bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 122: Stromal extracellular matrix is a microenvironmental cue promoting resistance to EGFR tyrosine kinase inhibitors in lung cancer cells
Authors: Zhang Ting , Guo Lixia , Wang Yuanyuan , Ren Tao , Yang Yanan
Published: August 2019

The acquisition of resistance to EGFR tyrosine kinase inhibitors (TKIs) remains a critical problem in lung cancer clinic, but the underlying mechanisms have remained incompletely understood. Although the TKI-induced or -selected genetic changes are known to drive resistance, resistance also occurs in tumor cells without genetic changes through poorly-characterized processes. Here, we show that the extracellular matrix (ECM) from various components of the tumor microenvironment, including neighboring tumor cells and fibroblasts, may be the driver of resistance in the absence of genetic changes. Unlike genetic changes, which may evolve during relatively long time of chronic EGFR TKI treatment to drive resistance, briefly culturing on de-cellularized ECM, or co-culturing with the ECM donor cells, immediately confers resistance to tumor cells that are otherwise sensitive to EGFR TKIs. We show evidence that collagen in the ECM may be its primary constituent driving resistance, at least partly through the collagen receptor Integrin-β1. Intriguingly, such effect of ECM and collagen is dose-dependent and reversible, suggesting a potential clinic-relevant application for targeting this effect. Collectively, our results reveal that the stromal ECM acts as a microenvironmental cue promoting EGFR TKI resistance in lung cancer cells, and targeting collagen and Integrin-β1 may be useful for treating resistance, especially the resistance without clearly-defined genetic changes, for which effective therapeutics are lacking.

Citation Format: Ting Zhang, Lixia Guo, Yuanyuan Wang, Tao Ren, Yanan Yang. Stromal extracellular matrix is a microenvironmental cue promoting resistance to EGFR tyrosine kinase inhibitors in lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 122.
Abstract 1220: High-resolution epitope mapping differentiates monoclonal antibodies in the crowded cancer immunotherapy space
Authors: Willis Sharon
Published: August 2019

Epitope mapping provides essential information for differentiating monoclonal antibodies (MAbs) that bind to proteins targeted by many competing drugs. In cancer immunotherapy, for instance, numerous approved or investigational MAbs target the immune checkpoint protein PD-1. Recent high-profile patent law cases have centered on epitope data, highlighting the importance of distinguishing MAb binding sites at amino-acid resolution. Additionally, epitope data can be used to distinguish the mechanisms by which MAbs exert their therapeutic effects, uncovering important differences between competing drugs. To achieve rapid and comprehensive conformational epitope mapping at the amino-acid level, we developed the Shotgun Mutagenesis Epitope Mapping platform. Every residue in a target protein is individually mutated by alanine scanning mutagenesis, expressed in human cells, and assayed by flow cytometry to determine the critical epitope residues. Data from our platform have been used to differentiate MAbs in terms of their binding residues and therapeutic mechanisms of action. We have used our platform to differentiate the unique binding residues and, in some cases, the mechanisms of action of novel MAbs on the crowded surfaces of cancer targets, such as HER-2, from those of benchmark MAbs.

Citation Format: Sharon Willis. High-resolution epitope mapping differentiates monoclonal antibodies in the crowded cancer immunotherapy space [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1220.
Abstract 1227: Immune modulatory effect of pramlintide for cutaneous squamous cell carcinoma treatment
Authors: Tordesillas Leticia , Hesterberg Rebecca , Ortiz-Rivera Ivannie , Sell Brittney R. , Chiang Omar Chavez , Nguyen Kimberly T. , Murphy Brian L. , Burnette Pearlie K. , Flores Elsa R. , Tsai Kenneth Y.
Published: August 2019

Cutaneous squamous cell carcinoma (cuSCC) accounts for 15-20% of skin cancers. Treatment of cuSCC with pramlintide, a synthetic analog of the hormone amylin, is currently under investigation in mouse models and human clinical trials. In cancer cells, pramlintide inhibits glycolysis, resulting in cell death and tumor regression. In order to investigate the potential use of pramlintide in combinatory therapies with checkpoint inhibitors, we aimed to study the immune modulatory effect of pramlintide in an immune competent mouse model of spontaneous ultraviolet radiation-induced cuSCC, as well as its effect on human T cells in vitro. To induce cuSCC, SKH-1 hairless mice were subjected to ultraviolet radiation until at least one tumor reached >4mm of diameter. Mice were then treated with 45 µg/kg of pramlintide or vehicle control for 10 days, every other day. Skin-draining lymph nodes, spleen and tumors were harvested for immunophenotyping of the T cell and myeloid compartment by flow cytometry. While the proportion of CD103+ dendritic cells (DCs) and CD3+ T cells remained constant after pramlintide treatment, a significant decrease in the proportion of both monocytic and granulocytic myeloid-derived suppressor cells (m-MDSCs and g-MDSCs) was observed relative to vehicle-control treated mice. Interestingly, pramlintide abolished m-MDSC differentiation from bone marrow progenitors in vitro suggesting that pramlintide may regulate a pathway necessary for m-MDSC development. Purified human CD3+ T cells activated in the presence of pramlintide exhibited a time-dependent increase in intracellular and secreted IFN-γ. Therefore, pramlintide may have immune modulatory effects based on both a reduction in myeloid-dependent suppression and a direct stimulatory effect on T cells that would culminate in superior anti-tumor T cell responses that augment immunotherapeutic approaches for the treatment of cuSCC.

Citation Format: Leticia Tordesillas, Rebecca Hesterberg, Ivannie Ortiz-Rivera, Brittney R. Sell, Omar Chavez Chiang, Kimberly T. Nguyen, Brian L. Murphy, Pearlie K. Burnette, Elsa R. Flores, Kenneth Y. Tsai. Immune modulatory effect of pramlintide for cutaneous squamous cell carcinoma treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1227.
Abstract 123: Evaluating cellular cytotoxicity and potency of antibody-drug conjugates within 3-dimensional tumor models
Authors: Mandavilli Bhaskar S. , Langsdorf Chris , Chen Aimei , Hu Yi-Zhen , Wickett Marcy , Clark Scott T.
Published: August 2019

3-dimensional tumor spheroids provide biochemical conditions that closely resemble the tumor microenvironment in an intact organism. Noninvasive approaches such as fluorescence microscopy and high content analysis are highly advantageous as they allow for the study of these 3-dimensional systems. Here we investigate the penetration and potency of natural killer cells, cytotoxic T cells, and antibody-drug conjugates in three-dimensional models of breast and lung cancer. Unstimulated T cells produced minimal cytotoxicity, similar to untreated spheroids. Activated T cells penetrated and produced significant cytotoxicity throughout cancer spheroids. SKBR3 breast cancer cells form a compact, viable spheroid. Addition of NK cells leads to moderate cytotoxicity, while addition of NK cells and trastuzumab results in substantial cytotoxicity and degradation of spheroid structure. Trastuzumab labeled with iFL pHrodo Red becomes brightly fluorescent following specific endosomal internalization into breast cancer cells, but minimal toxicity is observed. Trastuzumab conjugated with both iFL pHrodo Red and MMAE internalizes into cells and results in cell killing. Fluorescence microscopy combined with novel cell and antibody labeling methods permits investigation of the penetration and potency of natural killer cells, cytotoxic T cells, and antibody-drug conjugates in three-dimensional solid tumor models.

Citation Format: Bhaskar S. Mandavilli, Chris Langsdorf, Aimei Chen, Yi-Zhen Hu, Marcy Wickett, Scott T. Clark. Evaluating cellular cytotoxicity and potency of antibody-drug conjugates within 3-dimensional tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 123.
Abstract 124: Evaluating heterogeneity of tumor microenvironment using single cell RNA sequencing
Authors: Cho Untack , Song Yong Sang , Park Woong-Yang , Lee Hae-Ock , Han Youngjin
Published: August 2019

Tumor microenvironment interacts with cancer cells, causing tumor progression. Tumor microenvironment consists of cellular and acelluar components. We investigated cellular components of tumor microenvironment and heterogeneity of ovarian cancer. We performed single cell RNA sequencing analysis of cellular components of malignant ascites from a patient with advanced ovarian cancer. Among the various cell types including cancer cells, B cells, macrophages and fibroblasts, macrophage was the most dominant in malignant ascites. To determine the heterogeneity of ovarian cancer, we obtained primary ovarian cancer tissue from ovarian cancer patient. We performed single cell RNA sequencing analysis of cellular components derived from ovarian cancer tissue. We determined intra-heterogeneity of ovarian cancer and cellular components of ovarian cancer tissue. Taken together, we determined cellular components of ovarian cancer tissue and ascites and intra-heterogeneity of ovarian cancer. This study can provide insight of tumor microvironment and help understanding of ovarian cancer heterogeneity.

Citation Format: Untack Cho, Yong Sang Song, Woong-Yang Park, Hae-Ock Lee, Youngjin Han. Evaluating heterogeneity of tumor microenvironment using single cell RNA sequencing [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 124.
Abstract 1246: Comprehensive RNA interference induced by CRISPR/Cas13a gene editing system in human cancer cells
Authors: Wang Qixue , Wang Yunfei , Tan Yanli , Kang Chunsheng
Published: August 2019

RNA is the intermediate link of central dogma. However, RNA is rarely used as a therapeutic target for its flexible structure and instability. CRISPR/Cas13a is reported as a powerful tool for RNA knockdown. The potential amplification of CRISPR/Cas13a in cancer cells could be further studied. Here we overexpressed the Cas13a protein by lenti-virus vectors in glioma and other cancer cells, and found out that crRNA-EGFP could induce the “collateral effect” after knocking down the target gene. EGFRvIII is a unique EGFR mutant subtype in glioma. CRISPR/Cas13a system could induce cell death of EGFRvIII overexpression glioma cells. Single cell and RNA-seq analysis confirmed the comprehensive RNA interference by CRISPR/Cas13a treatment. Furthermore, CRISPR/Cas13a system could inhibit the formation of glioma intracranial tumor in mice. Our results exhibit the comprehensive RNA interference of CRISPR/Cas13a system in cancer cells, and demonstrated the powerful tumor elimination potential of this system.

Citation Format: Qixue Wang, Yunfei Wang, Yanli Tan, Chunsheng Kang. Comprehensive RNA interference induced by CRISPR/Cas13a gene editing system in human cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1246.
Abstract 125: Digital Spatial Molecular Profiling evidence from H&E stained FFPE that the tumor microenvironment confers gene expression phenotype specificity
Authors: Imler Elliot , Nagle Raymond , Seligmann Bruce
Published: August 2019

Identifying the molecular phenotype of cells within the context of their tumor tissue microenvironment may be critical to understanding cancer and improving patient treatments and outcomes. TempO-Seq® (DOI:10.1371/journal.pone.0178302, doi.org/10.1093/toxsci/kfx153) can be used to profile gene expression from lysates of scraped FFPE tissue down to 2 mm2 areas of 5 μm thick sections. We report use of an in situ TempO-Seq transcriptomic assay carried out on slide-mounted FFPE by an automated stainer (Leica BOND), followed by H&E staining and imaging to perform standard histology. Regions of interest (ROIs) down to 20 μm in diameter are selected at the same time. Probes from these ROIs are automatically eluted, obtaining a detailed gene expression profile. This process is called Digital Spatial Molecular Profiling (DSMP). DSMP was used to profile gene expression from histologically discrete areas within tumor sections. A pan cancer assay of 5,207 genes was used, containing biomarkers for cancer, immune responses and interactions, as well as the S1500v2 whole transcriptome surrogate assay (doi.org/10.1371/journal.pone.0191105), permitting all known molecular pathways to be mapped. Data analysis was carried out using the automated TempO-SeqR™ software package. Replicate areas of cancer, high grade PIN (prostatic intraepithelial neoplasia), normal epithelium, and stroma within prostate cancer FFPE sections were profiled and compared to the profiling of a 2 mm2 scraped area of tissue defined as 100% cancer. What was notable from the DSMP data was that key cancer and immune cell biomarkers were not detected at all in normal, key normal biomarkers were not detected at all in cancer, PIN expressed a collection of normal, cancer and immune biomarkers, and stromal biomarkers were not detected in normal, PIN or cancer glands, while housekeepers were detected in all tissue types. In contrast, housekeeper genes and key biomarkers of cancer, normal, stroma, and immune cells, were all expressed in the 100% pure area of cancer, lacking the biomarker specificity afforded by histologically focused DSMP profiling. The DSMP specificity obtained from the FFPE tissue was striking, leading us to hypothesize that factors within the tumor microenvironment (whether it is adjacent cells, the structural effects of tissue, or sequestration/gradients of extracellular modulators within the tissue) exert molecular specificity on cells within the tissue not present in cultured or dissociated cells devoid of context.

Citation Format: Elliot Imler, Raymond Nagle, Bruce Seligmann. Digital Spatial Molecular Profiling evidence from H&E stained FFPE that the tumor microenvironment confers gene expression phenotype specificity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 125.
Abstract 1265: Validation of the interaction between a candidate compound and the intended drug target by a phenotypic rescue approach
Authors: Geerts Lieke , Grandmoursel Laure , Aarbiou Jamil , DeGroot Jeroen , Schüler Julia , Waddell Ian , Zuurmond Anne-Marie
Published: August 2019

New targets for cancer treatment frequently emerge in literature, but the thorough target validation required to consider these targets for a drug discovery program is often lacking. In pharmacological or genetic perturbation studies using complex biological assays, undesired off-target effects cannot be easily distinguished from the intended mode of action at the desired target. This is especially evident in cancer drug development where it is important to discriminate on-target effects on cell viability from off-target effects resulting in non-specific loss of cellular fitness. Neglecting the possibility of being deceived by off-target effects can have tremendous scientific and financial impact on a drug discovery program. Ideally confidence in a preclinical drug target and a modulating compound is boosted in an early stage by more extensive analysis and validation of the actual drug-target relationship. Rescue of a disease-relevant phenotype by genetic restoration of a target mutation is a gold standard approach in drug discovery by which target validation can be achieved. We aim to follow this approach targeting the BRAF V600E mutation in a number of well described melanoma lines as well as the MAP2K1 Q56P mutation in non-small cell lung cancer cell line H1437. Target validation for both BRAF and MAP2K1 will be addressed by assessing viability, phenotypic changes and sensitivity to compound modulation upon CRISPR/Cas9 repair of the target mutation or by exogenous re-expression of the wildtype variant. Compounds tested will be Vemurafenib for BRAF and Trametinib for MAP2K1. Further investigation into target validity will be done using a physiologically relevant 3D spheroid based co-culture system. Mimicking the tumor microenvironment increases the knowledge about “drug-ability” of a target and sustainability of the target modulation at an early time point in the development process. Such early in-depth validation of the relationship between a compound and the drug target is vital to mitigate the risk of failure at later steps of drug development.

Citation Format: Lieke Geerts, Laure Grandmoursel, Jamil Aarbiou, Jeroen DeGroot, Julia Schüler, Ian Waddell, Anne-Marie Zuurmond. Validation of the interaction between a candidate compound and the intended drug target by a phenotypic rescue approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1265.
Abstract 127: CYP4A11 overexpression increases aggressiveness in glioblastoma and breast cancer
Authors: Borin Thaiz F. , Ali Sehar , Angara Kartik , Rashid Mohammad , Myers Stephanie , Chawla Divya , Ara Roxan , Lebedyeva Iryna , Achyut Bhagelu R. , Arbab Ali S.
Published: August 2019

Metastatic breast cancer (BC) and glioblastoma (GBM) are two highly invasive forms of cancer that contribute greatly to patient mortality. Due to the complex mechanisms underlying cancer growth and metastasis, novel therapeutic targets must be found to increase patient survival. One such target is the cytochrome P450 4 (CYP4) metabolic pathway leading to the production of 20-hydroxyeicosatetraenoic acid (20-HETE). 20-HETE is a lipid signaling molecule that promotes invasion, growth, and neovascularization in tumors by inducing the proliferation and migration of both, the endothelial cells and the tumor cells, therefore promoting BC and GBM tumor survival. N-hydroxy-N’-(4-butyl-2 methyl phenyl) formamidine (HET0016) is a selective inhibitor of 20-HETE synthesis and thus impairs tumor growth and neovascularization. In this study, we investigate the functional and phenotypic changes in BC (MDAMB231 and MCF7) and GBM (U251 and U87) cells overexpressing CYP4A11, a human CYP4 enzyme, with and without HET0016 treatment. Cells that overexpressed CYP4A11 were produced using lentivirus infection and confirmed via western blot. Tumor cell aggressiveness was assessed in vitro using migration and proliferation assays and in vivo by ascertaining the Ki67 levels and incidence of lung metastases foci. Tumor growth with and without CYP4A11 overexpression was followed for 21 days in vivo in athymic nude mice for all groups of cells and the acquisition of stem cell and angiogenic phenotypes by the tumor cells were determined by flow cytometry. The results demonstrated that cells overexpressing CYP4A11 showed protein levels 3-6 times higher than the non-modified cells. CYP4A11-overexpressing MDAMB231 and U87 cells demonstrated increased proliferation capacity, motility and significantly increased tumor growth. Interestingly, MCF7 and U251 cells overexpressing CYP4A11 did not show significant alterations in tumor growth compared to non-modified cells. Additionally, all cells treated with HET0016 showed decreased migration after 24 hours. U87 tumors showed increased stem cell and angiogenic phenotypes compared to non-modified U87 tumors. In conclusion, overexpression of CYP4A11 in Triple Negative BC and U87 GBM cells increased tumor aggressiveness proving that this pathway is an important target for therapeutic inhibition to control tumor growth and invasion.

Citation Format: Thaiz F. Borin, Sehar Ali, Kartik Angara, Mohammad Rashid, Stephanie Myers, Divya Chawla, Roxan Ara, Iryna Lebedyeva, Bhagelu R. Achyut, Ali S. Arbab. CYP4A11 overexpression increases aggressiveness in glioblastoma and breast cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 127.
Abstract 1270: Human P-glycoprotein-mediated drug transport: Two aromatic residues in the drug-binding pocket are critical for function
Authors: Murakami Megumi , Chufan Eduardo E. , Lusvarghi Sabrina , Ambudkar Suresh V.
Published: August 2019

P-glycoprotein (P-gp) is an efflux pump that transports cytotoxic agents, thereby affecting the pharmacokinetics of many drugs and conferring resistance to chemotherapeutic agents in many cancers. P-gp utilizes energy from ATP hydrolysis for the efflux of a number of amphipathic natural products or small molecule anticancer drugs. However, the nature and number of translocation pathways for the transport of substrate-drugs by P-gp is not yet known. Previously we showed that substitution of certain aromatic residues in the drug-binding pocket switched the modulation of ATPase activity from inhibition to stimulation. Molecular modeling studies led to identification of two T-shaped structural motifs formed by F728/Y310 and F978/Y953. In this study, we demonstrate that substitution of all four residues of the T-shaped structural motifs with alanine (Y310A/F728A/Y953A/F978A) results in complete loss of transport of ten fluorescent substrates. Furthermore, alanine substitution of one or two of these residues revealed that when F728 and F978 are both substituted, this has the greatest impact on the binding and/or translocation of drug-substrates. ATPase assays and in silico analyses were performed to gain insight into interactions of these residues with substrates and modulators. These results indicate that the F728 and F978 residues together are critical to maintaining the conformation of the drug-binding pocket for binding and translocation of substrates by P-gp.

Citation Format: Megumi Murakami, Eduardo E. Chufan, Sabrina Lusvarghi, Suresh V. Ambudkar. Human P-glycoprotein-mediated drug transport: Two aromatic residues in the drug-binding pocket are critical for function [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1270.
Abstract 1273: Molecular dynamics prediction of the mechanism of acquired resistance to EGFR inhibitors in EGFR-mutant lung cancer
Authors: Lee Youngjoo , Choi Yu-Ra
Published: August 2019

Background Acquired drug resistance is the most challenging problem in treating nonsmall cell lung cancer (NSCLC) patients with EGFR tyrosine kinase inhibitor (TKI). We evaluated whether resistance cell subclones significantly increased shortly after treatment with EGFR-TKIs could determine the final resistance phenotype for EGFR-TKIs in the tumor with EGFR mutations.

Methods We generated 2 EGFR-mutant lung cancer cell lines (PC9GR and HCC827GR) resistant to gefitinib and evaluated the resistance mechanisms in each cell line. In parent cell lines (PC9 and HCC827) the amounts of EGFR T790M mutation and C-MET and VIMENTIN mRNA expression were measured before and after 48 hours treatment with gefitinib or paclitaxel. Using a cell line derived directly from tumors of treatment-naïve NSCLC patient with EGFR mutation, the above tests were repeated. We prospectively collected the bloods from 4 NSCLC patients with EGFR mutations at baseline and eight weeks after EGFR-TKI treatment and measured the level of T790M mutation in circulating tumor DNAs (ctDNA) from their bloods by colourimetric assay.

Results EGFR T790M mutation was detected in PC9GR cells whereas C-MET amplification in HCC827GR cells. The proportion of EGFR T790M mutation significantly increased in the PC9 cells treated with gefitinib whereas not in the same cells treated with paclitaxel or not in the HCC827 treated with gefitinib or paclitaxel. The mRNA expression of C-MET was significantly increased in the HCC827 treated with gefitinib. The VIMENTIN mRNA expression was not changed in both cell lines. When we treated the patient-derived cells with gefitnib, the frequency of T790M significantly increased whereas C-MET mRNA expression decreased and VIMENTIN mRNA expression was not changed. In the corresponding resistance tumor tissue, only T790M mutation was detected without C-MET amplification and VIMENTIN overexpression. Two cases whose T790M mutation level in their ctDNAs increased at 8 weeks by more than 50% revealed T790M mutation in their final resistance tumor tissue. But, in other cases without an early surge of T790M mutation in their ctDNAs, T790M mutation was not detected in posttreatment biopsy sample.

Conclusions Early-on-treatment quantitative change of resistance-related gene alterations may predict the final mechanism of resistance to EGFR-TKI.

Citation Format: Youngjoo Lee, Yu-Ra Choi. Molecular dynamics prediction of the mechanism of acquired resistance to EGFR inhibitors in EGFR-mutant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1273.
Abstract 1281: T790M gatekeeper mutation emerges via de novo at the early stages of erlotinib treament in PC9 non-small cell lung cancer cells
Authors: Kim Sujin , Park Angela KJ , Cho Jeonghee
Published: August 2019

The emergence of the T790M gatekeeper mutation in the Epidermal Growth Factor Receptor (EGFR) gene is an important mechanism that can lead to the acquired resistance to EGFR-targeted tyrosine kinase inhibitors used in a subset of non-small cell lung cancer (NSCLC) patients harboring activating mutations. Here we investigated the paths leading to the acquisition of the T790M mutation by establishing an erlotinib resistant PC9 cell model harboring ectopically introduced EGFR cDNA. We detect the emergence of T790M mutation within the EGFR cDNA in a subset of erlotinib resistant PC9 cell models through Sanger sequencing and droplet digital PCR-based methods, confirming that T790M mutation can emerge via de novo events following treatment with erlotinib. In addition, we show that the de novo T790M bearing erlotinib resistant PC9 cells are sensitive to the 3rd generation EGFR-targeted drug, WZ4002. Furthermore, GFP-based competition cell proliferation assays reveal that PC9 cells ectopically expressing EGFR mutant become more dominantly resistant to erlotinib than parental PC9 cells by acquiring T790M mutation. Taken together, we believe that our findings expand upon the previous notion of evolutionary paths of T790M development, providing an important clue to designing a therapeutic strategy to overcome drug resistance.

Citation Format: Sujin Kim, Angela KJ Park, Jeonghee Cho. T790M gatekeeper mutation emerges via de novo at the early stages of erlotinib treament in PC9 non-small cell lung cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1281.
Abstract 129: Tumor-associated macrophages secrete CCL2 and induce the invasive phenotype of human breast epithelial cells through upregulation of ERO1-alpha and MMP-9
Authors: Lee Seungeun , Lee Eunhye , Ko EunYi , Ham Mina , Lee Hye Min , Kim Eun-Sook , Koh Minsoo , Lim Hyun Kyung , Jung Joohee , Park So Yeon , Moon Aree
Published: August 2019

Tumor-associated macrophages (TAMs) are major components of tumor microenvironment that promote invasion and metastasis of cancer cells. In this study, we investigated the effect of TAMs on phenotypic conversion of non-neoplastic MCF10A human breast epithelial cells using an indirect co-culture system. Co-culture with TAMs induced epithelial-to-mesenchymal transition, invasive phenotype, and MMP-9 upregulation in MCF10A cells. Comparative proteomic analysis revealed that endoplasmic reticulum oxidoreductase (ERO)1-alpha was increased in MCF10A cells co-cultured with TAMs compared to that in mono-cultured cells. ERO1-alpha was crucial for TAMs-induced invasive phenotype and MMP-9 upregulation involving transcription factors c-fos and c-Jun. Cytokine array analysis showed that levels of interleukin (IL)-6, C-X-C motif ligand (CXCL)1, C-C motif ligand (CCL)2, growth-regulated protein (GRO), IL-8, and granulocyte-macrophage colony-stimulating factor (GM-CSF) were increased in conditioned media of co-cultured cells. Among these cytokines increased in conditioned media of co-cultured cells, CCL2 was secreted from TAMs, leading to induction of ERO1-alpha, MMP-9 upregulation, and invasiveness in MCF10A cells. Our findings elucidated a molecular mechanism underlying the aggressive phenotypic change of non-neoplastic breast cells by co-culture with TAMs, providing useful information for prevention or treatment of recurrent breast cancer

Note: This abstract was not presented at the meeting.

Citation Format: Seungeun Lee, Eunhye Lee, EunYi Ko, Mina Ham, Hye Min Lee, Eun-Sook Kim, Minsoo Koh, Hyun Kyung Lim, Joohee Jung, So Yeon Park, Aree Moon. Tumor-associated macrophages secrete CCL2 and induce the invasive phenotype of human breast epithelial cells through upregulation of ERO1-alpha and MMP-9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 129.
Abstract 1290: Development of anti inflammatory agents targeting TonEBP for treatment of chronic inflammatory diseases
Authors: YE Byeongjin , Kwon Hyug Moo , Choi Soo Youn , Lee Jun Ho , Kang Hyun Je , Park Cheol-Min
Published: August 2019

Tonicity-responsive enhancer binding protein (TonEBP), also known as nuclear factor of activated T cells 5 (NFAT5), belongs to the Rel family of transcriptional factors which include NF-κB and NFAT. TonEBP is a key regulator in chronic inflammatory diseases such as rheumatoid arthritis, atherosclerosis, diabetic nephropathy and hepatocellular carcinoma. TonEBP is a rate-limiting component of “pro-inflammatory enhanceosome” in which TonEBP provides a physical tether between activated transcription factors NF-κB and AP-1 to p300 and RNA polymerase II. Assembled in response to inflammatory signals, the pro-inflammatory enhanceosome drives the transcription of TNF-α, IL-1β, IL-6, MCP-1, iNOS, and COX-2. We discovered a class of compounds (CXs) targeting TonEBP. CXs blocked the assembly of the pro-inflammatory enhancesome and potently suppressed the inflammation-induced expression of TNF-α, IL-1β, IL-6, MCP-1, iNOS, and COX-2. Orally administered CXs exhibited remarkable therapeutic activities in mouse models of inflammatory arthritis. Using an alkynyl derivative of a CX and click reaction, we found that CX was covalently attached to TonEBP by alkylation but not to other components of the enhanceosome. Cells harboring a site-directed mutant TonEBP molecule whose amino acid residues for the alkylation were removed exhibited a markedly reduced sensitivity to CXs. These data demonstrate that CXs are a new class of anti-inflammatory agents with a novel mode of action.

Citation Format: Byeongjin YE, Hyug Moo Kwon, Soo Youn Choi, Jun Ho Lee, Hyun Je Kang, Cheol-Min Park. Development of anti inflammatory agents targeting TonEBP for treatment of chronic inflammatory diseases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1290.
Abstract 1291: Notch2 inhibition as a therapeutic intervention in osteosarcoma
Authors: Kannan Sankaranarayanan , Livingston John A. , Roth Michael , Benjamin Jonathan , Wang Yifei , Zhang Zhongting , Zhang Wendong , Wu Chia-Chin , Beird Hannah , Futreal Andrew , Gorlick Richard
Published: August 2019

Notch signaling pathway is a mediator of cell differentiation and is critical for normal bone development. Four functional Notch ligands DLL1 and DLL4, and JAG1 and JAG2 showed various levels of affinity for Notch1-4 receptors. Dose dependent Notch signaling activation or blocking is attributed to anchored or soluble form of Notch ligands binding. Interestingly, Notch pathway is shown to play a dual role, either oncogenic or tumor suppressive, depending on signal dose and tissue-context. To understand Notch signaling activities in osteosarcoma (OS), we first studied the cell surface levels of Notch receptor expression in OS cell lines and patient derived xenograft (pdx) models. Cytometry based screening of NOTCH1-4 receptors showed NOTCH2 as a predominant surface expressed protein in most of the samples. Interestingly, the canonical Notch target genes HES1 and DTX1 were higher (~5-fold) in OS cells compared to mesenchymal cells (MSC). Subsequently, we evaluated selected Notch pathway gene expression in 48 patients with recurrent/metastatic OS by RNAseq analysis. High expression of Notch pathway-related genes was seen in a subset of patients. NOTCH2 expression was comparable between primary and metastatic OS specimens, highest as compared to other Notch receptors and was significantly higher than normal tissue. Thus we aimed to elucidate soluble Notch ligands mediated blocking of Notch signaling using immunoglobulin-G bound DLL-1, -3, and -4 and JAG-1, and -2 in in vitro studies using OS cells. Soluble DLL1-Fc treatment increased OS cell death as compared to the other Notch ligands as well as control-Fc cells. Addition of soluble DLL1 resulted in the decreased expression of Notch downstream genes such as HES1, DTX1 and HEY1. Taken together, these findings identify soluble DLL1 as a potent Notch pathway inhibitor in OS in vitro and support further exploration of NOTCH2 as a potential therapeutic target in OS.

Citation Format: Sankaranarayanan Kannan, John A. Livingston, Michael Roth, Jonathan Benjamin, Yifei Wang, Zhongting Zhang, Wendong Zhang, Chia-Chin Wu, Hannah Beird, Andrew Futreal, Richard Gorlick. Notch2 inhibition as a therapeutic intervention in osteosarcoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1291.
Abstract 1295: First-in-field small molecule inhibitors targeting BRN2 as a therapeutic strategy for small cell prostate cancer
Authors: Thaper Daksh , Munuganti Ravi , Nouruzi Shaghayegh , Kumar Sahil , Kim Soojin , Sivak Olena , Aguda Adeleke , Ganguli Dwaipayan , Vahid Sepideh , Puca Loredana , Beltran Himisha , Zoubeidi Amina
Published: August 2019

Introduction: Resistance to newly developed androgen receptor pathway inhibitors (ARPIs), such as Enzalutamide (ENZ), rapidly emerges and patients generally die within two years. In particular, a subset of patients who relapse following ARPI therapy exhibit lineage switching whereby tumours shed their dependence on AR signaling and emerge with neuroendocrine features. These tumours, termed treatment induced neuroendocrine prostate cancer (t-NEPC), carry an extremely poor prognosis and, to date, treatment remains decades old cytotoxic chemotherapy which carries a short-lived response at the cost of significant toxicity. Therefore, targeted therapies for this deadly disease are desperately needed. Thus, the need to develop targeted treatments for this devastating disease is of paramount importance. Recently our group identified the neural transcription factor BRN2 as a major clinically relevant driver of NEPC and aggressive tumor growth, both in vitro and in vivo, suggesting targeting BRN2 is a promising strategy to prevent neuroendocrine differentiation or treat NEPC.

Methods: Study the effects of BRN2 inhibition using siRNA, small molecule inhibitors and CRISPR K/O models.

Results: Inhibition of BRN2 by siRNA and by CRISPR/Cas9 knockout drastically reduced cell proliferation in 42DENZR (NEPC) cell lines. This data was re-capitulated in human NEPC NCI-H660 cells. Loss of BRN2 initiated drastic epigenetic changes in NEPC cell lines as well as in G1 arrest through up-regulation of CDKN1A/1B. This was confirmed using our first in field BRN2 inhibitors. Targeting BRN2 also lead to downregulation several known targets in NEPC like EZH2, AURKA, SOX2 and Peg10. Treatment with BRN2i reduced recruitment of BRN2 to the chromatin by approximately 93% within 16 hours. Moreover, these BRN2 inhibitors displayed adequate pharmacokinetic properties and reduced NEPC proliferation in vivo.

Conclusion: No therapies exist for highly lethal NEPC. Hence, the described work aims to verify BRN2, a central driver of NEPC, and lay the pre-clinical foundation for the integration of targeted therapies into the treatment landscape to improve survival and quality of life for patients suffering from deadly form of prostate cancer.

Citation Format: Daksh Thaper, Ravi Munuganti, Shaghayegh Nouruzi, Sahil Kumar, Soojin Kim, Olena Sivak, Adeleke Aguda, Dwaipayan Ganguli, Sepideh Vahid, Loredana Puca, Himisha Beltran, Amina Zoubeidi. First-in-field small molecule inhibitors targeting BRN2 as a therapeutic strategy for small cell prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1295.
Abstract 1296: Arylsulfonamide KCN1 suppresses primary and metastatic growth of uveal melanoma through anti-angiogenic and anti-invasion mechanisms
Authors: Kaluz Stefan , Zhang Qing , Yang Hua , Osuka Satoru , Mun Jiyoung , Devi Narra S. , Goodman Mark , Grossniklaus Hans E. , Meir Erwin G. Van
Published: August 2019

Uveal melanoma (UM) is the most prevalent primary intraocular malignancy in adults with a survival time less than 1 year for patients that develop metastases (about 50%). HIF-1 plays a critical role in UM adaptation to the hypoxic microenvironment, treatment failure, and metastasis. In this study, we evaluated biodistribution and anti-cancer efficacy of arylsulfonamide KCN1, a lead compound in a novel class of small-molecule inhibitors of the hypoxia-inducible factor (HIF-1) pathway in UM models. PET with 11C-labeled KCN1 showed that KCN1 preferentially localizes to the eye and liver, the organ where UM preferentially metastasizes. In survival studies, UM cells were injected into the suprachoroidal space of the right murine eye using a transscleral technique and the eye was enucleated 7-9 days post-inoculation. Starting on Day 1, KCN1 or vehicle only (cremophor EL/ethanol 1:1, diluted with sterile PBS 1:5) were administered daily i.p., 5 x week, until animals reached the endpoint. Main organs were collected and evaluated for metastatic load. We found that KCN1 potently inhibited the growth of primary eye tumors, VEGF expression, angiogenesis in the primary tumor, the number and size of hepatic metastases, and extended mice survival. KCN1 inhibited invasion of UM cells in in vitromodels, such as scratch-wound and Boyden chamber migration assays. To identify possible mediators of hypoxia-HIF-induced metastatic processes inhibited by KCN1 in UM, we tested a series of 20 candidates by qRT-PCR. Among the most consistently HIF-induced/KCN1-inhibited genes were prolyl-4-hydroxylases P4HA1 and 2 that modulate physical properties of the tumor microenvironment by post-translationally modifying collagen. Expression of P4HA1 and 2 is significantly higher in UM patients with metastatic disease and high level of expression correlates with poor prognosis. In summary, our studies suggest KCN1 exerts anti UM activity by inhibiting the primary tumor growth, reducing vascular density in the primary tumor, and the number and size of the hepatic metastases; KCN1 has therefore therapeutic potential for treatment of UM and further clinical development.

Citation Format: Stefan Kaluz, Qing Zhang, Hua Yang, Satoru Osuka, Jiyoung Mun, Narra S. Devi, Mark Goodman, Hans E. Grossniklaus, Erwin G. Van Meir. Arylsulfonamide KCN1 suppresses primary and metastatic growth of uveal melanoma through anti-angiogenic and anti-invasion mechanisms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1296.
Abstract 1299: Potential antitumor effects of a Golgi disrupting agent, M-COPA, via targeting cell-extracellular matrix interaction under the spheroid culture conditions
Authors: Ohashi Yoshimi , Takeuchi Kazuma , Okamura Mutsumi , Akatsuka Akinobu , Shiina Isamu , Yoshimatsu Kentaro , Dan Shingo
Published: August 2019

The Golgi apparatus plays an essential role in the transport, processing, and sorting of cell surface proteins. We previously demonstrated that a Golgi disrupting agent, 2-methylcoprophilinamide (M-COPA), exhibited antitumor effect in human cancer cells in vitro and in vivo; however, in vivo antitumor efficacy of M-COPA did not simply reflect its in vitro efficacy, probably due to the lack of microenvironmental features of tumor tissues in the monolayer culture conditions. To bridge the gap between in vitro and in vivo efficacies, we exploited 3-dimensional (3D) spheroid culture models, which can recapitulate physiological characteristics of tumor tissues; i.e., nutrient/oxygen gradient, cell-cell interaction and cell-extracellular matrix (ECM) interaction, if cancer cells synthesize their own ECM molecules. Interestingly, human gastric cancer MKN1 cells displayed hypersensitivity to M-COPA under the 3D culture conditions compared to those in the monolayer culture conditions. Especially, we found that M-COPA caused loss of spheroid integrity. We previously demonstrated that M-COPA exerted antitumor effects via inhibiting intracellular transport of receptor tyrosine kinases onto the cell surface. Therefore, we examined the involvement of cell adhesion molecules in the antitumor effect of M-COPA. As a result, we found that specific integrins, which serve as cell surface receptors responsible for cell-ECM adhesion, were overexpressed and their downstream signaling molecules such as Src, Akt and MEK/ERK were highly phosphorylated in the cells under the 3D culture conditions compared to those under the monolayer culture conditions. Upon exposure to M-COPA, cell surface expression of these integrins was efficiently decreased and the downstream signaling molecules were dephosphorylated along with loss of spheroid integrity. Moreover, expression knockdown or neutralizing antibody of the integrins clearly suppressed sphere formation of MKN1 cells. These results suggest that M-COPA exerted in vivo antitumor effects via targeting specific integrins that mediate cell-matrix adhesion and its downstream intracellular signaling pathway observed in 3D spheroid culture models.

Citation Format: Yoshimi Ohashi, Kazuma Takeuchi, Mutsumi Okamura, Akinobu Akatsuka, Isamu Shiina, Kentaro Yoshimatsu, Shingo Dan. Potential antitumor effects of a Golgi disrupting agent, M-COPA, via targeting cell-extracellular matrix interaction under the spheroid culture conditions [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1299.
Abstract 1303: Targeted inhibition of galectin-1 suppresses glioblastoma growth
Authors: Cachia David , Chatterjee Arindam Rano , Vandergrift William Alex , Patel Sunil J. , Rabinovich Gabriel A. , Das Arabinda
Published: August 2019

Background: Galectin-1 (Gal1), a carbohydrate-binding protein is implicated in cancer cell proliferation, invasion and tumor angiogenesis. An earlier report from our laboratory demonstrated that glioblastoma (GB) cells express high levels of Gal-1, which promotes tumor progression through upregulation of CXCR4 viaNF-κB in in vitro GB models. Recently, several Gal1-targeting compounds have emerged. OTX008 is a calixarene derivative designed to bind the Gal1 amphipathic β-sheet conformation. Our study aimed to validate the effect of the non-peptidic galectin-1 inhibitor OTX008 in human GB in relevant pre-clinical models.

Methods: In vitro cell culture and in vivo GL261 murine models of GB were utilized to assess efficacy of treatment with OTX008 via Alzet osmotic pumps. To investigate the mechanistic effect of OTX008 on GB cells, we used RT-PCR and western blots techniques. Magnetic resonance imaging (MRI), and immunohistochemistry (IHC)-staining methods were used to detect tumor growth in in vivo GL261 murine model following OTX008 treatments.

Results: In cultured human and mouse GB cells, OTX008 inhibited proliferation and invasion at micromolar concentrations. We also found that the anti-proliferative effects correlated with Gal1 expression in human and mouse GB cell lines. Furthermore, OTX008 inhibited Gal1 expression and AKT-dependent survival pathways, and decreased CXCR4 expression. In vivo, continued 100ng/kg OTX008 treatment via Alzet osmotic pumps over 14 days reduced tumor growth of GL261 murine model after 3 weeks of implantation. OTX008 treatment was associated with downregulation of Gal1 and Ki67 in treated tumors, as well as decreased micro-vessel density and VEGFR2 expression.

Conclusion: These findings suggest that targeting galectin-1 may be an effective approach in the treatment of GB patients.

Citation Format: David Cachia, Arindam Rano Chatterjee, William Alex Vandergrift, Sunil J. Patel, Gabriel A. Rabinovich, Arabinda Das. Targeted inhibition of galectin-1 suppresses glioblastoma growth [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1303.