D/+; LSL-Trp53R172H; Rosa26LSL-YFP herein KPCY), whereby we studied the molecular consequences of losing the tumor suppressor P120-CATENIN (encoded by the gene Ctnnd1). We demonstrated that pancreas-specific Ctnnd1 heterozygous deletion (herein P120-het) in KPCY mice dramatically increased metastatic potential relative to KPCY control animals. Upon further investigation with co-immunofluorescence, we found that the metastatic lesions were largely devoid of any P120 protein. The metastatic cells with complete P120 loss appear more mesenchymal, a phenotype associated with tumor cells undergoing epithelial-to-mesenchymal transition (EMT) and with increased metastatic potential. To determine the underlying processes downstream of P120 loss that may be driving EMT and metastasis, we performed RNA-sequencing on P120-WT (nonmetastatic) and P120-null (highly metastatic) tumor cells. Compared to the P120-WT tumor cells, the P120-null tumor cells revealed significant upregulation of several genes involved in extracellular adenosine signaling, Enpp3, Nt5e, and Adora2B. More recently, we confirmed upregulation of ENPP3, NT5E, and ADORA2B at the protein level, compared to healthy pancreas tissue, using immunohistochemistry (IHC). In the highly metastatic cells, Enpp3 (Ectonucleotide pyrophosphatase/phosphodiesterase-3) expression was increased 57-fold. ENPP3 is a type II glycoprotein involved in binding to and converting extracellular ATP into AMP. Nt5e/CD73 was increased 9-fold and also has ecto-nucleotidase activity to convert AMP to adenosine. Increases in extracellular adenosine can activate ADORA2B (a cell membrane receptor that binds adenosine and initiates downstream intracellular signaling), which was activated 4-fold. While this pathway has been shown to be important and therapeutically relevant in other cancers, it has yet to be fully characterized in the context of pancreatic cancer. In summary, using a model with increased metastatic capability, we have unraveled a novel panel of upregulated and functionally related genes that are related to increasing and sensing extracellular adenosine levels. Our recent data suggest that extracellular adenosine signaling may be a previously undiscovered mechanism for cells to acquire metastatic ability.

Citation Format: Anna M. Chiarella, Rustgi K. Rustgi. Elucidation of the extracellular adenosine pathway in metastatic pancreatic cancer [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 A06.
Abstract A12: The establishment, maintenance, and maladaptive role of epigenetic memory in mediating pancreatic tumorigenesis
Authors: Falvo David J. , Chandwani Rohit
Published: March 2020

Cellular decision-making is highly dependent on cues present in the environment. As such, biologic systems have evolved efficient ways to respond to salient and often transient stimuli by encoding and transducing relevant extracellular information to chromatin. Additionally, accumulating evidence suggests that cells are capable of remembering previously experienced stimuli, allowing them to adjust their behaviors appropriately when confronted in the future with a similar or different stimulus. However, the ability of cells to respond to a transient stimulus and to encode a persistent record of that experience following its resolution has yet to be explored comprehensively in a preneoplastic context. Here we determine the lasting effect in the pancreas of an acinar-to-ductal metaplasia (ADM) event, to elucidate the impact of prior inflammation on tissue function and subsequent tumorigenesis. In a lineage-traced model of acinar cell dedifferentiation, we demonstrate that following ADM, histologically normal acinar cells exhibit broad gains in chromatin accessibility that persist well after exposure to the initial ADM-inducing stimulus. We show that the effect on chromatin is not reflected in the transcriptional state of acinar cells. Moreover, we employ extensive flow-cytometry based analysis of immune cell and niche acinar populations to show there is no long-term cell-extrinsic population, nor a significant expansion of either the Dclk1+ or Nk1r+ acinar populations. Finally, we employ single-cell RNA-seq analyses to define the alterations to epithelial cells and the associated microenvironment long after regeneration following an episode of pancreatitis. These data suggest that inflammation-exposed acinar cells are capable of establishing an altered, persistently accessible chromatin state that lasts long after the initial lineage destabilizing event. Currently we are defining if, following resolution of pancreatitis, activation of mutant Kras will be sufficient to induce premalignant transformation within the acinar compartment that manifests as pancreatic intraepithelial neoplasia (PanIN)—the most common precursor lesion to pancreatic ductal adenocarcinoma (PDAC). Understanding how this postinflammatory chromatin state synergizes with oncogenic stress will further explain why inflammation is a hallmark risk factor for cancer development. Together, our studies will characterize the establishment, maintenance, and maladaptive role of epigenetic memory in the development of pancreatic cancer.

Citation Format: David J. Falvo, Rohit Chandwani. The establishment, maintenance, and maladaptive role of epigenetic memory in mediating pancreatic tumorigenesis [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 A12.
Abstract A15: Elucidating the role of p53 in the cellular origins of pancreatic cancer development
Authors: Flowers Brittany M. , Xu Hang , Hanson Kathryn , Curtis Christina , Vogel Hannes , Wood Laura D. , Attardi Laura D.
Published: March 2020

The tumor suppressor gene TP53 encodes a transcriptional activator critical for suppressing pancreatic ductal adenocarcinoma (PDAC). TP53 mutations, which are most frequently missense mutations in the DNA binding domain, result in both loss of wild-type p53 function and gain of novel, oncogenic functions. Mouse models of PDAC relying on oncogenic Kras and p53 mutation have suggested that either pancreatic acinar or ductal cells, the two major epithelial cell types in the pancreas, may be the cell of origin for PDAC, although there is very limited understanding of the molecular pathways through which p53 mutations contribute to PDAC driven by oncogenic Kras. Moreover, the cellular functions of p53 involved in tumor suppression remain enigmatic. To set up a platform for deconstructing p53 loss-of-function (LOF) and gain-of-function (GOF) pathways, we have generated mouse models to study p53 function in adult mice, mimicking the context of human cancer, using tamoxifen-inducible Cre recombinases to activate Kras (KrasLSL-G12D) in adult mouse pancreatic acinar (Ptf1aCreER) and ductal (Sox9CreER) compartments, in the backdrop of different p53 alleles. To investigate p53 LOF, we generated mouse cohorts with wild-type p53 expression or conditional p53 knockout (p53fl/fl). To examine p53 GOF, we generated mouse cohorts with conditional expression of structural mutant p53R172H (p53LSL-R172H/fl), contact mutant p53R270H (p53LSL-R270H/fl) and conditional p53 knockout (p53null/fl) for comparison in a uniform p53 heterozygous genetic background of stromal tissues. Our tumor studies have demonstrated that oncogenic Kras expression and p53 knockout or mutation in acinar cells of adult mice leads to metastatic PDAC development with complete penetrance. This suggests a novel role for p53 in tumor suppression by blocking cellular reprogramming of acinar cells to ductal-like cells. In contrast, these mutations drive PDAC from ductal cells less efficiently. To understand the origins of human PDAC, we are investigating the molecular pathways altered in mouse acinar and ductal cell-derived tumors. Through comparison with human pancreatic cancer data, our studies will elucidate the cellular origins of different molecular subtypes of human PDAC. Elucidating the mechanisms of PDAC development is critical to improving detection and therapy in PDAC patients.

Citation Format: Brittany M. Flowers, Hang Xu, Kathryn Hanson, Christina Curtis, Hannes Vogel, Laura D. Wood, Laura D. Attardi. Elucidating the role of p53 in the cellular origins of pancreatic cancer development [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 A15.
Abstract A20: Gemcitabine primes the pancreatic tumor microenvironment for second-line immunotherapy
Authors: Principe Daniel R. , Narbutis Matthew , Kumar Sandeep , Park Alex , Viswakarma Navin , Dorman Matthew J. , Kamath Suneel D. , Munshi Hidayatullah G. , Rana Ajay
Published: March 2020

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death with a median survival time of 6-12 months. As most patients present with disseminated disease, the majority are offered palliative chemotherapy. As there are no treatment strategies for patients who progress on chemotherapy, we explored the effects of long-term gemcitabine on the tumor microenvironment in order to identify potential therapeutic options in chemorefractory PDAC. Using a combination of mouse models and human-derived PDAC cell lines, we first evaluated chemotherapy-induced alterations in the tumor secretome and immune surface proteins by high-throughput proteomic arrays. In addition to enhancing antigen presentation and immune checkpoint expression, gemcitabine led to large-scale alterations in the tumor secretome, namely by increasing the synthesis of CCL/CXCL chemokines and TGFβ-associated proteins. These secreted factors altered the composition of the tumor stroma, conferring drug resistance to cancer-associated fibroblasts in vitro. We therefore combined gemcitabine with anti-PD-1 in transgenic models of murine PDAC, which failed to alter disease course unless mice also underwent genetic or pharmacologic ablation of TGFβ signaling. In the setting of TGFβ signal deficiency, gemcitabine and anti-PD-1 led to a robust CD8+ T cell-response and decrease in tumor burden. These results suggest that gemcitabine may prime PDAC tumors for immune checkpoint inhibition by enhancing antigen presentation, but requires the inhibition of a TGFβ cytokine barrier. Given the current lack of third-line treatment options, this approach warrants consideration in the management of gemcitabine-refractory PDAC.

Citation Format: Daniel R. Principe, Matthew Narbutis, Sandeep Kumar, Alex Park, Navin Viswakarma, Matthew J. Dorman, Suneel D. Kamath, Hidayatullah G. Munshi, Ajay Rana. Gemcitabine primes the pancreatic tumor microenvironment for second-line immunotherapy [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 A20.
Abstract A32: Adipose-derived mesenchymal stem cell has the differentiation/reprogramming capacity towards two distinct cancer-associated fibroblasts
Authors: Miyazaki Yoshihiro , Kumagai Yutato , Kushige Hiroko , Shimomura Osamu , Kida Yasuyuki , Oda Tatsuya
Published: March 2020

Cancer-associated fibroblasts (CAFs) are well known of its heterogeneity with distinct phenotypes and functions in tumor progression. CAFs are believed to be composed mainly of resident tissue fibroblast, such as pancreas stellate cells, but bone marrow-derived mesenchymal stem cells (MSC) are also known to be their source (Ishii G et al., Adv Drug Deliv Rev 2016). Herein, we focus on adipose-derived MSC (AD-MSC) as an alternative source of CAF, as one has high reactivity of differentiation and reprogramming to the many cell types (Ronnov-Jessen L et al., J Clin Invest 1995; Sugii/Kida et al., PNAS 2010). It was reported that CAFs include myofibroblastic CAFs (myCAFs), which are found adjacent to tumor cells, and inflammatory CAFs (iCAFs), which localize farther away within the dense stroma (Ohlund, Tuveson et al., J Exp Med 2017). We investigated whether AD-MSC can be a CAF progenitor and differentiated to the different subpopulations of CAFs by employing different culture condition. To evaluate the differentiation capacity, a GFP labeled AD-MSC cell line and the human pancreatic-cancer cell line (Capan-1) were co-cultured in two condition, including direct (DR) co-culture or indirect transwell (TW) co-culture. We then performed qRT-PCR analysis and immunocytochemistry to examine their myCAF marker (ACTA-2, CTGF) or iCAF markers (IL-6, CXCL1, LIF) and compared with those in monocultured AD-MSC. We also analyzed in vitro tumor formation in DR co-culture using time-lapse imaging. In DR co-culture, the morphology of Capan-1 and AD-MSC recapitulated the clinical pancreatic cancer, with those CAF markers expressions. In the course of co-culture, sequential morphologic alterations were observed in AD-MSCs. The AD-MSC with stellate shape changed to large and small spindle-like cells through crosstalk with cancer cells. In sharp contrast, only iCAF marker expression was elevated in TW co-culture. These results collectively suggest that the AD-MSC have a potential to be progenitors of both myCAF and iCAF, explaining the heterogeneity of CAFs under the direct crosstalk with cancer cells. In the presentation, we discuss the differentiation/reprogramming capacity of AD-MSC towards two distinct CAFs (i.e., myCAF and iCAF), focusing on the cell morphology and RNA-seq analysis.

Citation Format: Yoshihiro Miyazaki, Yutato Kumagai, Hiroko Kushige, Osamu Shimomura, Yasuyuki Kida, Tatsuya Oda. Adipose-derived mesenchymal stem cell has the differentiation/reprogramming capacity towards two distinct cancer-associated fibroblasts [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 A32.
Abstract A33: Single-cell profiling reveals subclonal vulnerabilities to therapy in patient-derived 2D and organoid models
Authors: Monberg Maria E. , Pagan Vincent B. , Semaan Alexander , Lee Jaewon J. , Guerrero Paola A. , Muthuswamy Senthil K. , Maitra Anirban
Published: March 2020

Pancreatic ductal adenocarcinoma (PDAC) is the 3rd leading cause of cancer-related deaths in the United States. This is largely due to the fact of high propensity of patients presenting at metastatic stages resulting from symptom ambiguity and a complete lack of general population screening programs. As the overwhelming majority of patients (85%) present with surgically unresectable disease, the 5-year survival rate is dire at only ~8%. Outside of what is known of the commonly mutated genes in PDAC, which include KRAS, TP53, SMAD4, and CDKN2A, little molecular information exists that has yielded clinical knowledge that may have an impact on survival outcomes. Therefore, a concerted clinical and research paradigm shift towards higher-resolution investigations of the genomics of PDAC is an area of highest public health significance. In our laboratory, we have leveraged single-cell sequencing approaches in both cell lines and patient-derived tissues to develop models for molecular underpinnings of PDAC and as potential tools for therapeutic stratification. First, to demonstrate the ability to model cellular heterogeneity of tumor populations, single-cell RNA and DNA sequencing was applied to widely used patient-derived PDAC cell lines. Using a bioinformatics pipeline, were able to differentiate between supposedly identical cell lines that had evolved rich subclonal architecture that may provide insight into complex in silico landscapes that naturally evolve as these cells are continuously used by laboratories. We then characterized single-cell gene expression profiles of patient-derived organoids (PDOs) to understand their heterogeneous composition and applied an in silico therapeutic drug prediction model to identify and validate combinatorial strategies targeting subpopulations. These present multiple phenotypic traits similar to that of the parental tumor including histoarchitecture, oxygen biology, epigenetic marking, and differentiation status. Orthogonal validation of predicted drugs was confirmed through a high-throughput drug screen of 764 agency-approved candidates on two PDOs. This strategy demonstrated the utility of single-cell transcriptomic profiling from PDOs in effectively identifying heterogeneous enriched pathways and molecular subtypes within cultures to predict viable treatment options using a pharmacogenomic approach.

Citation Format: Maria E. Monberg, Vincent B. Pagan, Alexander Semaan, Jaewon J. Lee, Paola A. Guerrero, Senthil K. Muthuswamy, Anirban Maitra. Single-cell profiling reveals subclonal vulnerabilities to therapy in patient-derived 2D and organoid models [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 A33.
Abstract A35: Microenvironmental adaptations drive obesity-associated pancreatic cancer
Authors: Chung Katherine Minjee , Singh Jaffarguribal , Lawres Lauren , Dorans Kimberly Judith , Robbins Rebecca , Bhutkar Arjun , Babic Ana , Vayrynen Sara A. , Costa Andressa D. , Nowak Jonathan A. , Chang Daniel T. , Dunne Richard F. , Hezel Aram F. , Koong Albert C. , Wilhelm Joshua J. , Bellin Melena D. , Nylander Vibe , Gloyn Anna L. , McCarthy Mark I. , Wolpin Brian M. , Jacks Tyler , Fuchs Charles S. , Muzumdar Mandar Deepak
Published: March 2020

Obesity is a major modifiable risk factor for pancreatic ductal adenocarcinoma (PDAC), yet how obesity contributes to pancreatic cancer development is not well understood. Moreover, whether and at what point weight loss or other obesity-related interventions may impact PDAC progression remains largely unexplored, with significant implications for prevention and treatment. Leveraging an autochthonous genetically engineered model of PDAC, we demonstrate that obesity plays a stage-specific causal and reversible role in early PDAC progression. Molecular and histologic analyses reveal prominent microenvironmental alterations in tumors from obese mice, including increased inflammation and fibrosis and evidence for marked pancreatic islet cell adaptation. In particular, we identify aberrant expression of the peptide hormone cholecystokinin (CCK) in pancreatic islets in tumors as an adaptive response to obesity and show that islet CCK expression promotes oncogenic Kras-driven pancreatic ductal tumorigenesis. Together, these studies link obesity, changes in the local microenvironment, and tumorigenesis and implicate islet-derived hormones beyond insulin in PDAC development. Furthermore, the reversible nature of these adaptations supports the potential of antiobesity strategies to intercept PDAC early during progression.

Citation Format: Katherine Minjee Chung, Jaffarguribal Singh, Lauren Lawres, Kimberly Judith Dorans, Rebecca Robbins, Arjun Bhutkar, Ana Babic, Sara A. Vayrynen, Andressa D. Costa, Jonathan A. Nowak, Daniel T. Chang, Richard F. Dunne, Aram F. Hezel, Albert C. Koong, Joshua J. Wilhelm, Melena D. Bellin, Vibe Nylander, Anna L. Gloyn, Mark I. McCarthy, Brian M. Wolpin, Tyler Jacks, Charles S. Fuchs, Mandar Deepak Muzumdar. Microenvironmental adaptations drive obesity-associated pancreatic cancer [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 A35.
Abstract B18: Defining the role of chromatin remodeling complexes in pancreatic cancer stem cells
Authors: Ferguson Lesley Paige , Gatchalian Jovylyn , Lytle Nikki K. , Rajbhandari Nirakar , Chambers Kendall , Albini Sonia , Bruneau Benoit , Puri Lorenzo , Sander Maike , Lowy Andrew , Hargreaves Diana , Reya Tannishtha
Published: March 2020

Pancreatic adenocarcinoma (PDAC) is a devastating disease characterized by high rates of metastasis and poor therapeutic response. It is currently the 4th leading cause of cancer-related deaths in developed countries, and despite efforts to improve therapy, the five-year survival rate remains at 9%. Therefore, it is critical to identify new programs that drive pancreatic cancer progression and therapeutic resistance. To define new cancer dependencies, work in the Reya lab has focused on characterizing stem cell programs that drive cancer initiation, propagation, and relapse. Previously published studies have demonstrated that the fate determinant Musashi2 (Msi2) functionally marks a drug-resistant stem population in pancreatic cancer. More recent work has revealed that this aggressive subpopulation is characterized by a unique transcriptional and epigenetic profile. Given the crucial role for epigenetic regulation in development, we hypothesized that differential expression of epigenetic regulatory factors could be responsible for the establishment or maintenance of a stem cell state in pancreatic cancer. To identify epigenetic factors critical to stem function, we used a functional shRNA screen to profile the impact of inhibition of stem-enriched candidate factors on 3D growth of Msi2+ pancreatic cancer cells in vitro. From this screen, we identified the gene Smarcd3 as a critical mediator of stem cell growth in pancreatic cancer. Smarcd3 encodes Baf60c, a component of the SWI/SNF nucleosome remodeling complex with known functions in cardiac and neural development. Though Smarcd3 function has not been studied in PDAC, the SWI/SNF complex at large is known to be dysregulated in pancreatic cancer. Preliminary studies have shown that Smarcd3 inhibition leads to reduced growth of both mouse and human pancreatic cancer cells in vitro and in vivo. Current studies are ongoing to 1) elucidate the mechanism by which Smarcd3 controls growth of pancreatic cancer cells through binding of developmental transcription factors, and 2) use genetically engineered mouse models of pancreatic cancer to define the role of Smarcd3 in pancreatic cancer initiation and progression.

Citation Format: Lesley Paige Ferguson, Jovylyn Gatchalian, Nikki K. Lytle, Nirakar Rajbhandari, Kendall Chambers, Sonia Albini, Benoit Bruneau, Lorenzo Puri, Maike Sander, Andrew Lowy, Diana Hargreaves, Tannishtha Reya. Defining the role of chromatin remodeling complexes in pancreatic cancer stem cells [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 B18.
Abstract B20: KrasG12D effector dependencies in the maintenance of pancreatic ductal adenocarcinoma
Authors: Grimont Adrien , Zhao Zhen , Leach Steven D. , Chandwani Rohit
Published: March 2020

More than 90% of pancreatic ductal adenocarcinoma (PDAC) tumors in humans feature activating mutations in the proto-oncogene KRAS. In mice, KrasG12D mutation alone is sufficient for PanIN formation; when coupled with mutational inactivation of TP53 (Tp53R172H), PanIN formation is accelerated and these neoplastic lesions progress to invasive and metastatic disease at high frequency. Several groups have demonstrated that inactivation of mutant Kras in established PanIN and PDAC leads to redifferentiation and apoptosis of the tumor, respectively. More recently, several combinatorial genetic and pharmacologic strategies have been employed that highlight RAF kinases as key oncoeffectors for KRAS addiction, but that RAF inhibition alone demonstrates little efficiency against KRAS mutant tumors. Additionally, previous studies suggest a requirement for both C-raf and EGFR blockade or the combination of RAF-MEK or RAF-PI3K inhibitors to inhibit to tumor growth. However, a systematic evaluation of dependencies of Kras effectors in tumor maintenance has not been explicitly performed. To investigate effector dependencies in the KrasG12D setting, we have developed an organoid platform to generate isogenic lines bearing doxycycline-inducible small hairpin (sh)RNAs coupled to GFP inserted into mouse pancreatic tumor organoids derived from KPC mice. These shRNAs are directed against each of the major Kras effector molecules, including B-raf, C-raf, Pi3k, RalA, RalB, and Rac1, with Kras shRNAs and Renilla shRNA used as controls. We identified 2 effective shRNAs per target, with strong and durable inhibition of each protein observed after doxycycline treatment. To investigate the hierarchy of Kras effectors, we employed a competition assay of the shRNA organoids, and observed the drop-out of Kras, C-raf, and Rac1 shRNA-bearing tumor organoid lines. Cell number and volumetric analyses confirmed strong dependencies on Kras, C-raf, and Rac1. Chromatin accessibility and transcriptomic analyses of Kras- , C-raf- , and Rac1-depleted organoids revealed consensus features of abrogated tumor maintenance and key molecular nodes in tumor maintenance. Together, we have employed a mouse organoid platform to create a rigorous map of the requirements for the key effectors of mutant Kras, and to dissect the biologic mechanisms shared by effectors disruptive of the mutant Kras epigenetic and transcriptional landscape. In turn, we anticipate these findings can inform the subsequent development of novel therapies to address these vulnerabilities.

Citation Format: Adrien Grimont, Zhen Zhao, Steven D. Leach, Rohit Chandwani. KrasG12D effector dependencies in the maintenance of pancreatic ductal adenocarcinoma [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 B20.