bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1510: Immune regulators identified in undifferentiated pleomorphic sarcoma (UPS) subpopulations
Authors: Haddox Candace Lynn , Bao Junjie , Barrientos Tomasa , Alman Benjamin A.
Published: September 2020

Introduction: UPS is a common subtype of soft tissue sarcoma with limited effective systemic therapies. We previously showed that a murine model of UPS has various subpopulations (SPs), characterized by distinct morphology, molecular changes, and oncogenic behaviors that likely contribute to the overall behavior of UPS. Here, we aimed to characterize interactions between UPS SPs to identify pathways important in UPS progression.

Methods: We used a murine model of UPS that recapitulates the human disease. Cre recombinase drives oncogenic KRAS-G12D, P53 deletion, and expression of confetti fluorescent protein to allow for in vivo lineage tracing. Individual SPs were isolated via FACS and cultured in the presence of conditioned media (CM) from other individual SPs. Growth rate was assayed using the IncuCyte system (Essen BioScience), and cell proliferation was measured using the Click-iT EdU flow cytometry assay kit. Invasion and migration were assayed using a cell culture insert with or without Matrigel, with CM or non-CM in the lower chamber. Cells migrating/invading into the lower chamber were counted. We performed proteomics analysis on the CM from the SP that potentiated growth and invasion to define the secretome of this SP. The CCL2/CCL7/CCL8 inhibitor, bindarit, was added to CM and cell confluency was assessed as above. RNAseq data from The Cancer Genome Atlas (TCGA) were used to generate immune infiltration scores in human UPS samples. These scores were correlated with expression of lead candidates from our secretome analysis.

Results: CM from one UPS SP (SP-1) increased the growth rate and invasion/migration of one other SP (SP-2). Proteomics analysis identified 233 secreted proteins in the CM from SP-1, including 37 with two-fold higher abundance compared to SP-2. Among these, several immunoregulatory proteins were identified, including serpine 1, CCL2, CCL7, CXCL1, and CX3CL1. Culture with bindarit abolished the effect of SP-1 CM on growth rate. Additionally, we found several semaphorins in the secretomes of SP-1 and SP-2, and emerging evidence suggests a key role for semaphorins in cancer progression and immune regulation. Semaphorin expression correlated with immune infiltration scores in human UPS samples.

Conclusions: We identified a UPS SP that positively regulates oncogenic behaviors of other SPs through secreted factors. Additionally, several of these secreted proteins play key roles in immune regulation. Given promising activity of immunotherapy in UPS, these proteins warrant further investigation.

Citation Format: Candace Lynn Haddox, Junjie Bao, Tomasa Barrientos, Benjamin A. Alman. Immune regulators identified in undifferentiated pleomorphic sarcoma (UPS) subpopulations [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1510.
Abstract 1535: Identification of novel epithelial-mesenchymal transition inducers and their clinical significance in colorectal cancer
Authors: Ishikawa Satoshi , Nishida Naohiro , Fujino Shiki , Ogino Takayuki , Takahashi Hidekazu , Miyoshi Norikatsu , Uemura Mamoru , Matsuda Chu , Mizusima Tsunekazu , Yamamoto Hirofumi , Mimori Koshi , Mori Masaki , Doki Yuichiro , Eguchi Hidedoshi
Published: September 2020

Purpose. Cancer tissues contain a substantial amount of stromal tissues, and transcriptome derived from bulk samples originates from a mixture of the cancer epithelium and stroma. Gene signature of epithelial-mesenchymal transition (EMT) cells is similar to that of stromal cells; therefore, gene signature that we considered to be related EMT might be only contamination of stroma. Here, we identify novel EMT inducers by analysis of independent gene expression profiles in the colorectal cancer epithelium and stroma.

Materials and Methods. Gene expression data of pure epithelial tissue was obtained using laser microdissection technique from 13 colorectal cancer tissues. Next, the ratio of each gene expression in the stroma to in the epithelium and correlation of each gene expression with a target gene derived from The Cancer Genome Atlas (TCGA) is plotted in two dimensions, and correlation coefficients are calculated. We regard genes with high correlation coefficients but highly expressed in the epithelium as candidates of EMT inducers. We manipulate the expression of some candidate genes in colorectal cancer cell lines to investigate the alterations of EMT markers and migration ability in vitro.

Results. We extract 91 new putative EMT-related genes in silico. They are highly expressed in similar cell lines to known EMT markers in a public database. Furthermore, some candidates have reported having a relation to EMT, and that indicates the validity of this procedure. The knockdown of three candidate genes upregulates epithelial gene expression and downregulates mesenchymal gene expression. The knockdown of one of the three genes reduces cell migration. In contrast, overexpression of the gene downregulates epithelial gene expression and leads to cell migration.

Conclusion. We identify novel EMT inducers successfully using purified gene expression data. They could be new prognostic markers and therapeutic targets of colorectal cancer.

Citation Format: Satoshi Ishikawa, Naohiro Nishida, Shiki Fujino, Takayuki Ogino, Hidekazu Takahashi, Norikatsu Miyoshi, Mamoru Uemura, Chu Matsuda, Tsunekazu Mizusima, Hirofumi Yamamoto, Koshi Mimori, Masaki Mori, Yuichiro Doki, Hidedoshi Eguchi. Identification of novel epithelial-mesenchymal transition inducers and their clinical significance in colorectal cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1535.
Abstract 1542: Roles of extracellular ATP in inducing EMT and CSC-like changes to promote early steps of metastasis in non-small lung cancer cell lines
Authors: Song Jingwen , Evers Maria , Chen Xiaozhuo
Published: September 2020

Cancer is a major death-causing disease worldwide, and metastasis, a hallmark of cancer, is associated with up to 90% of all cancer-related deaths. Metastasis requires ATP, and the tumor microenvironment (TME) has much higher concentrations of intratumoral and extracellular ATP (eATP) than healthy tissue, implying that eATP may have potential roles in tumor development and progression. Our previous ATP studies, cited by a Nature Review Cancer paper in 20181 showed that eATP is internalized by cancer cell through macropinocytosis, and it promotes cancer cell growth, survival, and drug resistance. However, how intratumoral eATP plays roles in metastasis remains largely unclear. Our 2019 published results show that eATP promotes cancer migration and invasion, initiates epithelial to mesenchymal transition (EMT), alters expression level of EMT related protein markers such as ZEB1, SNAIL, SLUG in human non-small lung cancer cell (NSCLC) lines2. It has been reported that the EMT and cancer stem cells (CSCs) share some molecular links, but the detailed mechanisms involved are still unknown. Recently, we have found the eATP significantly induces the gene expression level of CSC transcription factors (TFs): SOX2, NANOG and OCT4 in A549 cells in a time-dependent manner. We have also found that, after A549 cells were pretreated with 0.5mM for 6 hrs, the ATP-pretreated A549 cells were able to show significantly increased migration and invasion even in the absence of ATP addition. Based on all these results, we hypothesize that extracellular ATP functions as a messenger, a signal facilitator, an energy molecule, and a transcription cofactor to induce EMT and CSC-like features to promote metastasis in NSCLC. RNAseq and metabolomics studies are being used to determine the transcriptional and metabolic changes associated with the ATP induced EMT and CSC process in NSCLC. The completion of the project will not only unravel eATP's roles in EMT and CSC induction and early steps of tumor metastasis, but also potentially provide novel promising therapeutic targets for inhibiting tumor metastasis.

Citation Format: Jingwen Song, Maria Evers, Xiaozhuo Chen. Roles of extracellular ATP in inducing EMT and CSC-like changes to promote early steps of metastasis in non-small lung cancer cell lines [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1542.
Abstract 1543: Development of a novel ECAD-EmGFP reporter line for pancreatic cancer MET study and drug discovery
Authors: Douglas Diana , Kumari Sangeeta , Huuskonen Jarkko , Shu Weiguo
Published: September 2020

World-wide, metastasis continues to be the leading cause of death in cancer patients1. Although epithelial-to-mesenchymal transition (EMT) and mesenchymal-to-epithelial transition (MET) have been implicated in the progression of cancer metastasis and drug resistance, their mechanistic pathway and specific role in disease progression is not fully understood2. As such, the development of a reporter line that enables the real-time monitoring of the changing status of cells will not only aid in dissecting the EMT/MET pathway in the research field, but could also become a robust platform for new cancer drug development.

E-cadherin, an adhesion protein expressed in epithelial cells, is upregulated in pancreatic cancer cells during MET and is associated with an increase in tight junctions and apico-basal polarity, as well as a change in morphology3,4. Here, we developed a novel PANC-1 ECAD-EmGFP reporter line using CRISPR/Cas9 genome-editing technology. In this cell line, the emerald green fluorescent protein (EmGFP) reporter was incorporated into the last exon of the endogenous E-cadherin gene, enabling real-time monitoring of MET status in live cells. The ECAD-EmGFP knock-in allele was confirmed at genomic, transcriptional, and translational levels. Functional data revealed that miRNA-200 treatment induced the increased expression of ECAD-EmGFP, and decreased expression of Snail, a transcription factor associated with mesenchymal traits. These changes in marker gene expression, as well as the decrease in invasive capacity upon induction, suggest that cells have undergone MET. This cell line is a valuable tool for dissecting the molecular mechanisms underlying EMT and MET and for evaluating or screening compounds targeting EMT and MET in pancreatic cancer.

1. Dizon DS, et al. Clinical Cancer Advances 2016: Annual Report on Progress Against Cancer from the American Society of Clinical Oncology. J Clin Oncol 34: 987-1011, 2016. 2. Nieto MA, et al. EMT: 2016. Cell 166: 21-45, 2016. 3. Polireddy K, et al, Targeting Epithelial-Mesenchymal Transition for Identification of Inhibitors for Pancreatic Cancer Cell Invasion and Tumor Spheres Formation. PLOS ONE 11(10): e0164811, 2016 4. Wong S, et al. E-cadherin: Its dysregulation in carcinogenesis and clinical implications. Critical Reviews in Oncology / Hematology 121: 11- 22, 2018

Citation Format: Diana Douglas, Sangeeta Kumari, Jarkko Huuskonen, Weiguo Shu. Development of a novel ECAD-EmGFP reporter line for pancreatic cancer MET study and drug discovery [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1543.
Abstract 1598: Reprogramming of immunosuppressive tumor microenvironments by multifunctional nanoemulsion and enhance immune checkpoint blockade therapy
Authors: Jin Seung Mo , Lee Sang Nam , Lim Yong Taik
Published: September 2020

The low response rate of current cancer immunotherapy suggests the presence of few antigen-specific T cells and a high number of immunosuppressive factors in tumor microenvironment (TME). Toll-like receptor (TLR) 7/8 agonist-based adjuvants have been widely used to activate antigen-presenting cells (APCs) and induce antigen-specific therapeutic T cells. However, the incorporation of TLR 7/8 agonists also induces immunosuppressive factors, such as Indoleamine 2,3-dioxygenase (IDO) in APCs and tumor cells, which results in the formation of immunosuppressive TME. In this research, we report the assemblable immune-modulating suspension (AIMS) consisting of TLR 7/8 agonist and IDO inhibitor, which was designed to down-regulate the immune-suppressive factors (TGF-β, IL-10, myeloid-derived suppressor cells, regulatory T cells) and to increase the infiltration of cytotoxic T lymphocytes. The best combination of immune-modulating agents in AIMS, we called supra-adjuvant, was selected by screening possible combinations of commercialized TLR 7/8 agonists and IDO inhibitors based on the ratio of pro- and anti-inflammatory cytokines. Paclitaxel was also introduced into AIMS to induce immunogenic cell death (ICD) and in situ vaccination efficacy with TLR 7/8 agonist at the tumor bed. Local administration of AIMS increased infiltrated T cells in both local and distant tumors, and inhibited the metastasis of tumors to lung significantly. AIMS also reshaped the TME from ‘cold tumor' to ‘hot tumor' successfully, and increased the responsive rate of immune checkpoint blockade therapy (ICBT) significantly. Taken together, the AIMS composed of supra-adjuvant and ICD-inducer is expected to be used as a platform for the re-programming of immunosuppressive TME and the induction of synergistic cancer immunotherapy with ICBT.

Citation Format: Seung Mo Jin, Sang Nam Lee, Yong Taik Lim. Reprogramming of immunosuppressive tumor microenvironments by multifunctional nanoemulsion and enhance immune checkpoint blockade therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1598.
Abstract 1620: MDM2 P1 and P2 promoters have different roles in tumorigenesis
Authors: Phinichkusolchit Narisa , Jamieson Thomas , Ridgway Rachel , Athineos Dimitris , Crawford Kerryanne , Vlatkovic Nikolina , Boyd Mark , Sansom Owen , Blyth Karen
Published: September 2020

The murine double minute 2 (MDM2) protein is an E3 ubiquitin ligase most notable for priming tumour-suppressor p53 for degradation. MDM2 contains two promoters - the constitutive P1 and the inducible P2. The p53-independent P1 promoter is responsible for basal levels of MDM2, whilst the p53-dependent P2 promoter is activated upon stress responses. Although the oncogenic functions of MDM2 are well understood, less is known about the individual roles of the promoters and their contribution to tumorigenesis and stress responses. To tease apart the roles of the promoters, we have used genetically engineered mouse (GEM) models null for the P1-promoter and mutated for the P2-promoter in the context of intestinal cancer, lymphoma and hepatocellular carcinoma (HCC). Here, we have shown that mice with heterozygous or homozygous loss of the P1-promoter in the APCMin/+ intestinal model have extended survival and reduced intestinal tumour burden in comparison to the P1- wild type (WT), APCMin/+ mice. Surprisingly, none of the P1-null APCMin/+ mice succumbed to intestinal tumours, but rather to thymic lymphomas. In the P1-null APCMin/+ lymphoma samples, we investigated the p53 functionality by examining the expression levels of p21, a p53 downstream target. Immunohistochemistry analysis clearly revealed that p53 is non-functional as p21 was not expressed, despite variations in p53 expression levels itself. This uncommon occurrence in the APCMin/+ model led us to investigate the role of the P1 and P2 promoters in the Eµ-Myc lymphoma model. Interestingly, in this model, heterozygous loss of the P1 promoter improves survival, while heterozygous loss of the P2 promoter accelerates lymphomagenesis. Our findings thus far indicate a differential role for the P1 and P2 MDM2 promoters in tumorigenesis. In addition to using GEM models, we are investigating the role of the MDM2 promoters in stress-induced HCC. These studies will allow for a better understanding of MDM2 functions, but also perhaps provide an alternative therapeutic strategy for cancers with aberrations in the p53-MDM2 signalling axis.

Citation Format: Narisa Phinichkusolchit, Thomas Jamieson, Rachel Ridgway, Dimitris Athineos, Kerryanne Crawford, Nikolina Vlatkovic, Mark Boyd, Owen Sansom, Karen Blyth. MDM2 P1 and P2 promoters have different roles in tumorigenesis [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1620.
Abstract 1658: Anti-CEACAM binding protein for fluorescence visualization of primary colon cancer in patient-derived orthotopic xenograph mouse models
Authors: Hollandsworth Hannah M. , Amirfakhri Siamak , Filemoni Filemoni , Schmitt Verena , Wennemuth Gunther , Schmidt Alexej , Hoffman Robert M. , Singer Bernhard B. , Bouvet Michael
Published: September 2020

Background The Helicobacter outer protein (HopQ) is a novel surface-located Helicobacter pylori adhesion protein that binds to various human carcinoembryonic antigen-related cell adhesion molecules (CEACAM1, 3, 5, 6). Our aim was to validate fluorescence targeting of colorectal tumors in patient derived orthotopic xenograft models with fluorescently labeled HopQ.

Materials/Methods CEACAM binding protein HopQ was conjugated to near-infrared dye IR800CW (LI-COR). Western blotting was performed with various colon cancer cell lysates to determine CEACAM expression. Nude mice (n=4) received orthotopic implantation of patient-derived primary colon tumors and patient-derived colon cancer metastases. Mice were administered either 25 ug or 50 ug of HopQ-IR800CW via tail vein injection and imaged 48 hours later. Intravital images were analyzed using the Image Studio Software Small Animal Imaging Analysis to determine tumor-to-background ratio (TBR).

Results Western blotting demonstrated varied expression of CEACAM binding in all 15 patient derived colon cancer lysates. Dose response intravital imaging demonstrated TBR of 1.41 when administered 25 ug of HopQ-IR800CW and TBR 3.78 when administered 50 ug of HopQ-IR800CW.

Conclusion CEACAM binding protein HopQ shows promising use for fluorescence imaging of patient derived orthotopic colon cancer. HopQ may be useful for tumor detection for pre-surgical diagnosis and fluorescence-guided surgery.

Citation Format: Hannah M. Hollandsworth, Siamak Amirfakhri, Filemoni Filemoni, Verena Schmitt, Gunther Wennemuth, Alexej Schmidt, Robert M. Hoffman, Bernhard B. Singer, Michael Bouvet. Anti-CEACAM binding protein for fluorescence visualization of primary colon cancer in patient-derived orthotopic xenograph mouse models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1658.
Abstract 166: Absolute quantification of DNA methylation markers using methylation-sensitive restriction enzymes and multiplex digital PCR
Authors: Nell Rogier J. , Van der Velden Pieter A.
Published: September 2020

Aberrant DNA methylation plays important roles in the initiation and progression of cancer. Therefore, DNA methylation markers have important diagnostic and prognostic potential. However, accurate quantification of these markers remains challenging in clinical samples of limited quality or quantity. We introduce a novel, digital approach for the absolute quantification of the amount, density and allele-specificity of DNA methylation.

We designed an experimental setup combining efficient methylation-sensitive restriction enzymes with quantitative digital PCR. This allowed to measure a targeted density of DNA methylation independent of bisulphite conversion. In-silico simulations were performed to demonstrate the mathematical validity of the setup. A practical validation was carried out by quantifying RASSF1A promotor methylation in a variety of healthy and malignant reference samples and in an innovative calibration curve.

Furthermore, we extended our setup to a multiplex digital PCR experiment. Hereby, methylation markers may be analyzed together with physically linked genetic markers to determine the allele-specificity of the methylation.

Overall, our results show an accurate quantification of DNA methylation markers with digital PCR, independent of bisulphite conversion. Effective measurements with high precision could be obtained, even in minute amounts of DNA. Moreover, as the context-density and allele-specificity of methylation can also be determined, mechanisms in cancer biology can now be quantitatively assessed in a digital experiment.

Citation Format: Rogier J. Nell, Pieter A. Van der Velden. Absolute quantification of DNA methylation markers using methylation-sensitive restriction enzymes and multiplex digital PCR [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 166.
Abstract 1666: A comprehensive DLBCL lymphoma PDX collection to evaluate the efficacy and resistant mechanism of rituximab
Authors: Qi Hui , Tang Xuzhen , Zou Zhiyuan , Fang Ying , Wang Li , Zhao Weili , Gu Qingyang , Ji Qunsheng
Published: September 2020

Rituximab has shown great efficacy in B-cell non-Hodgkin's lymphoma (NHL), by binding CD20 to induce cell destruction through activating apoptosis, complement and cell-mediated cytotoxity. However, the response rate of patients with relapsed or refractory CD20-positive NHL to standard rituximab treatment is less than 50%, and the non-responders, especially DLBCL patients, still could express high levels of CD20. Therefore, a translational in vivo platform to understand the mechanisms behind Rituximab resistance is in urgent need. We established and characterized a series of DLBCL lymphoma PDX. Eleven non-GCB DLBCL PDX models were selected for the Rituximab efficacy study, of which 3 are relapse DLBCL. IHC and RNAseq were conducted to evaluate CD20 status and expression of other genes. The anti-tumor efficacy of Rituximab in 11 PDX models revealed that 5 of them were sensitive to rituximab and 6 had a poor response. All these models are CD20 positive judged by IHC. By analyzing the correlation of tumor growth inhibition (TGI) and the genomic background of these PDX models, we identified several candidate genes such as BCL2L1, PDK1, TGF-β, Lgalsl and p56/p53Lyn that may contribute to drug resistance mediated by elevated apoptotic threshold, modulation of complement activity or diminished cellular cytotoxicity. Further validation studies are in progress. We established a series of DLBCL lymphoma PDX; with advanced genomic profiling method and validation, these models can be precisely used for subtype-specific therapy evaluation and the investigation of the mechanism of drug resistance.

Citation Format: Hui Qi, Xuzhen Tang, Zhiyuan Zou, Ying Fang, Li Wang, Weili Zhao, Qingyang Gu, Qunsheng Ji. A comprehensive DLBCL lymphoma PDX collection to evaluate the efficacy and resistant mechanism of rituximab [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1666.
Abstract 1752: Inhibition of histone demethylase KDM6B as a promising target for non-small cell lung cancer
Authors: Lesbon Jessika C. , Xavier Pedro L. , Garnica Taismara K. , Rochetti Arina L. , Reis Rui M. , Müller Susanne , Fukumasu Heidge
Published: September 2020

Despite advances in diagnostic and therapeutic approaches, the majority of lung cancer patients still progress to advanced stages with metastatic lesions. Abnormalities in epigenetic mechanisms are related to the most malignant cancer phenotypes. Thus, there is an increasing interest in the development and validation of drugs that targets epigenetic proteins. Trimethylation of H3K27 is an important post-translational modification and some studies have shown that KDM6B (lysine (K)-specific demethylase 6B), an H3K27 demethylase, is a crucial regulator involved in tumorigenesis and play important roles in various types of cancers. In this study, our goal was to determine potential epigenetic targets to control invasion and migration of NSCLC cancer cells. Initially, KDM6B was found a promise target through in silico analysis for the inverse correlation between gene expression and overall survival on 2438 NSCLC cases from GEO, EGA and TCGA using KMplotter tool and selecting the genes according to HR and p-values (HR > 1 and p < 0.05). Next, NSCLC cell lines (A549, H23, H2126 and H1568) were evaluated for the expression of the KDM6B using CellExpress tool and next by real-time PCR and one cell was chosen for cytotoxicity of the KDM6B inhibitor (GSK-J4) for 72h by MTT assay. The IC50 value and the regression curve were calculated with 6.0 Prism (GraphPad Software, USA). KDM6B gene expression was inversely correlated with patient survival rate for pulmonary adenocarcinoma (HR= 2.81; p= 6,3E-09). All pulmonary adenocarcinoma cell lines expressed KDM6B gene and low expression in healthy lung tissue (Z-score= 2.8). The KDM6B IC50 for A549 cells was 2.78±0.09μM. These early findings report that KDM6B as a promising target for epigenetic therapy for pulmonary adenocarcinoma. Therefore, our next step will be to evaluate the potential of KDM6B inhibition on the phenotype of cancer cell migration and invasion and global analysis of gene expression to understand genes and mechanisms associated with possible inhibitory effects of KDM6B.

Citation Format: Jessika C. Lesbon, Pedro L. Xavier, Taismara K. Garnica, Arina L. Rochetti, Rui M. Reis, Susanne Müller, Heidge Fukumasu. Inhibition of histone demethylase KDM6B as a promising target for non-small cell lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1752.
Abstract 1758: Discovery of novel potent and selective inhibitors of PRMT5 with anti-tumor activity in hepatocellular carcinoma and lung pre-clinical models
Authors: Gonzalvez Francois , Vandyck Koen , Debing Yannick , Gupta Kusum , Misner Dinah , Zhang Qingling , Liu Jyanwei , Stoycheva Antitsa , Stevens Sarah , Symons Julian , Beigelman Leonid , Raboisson Pierre , Deval Jerome
Published: September 2020

Protein arginine methyltransferase-5 (PRMT5) regulates many essential biological processes by catalyzing the symmetric di-methylation of arginine (sDMA) on a variety of histones and non-histone proteins. PRMT5 activity is dysregulated across many hematological and solid tumor and its overexpression is associated with poor patient prognosis. PRMT5 is also often overexpressed in cancer cells. Genetic and pharmacological inactivation have confirmed the importance of PRMT5 activity to the survival of many cancer cells, including hepatocellular carcinoma (HCC) and non-small cell lung cancer (NSCLC). Thus, inhibiting PRTMT5 activity has emerged as an attractive therapeutic strategy in oncology.

We used structure-based drug design to identify novel small molecule inhibitors of PRMT5. ALG-070005, ALG-070043 and ALG-070017 inhibited PRMT5 enzymatic activity in biochemical assays with half-maximal inhibitory concentrations (IC50s) values between 50-150 pM and showed high selectivity against a panel of six other PRMTs. In HepG2 cells (HCC), ALG-070005, ALG-070043 and ALG-070017 potently decreased sDMA levels and inhibited cell proliferation with IC50 values of 1, 3, and 0.2 nM, respectively. These compounds also exhibited similar antiproliferative activity in A549 NSCLC cells. The three novel PRMT5 inhibitors demonstrated favorable ADME/PK profiles with exposure levels in target organs well above IC50 values, even 24 hours after dosing. When dosed orally in mice, these Aligos compounds inhibited tumor growth in HCC and NSCLC xenograft models, with concomitant inhibition of sDMA in the tumor.

In conclusion, we identified and characterized novel potent and selective inhibitors of PRMT5 with antiproliferative effects in various preclinical models of HCC and lung cancers. These drug candidates also have a broad therapeutic potential in oncology beyond HCC and lung cancer.

Citation Format: Francois Gonzalvez, Koen Vandyck, Yannick Debing, Kusum Gupta, Dinah Misner, Qingling Zhang, Jyanwei Liu, Antitsa Stoycheva, Sarah Stevens, Julian Symons, Leonid Beigelman, Pierre Raboisson, Jerome Deval. Discovery of novel potent and selective inhibitors of PRMT5 with anti-tumor activity in hepatocellular carcinoma and lung pre-clinical models [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1758.
Abstract 180: Systematic genomic analysis of matched PDX, PDX-derived organoids (PDXO), and PDX-derived cell lines (PDXC)
Authors: Xu Xiaoxi , Qian Wubin , Guo Sheng , Li Henry Q.
Published: September 2020

INTRODUCTION

Patient-derived xenografts (PDX) are considered predictive in vivo models for assessing cancer therapeutics due to their close genomic and histopathological resemblance to primary patient tumors. PDX can also be used to derive in vitro organoids (PDXO) and cell lines (PDXC), as we recently proposed and demonstrated, essentially creating matched pairs of in vitro and in vivo models. Cell lines serve as a conventional solution for fast and affordable evaluation of chemo- and targeted therapies, but they suffer from reduced clinical translatability. Organoids maintain organ structures and relevant functions in 3D culture, and are considered to be more physiologically relevant cancer models1. It is possible that the three experimental models have different genomic profiles, caused by different growing conditions, and that this will affect therapeutic evaluation, yet a systematic analysis is so far lacking.

METHODS

We developed a set of PDX-PDXO-PDXC model trios. Models were characterized for morphology, histopathology, genomics, and response to anticancer drugs, followed by a comparative analysis with an emphasis on genomic profiles.

RESULTS

We found that PDXO and PDXC have varied model establishment rates from PDX depending on cancer type. PDXO and PDXC show characteristic morphology. Like PDX, they exhibit model-specific growth kinetics and drug response. A high concordance of drug response was observed between PDX and matched PDXO. Genome-level mutation analysis showed good conservation of both somatic and driver mutations among the trios, indicating largely identical tumorigenesis mechanisms. However, significant transcriptomic differences were observed in pairwise comparisons between PDX-PDXO, PDX-PDXC, and PDXO-PDXC. We performed an in-depth analysis of differentially expressed genes, pathways, and gene networks to understand the underlying molecular mechanisms and their potential impact on drug evaluation.

CONCLUSIONS

Our study is the first large-scale comparative study to help understand the relatedness and uniqueness of PDX, PDXO, and PDXC on genomics.

REFERENCES

[1] Tuveson, D. & Clevers, H. Cancer modeling meets human organoid technology. Science 364, 952-955 (2019).

Citation Format: Xiaoxi Xu, Wubin Qian, Sheng Guo, Henry Q. Li. Systematic genomic analysis of matched PDX, PDX-derived organoids (PDXO), and PDX-derived cell lines (PDXC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 180.
Abstract 1811: Sorafenib-mediated apoptotic and autophagic cell death is increased by carnosic acid and a vitamin D2 analog in hepatocellular carcinoma (HCC)
Authors: Wang Xuening , Wu Qunfeng , Pham Kien , Luna Aesis , Danilenko Michael , Liu Chen , Studzinski George P.
Published: September 2020

Hepatocellular carcinoma (HCC) is the third most common cause of cancer mortality worldwide. Its incidence is increasing, and currently there is no curative therapy for it in advanced cases. Sorafenib (Sf), a multikinase inhibitor, is an FDA approved drug for the treatment of unresectable hepatomas, but the improvement in patient survival is very short, so new approaches are needed to supplement the existing regimens. In this study we show that the addition of a plant antioxidant Carnosic acid (CA) and a vitamin D2 derivative Doxercalciferol (D2) to Sf can markedly enhance its cytotoxic effect on HCC cell lines Huh7 (Sf-sensitive) and HCO2 (partially Sf-resistant). In contrast, similar combinatorial treatment of Hu1545 cells (immortalized human hepatocytes) results in only a minimal increase in cell death (CD). The combination Sf-CA/D2 increases HCC cell death by enhancing both apoptosis and cytotoxic autophagy (AuPh). Apoptosis was shown by Annexin V binding analysis and by the increased levels of apoptosis-related proteins BIM, cleaved caspases 9 and 3. AuPh was demonstrated by increased cytoplasmic vacuolation and elevated protein levels of Beclin1, Atg3, and LC3-II. Individually, both CA and D2 enhance Sf-induced CD, but CA produces the predominant effect. These effects were significantly reduced by the addition of the Caspase 3 inhibitor Z-DEVD-fmk or the AuPh inhibitor Chloroquine, and by Beclin1 depletion. A pilot experiment with mice bearing a Huh7 HCC cell xenograft showed markedly increased survival of the animals when CA was added to Sf. These results suggest that CA alone or with D2 may increase the survival of Sf-treated HCC patients. Supported by NIH grants 1R01DK110024 (to CL) and 5R01 44722-27 (to GPS).

Citation Format: Xuening Wang, Qunfeng Wu, Kien Pham, Aesis Luna, Michael Danilenko, Chen Liu, George P. Studzinski. Sorafenib-mediated apoptotic and autophagic cell death is increased by carnosic acid and a vitamin D2 analog in hepatocellular carcinoma (HCC) [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1811.
Abstract 1831: Mistletoe lectin induces apoptosis in MYC-driven cancers through post-translational downregulation of c-MYC protein
Authors: Kennelley Gabrielle , Yuan Eric , Shatat Mohammad , Gauthier Betsy , Yang Peiying , Difeo Analisa , Lee Richard
Published: September 2020

c-Myc is part of the Myc family proteins associated with cellular proliferation, differentiation, and apoptosis. Dysregulated c-Myc expression is associated with tumorigenesis, and cancers with c-Myc overexpression are often associated with poor prognosis, partly due to difficulty in targeting c-Myc. Mistletoe lectin (ML), derived from the European mistletoe plant, has shown variable efficacy in cancer patients. One case series following 12 patients with advanced hepatocellular carcinoma (HCC) showed 2 patients with >30% reduction in the tumor marker alpha-fetoprotein and 1 patient who had stable disease for 9 months. These cases indicate promising results. ML consists of a heteroprotein complex of an A- and B-chain connected by a disulfide bond. The A-chain was previously described to inhibit translation by 28s rRNA cleavage, while the B-chain binds cell surface glycoproteins to trigger internalization of the lectin. We found that purified mistletoe extract, consisting primarily of ML, downregulates c-Myc protein and induces apoptosis in in vitro and in vivo models of human-derived cancer, and that the efficacy of ML treatment is strongly correlated with MYC gene expression and c-Myc protein abundance in in vitro human models of HCC, ovarian cancer, small cell lung cancer, and lymphoma. MTS assay data demonstrated that exogenous MYC expression can sensitize low-MYC cell lines to ML treatment, suggesting that anti-cancer effects of ML are dependent on its ability to downregulate c-Myc. While ML may be able to downregulate many proteins via general translational inhibition, we found that of 296 proteins profiled by RPPA, c-Myc was one of 11 proteins decreased by ML with at least a 2-fold change (p<0.01). We also found that ML promotes c-Myc ubiquitylation, suggesting that ML regulates c-Myc protein stability, rather than synthesis. In summary, here we describe a novel mechanism of action for ML in downregulating c-Myc and promoting apoptotic cell death in various models of MYC-driven cancer, and provide evidence for MYC gene expression as a predictive marker for response. This research supports the development of ML-based therapies that may provide a c-Myc-targeting treatment in multiple cancer types.

Citation Format: Gabrielle Kennelley, Eric Yuan, Mohammad Shatat, Betsy Gauthier, Peiying Yang, Analisa Difeo, Richard Lee. Mistletoe lectin induces apoptosis in MYC-driven cancers through post-translational downregulation of c-MYC protein [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1831.
Abstract 1881: Immune mediated interaction between radiation and cetuximab in a syngeneic murine model of head and neck squamous cell carcinoma
Authors: Jin Wonjong , Erbe Amy K. , Schwarz Ciara , Jaquish Abigail , Anderson Bryce R. , Sriramaneni Raghava N. , Jagodinsky Justin C. , Clark Paul A. , Morris Zachary S.
Published: September 2020

Purpose: Patients with head and neck squamous cell carcinoma (HNSCC) treated with anti-EGFR (cetuximab) mAb-based immunotherapy often acquire therapy-resistance, reducing clinical benefit. We evaluated the efficacy of a combined modality in situ vaccination regimen aimed at overcoming resistance to cetuximab using a syngeneic tumor model.

Experimental Design: The murine HNSCC cell line MOC2 was transduced to express human EGFR (MOC2-huEGFR) and used as a syngeneic model of therapeutic resistance to cetuximab. The cells were tested for viability, radiosensitivity, and huEGFR-specific mAb binding efficacy in the presence of cetuximab. Antibody-dependent cellular cytotoxicity (ADCC) was tested by coculture of 51chromium-labeld MOC2-huEGFR with PBMC. Mice bearing these syngeneic tumors were treated with therapeutic regimens that included local radiation (RT), intratumoral (IT) cetuximab, and/or systemic immune checkpoint inhibitors, and tumor growth/survival rate were monitored.

Result: Cetuximab induced antibody-dependent cellular cytotoxicity (ADCC) in MOC2-huEGFR and PBMC co-cultures, but showed no effect on cell proliferation or radiosensitivity. Combined RT and IT-cetuximab treatment induced tumor growth delay, which required huEGFR expression, NK cells, and FcγR expression. In addition, combined RT+IT-cetuximab therapy greatly induced tumor infiltration by both CD8 T cells and NK cells. RT promoted NK cell-mediated ADCC effects against 51chromium-labeld MOC2-huEGFR with increased IFNγ expression. Proliferating tumor cells gradually increased Pd-l1 mRNA expression, and RT enhanced cell surface PD-L1 in MOC2-huEGFR cells. Systemic anti-PD-L1 antibody or IT-cetuximab alone did not show antitumor response against MOC2-huEGFR bearing mice; however, significant tumor growth inhibition was observed with combined anti-PD-L1, RT, and IT-cetuximab.

Conclusion: The MOC2-huEGFR HNSCC tumor model is immunologically “cold” and does not respond to either cetuximab or anti-PD-L1 therapies alone. In this model, we show that a combination of RT+cetuximab+anti-PD-L1 elicits a cooperative in situ vaccine effect. Combining RT, cetuximab and immune checkpoint blockade may enable anti-tumor immune response in HNSCC patients with immunologically cold, huEGFR-expressing tumors.

Citation Format: Wonjong Jin, Amy K. Erbe, Ciara Schwarz, Abigail Jaquish, Bryce R. Anderson, Raghava N. Sriramaneni, Justin C. Jagodinsky, Paul A. Clark, Zachary S. Morris. Immune mediated interaction between radiation and cetuximab in a syngeneic murine model of head and neck squamous cell carcinoma [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1881.
Abstract 1936: Immunoproteasome inhibitors for the treatment of ALL
Authors: Jenkins Tyler W. , Downey-Kopyscinski Sondra , Colley William C. , Fields Jennifer L. , Fiering Steven N. , Kisselev Alexei F.
Published: September 2020

The t(4;11)(q21;q23) chromosomal translocation that creates MLL-AF4 fusion protein confers a poor prognosis protein in infant acute lymphoblastic leukemia (ALL). This translocation also sensitizes cells to proteasome inhibitors bortezomib and carfilzomib, which are approved by the FDA for the treatment of multiple myeloma. Clinical activity of bortezomib in combination with standard chemotherapy has been documented in several clinical trials of ALL patients, and a case of a single-agent activity against relapsed leukemia driven by MLL-AF4 translocation has been described. However, toxicities of bortezomib and carfilzomib may be unacceptable to pediatric patients. We found that the overwhelming majority of proteasomes in this subtype of ALL are lymphoid tissue specific immunoproteasomes. Cells with MLL-AF4 translocations were sensitive to pharmacologically relevant concentrations of specific immunoproteasome inhibitor ONX-0914. Furthermore, ONX-0914 dramatically delayed growth of orthotopic xenograft tumors in mice. Thus, immunoproteasomes are therapeutic targets in ALL; replacing bortezomib and carfilzomib with immunoproteasome inhibitors in ALL should reduce toxicities associated with inhibition of the proteasomes in non-lymphoid tissues.

Citation Format: Tyler W. Jenkins, Sondra Downey-Kopyscinski, William C. Colley, Jennifer L. Fields, Steven N. Fiering, Alexei F. Kisselev. Immunoproteasome inhibitors for the treatment of ALL [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1936.
Abstract 1955: Inhibition of eIF4A by eFT226 blocks KRAS mutant tumor growth
Authors: Stumpf Craig R. , Goel Vikas K. , Chen Joan , Staunton Jocelyn , Santori Emily M. , Barrera Maria , Howard Haleigh , Webster Kevin R. , Chiang Gary G. , Thompson Peggy A.
Published: September 2020

Mutations in KRAS are among the most common oncogenic lesions across a variety of human cancers. Activation of KRAS directs signaling via the MAPK and PI3K pathways to promote tumor growth. One outcome of enhanced KRAS signaling is the induction of mRNA translation by eIF4A, eIF4E, and eIF4G, which together comprise the eIF4F complex. eIF4A is an RNA helicase that functions to unwind elements in the 5'-untranslated region (UTR) of mRNAs to facilitate scanning of the 40S ribosomal subunit. eFT226 is a highly potent and selective inhibitor of eIF4A that functions by forming a ternary complex between eIF4A, eFT226 and specific polypurine motifs in the 5'-UTR of select mRNAs, thus blocking ribosome scanning and inhibiting mRNA translation. This polypurine sequence motif is highly enriched in the 5'-UTR of eFT226 target genes, many of which are known proto-oncogenes. Translational profiling revealed KRAS to be a target of eFT226 and 5'-UTR sequence analysis of KRAS mRNA identified the polypurine regulatory motif, which imparts sensitivity to eFT226 mediated inhibition of translation. In cell-based reporter assays used to monitor translation, mutation of the KRAS 5'-UTR polypurine motifs resulted in a 10-fold decrease in sensitivity to eFT226 relative to the wild-type sequence. In a cell panel screen for in vitro apoptosis induction by eFT226, the most sensitive models were enriched for cell lines driven by KRAS mutations. Consistent with these results, treatment with eFT226 decreased KRAS protein levels, repressed downstream MAPK signaling, inhibited cell proliferation, and induced apoptosis in a collection of non-small cell lung, colorectal and pancreatic KRAS mutant cancer cell lines. The ability of eFT226 to block tumor cell growth and induce apoptosis is independent of the specific KRAS mutation present (e.g. G12C/V, G13D, Q61H/L) suggesting that eFT226 could be broadly efficacious in treating tumors with activating KRAS mutations. Treatment of KRAS mutant solid tumor xenografts with eFT226 as a monotherapy significantly inhibited tumor growth. Together, these results highlight the ability of eFT226 to inhibit growth and promote apoptosis in KRAS mutant tumors and support the clinical development of eFT226 in KRAS driven tumors. A clinical trial evaluating eFT226 in patients with solid tumor malignancies has initiated.

Citation Format: Craig R. Stumpf, Vikas K. Goel, Joan Chen, Jocelyn Staunton, Emily M. Santori, Maria Barrera, Haleigh Howard, Kevin R. Webster, Gary G. Chiang, Peggy A. Thompson. Inhibition of eIF4A by eFT226 blocks KRAS mutant tumor growth [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1955.
Abstract 1981: Cell free DNA promotes malignant transformation in non-tumor cells
Authors: Souza Aline Gomes , Martins Mario Machado , Bastos Victor Alexandre , Resende Natassia Caroline , Santos Paula Sousa , Marangoni Karina , Goulart Luiz Ricardo , Goulart Vivian Alonso
Published: September 2020

Cell free DNA (cfDNA) are present at various biological fluids and when released by tumor cells it can contribute with the induction of pro-tumorigenic events, such as malignant transformations and metastasis. In this work we evaluated the effects of tumor derived cfDNA, isolated from blood of prostate cancer patients, in the non-tumoral prostate cellular linages RWPE-1 and PNT-2. We quantified, characterized and evaluated the function of the isolated cfDNA by qPCR of tumor progression related genes and MS-metabolomics. The results showed that the isolated cfDNA was able to induce the increase expression of MMP9 and CD44 in the normal prostate cellular linages, as well as to alter the consumption of tryptophan by the cells, these changes can indicate the sifting of the cells metabolism in favor of a tumoral profile. Those results demonstrate the potential of tumor derived cfDNA to alter receptor cells profiles, triggering malignant transformation events, making them a good target for new therapies and molecular targets for diagnosis.

Citation Format: Aline Gomes Souza, Mario Machado Martins, Victor Alexandre Bastos, Natassia Caroline Resende, Paula Sousa Santos, Karina Marangoni, Luiz Ricardo Goulart, Vivian Alonso Goulart. Cell free DNA promotes malignant transformation in non-tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1981.