bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 743: Fusion gene identification from common cancer cell lines and comparison to primary tumors
Authors: Vellichirammal Neetha Nanoth , Albahrani Abrar , Banwait Jasjit K. , Li You , Guda Babu
Published: August 2019

Fusion transcripts, frequently observed in cancer, contribute to oncogenicity by either altering the expression of tumor suppressors/proto-oncogenes or modifying the original function of a protein resulting in an abnormal chimeric protein that stimulates tumorigenesis. Cancer cell lines, extensively used as in-vitro molecular models to study biological processes related to cancer, often do not represent the genomic profiles of primary tumors. To examine the true representation of the cancer cell lines, we examined fusions in tumor- derived cancer cell lines from 20 cancer types in the Cancer Cell Line Encyclopedia (CCLE) and compared them to fusions present in the primary tumors from the TCGA (The Cancer Genome Atlas). ChimeRScope, an alignment-free algorithm developed by our group, was used to screen fusion genes using level 1 paired-end RNA sequencing read data from CCLE and TCGA (GDC data portal). A total of 8,883 primary tumor samples, 730 normal samples spanning 33 cancer types from TCGA, and 802 common laboratory cell lines from CCLE were analyzed in this study. The pool of recurrent fusions identified from CCLE was vastly different from those identified in primary tumors with merely 2% of an overlap. Canonical fusions were found to be more common than non-canonical fusions (fusions containing genes in antisense strand) between TCGA and CCLE. Hierarchical clustering analysis of both canonical and non-canonical recurrent fusions in CCLE and TCGA clustered the primary tumors and cell lines into separate clusters, revealing that fusions in cell lines are vastly different from primary tumors. However, canonical and non-canonical fusion profiles of endometrial cancer cell lines were very similar to female primary tumors (ovarian and breast) and thyroid cancer cell lines were similar to bladder cancer in TCGA. The results from this study indicate that the recurrent fusions in common cancer cell lines harbor fusions that are significantly different from primary tumors.

Citation Format: Neetha Nanoth Vellichirammal, Abrar Albahrani, Jasjit K. Banwait, You Li, Babu Guda. Fusion gene identification from common cancer cell lines and comparison to primary tumors [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 743.
Abstract 747: Characteristic molecular signature of pericardial effusion identifies malignant cancer in pericardial disorder patients
Authors: Nam Suk Woo , Kim Sang Yean , Kim Hyung Seok , Yang Hee Doo , Na Min Jeong
Published: August 2019

Pericardial effusion (PE) can develop in patients with virtually any condition that affects the pericardium, including acute pericarditis and a variety of systemic disorders. PE is also common in cancer patients, and that may develop by several different mechanisms by direct or metastatic spread of the primary process or as a complication of antineoplastic therapies. Thus, definite differentiation of malignant PE and rapid diagnosis are of particular therapeutic and prognostic importance. We aim to determine etiology-based characteristic molecular changes of PE in pericardial disorder patients, and to identify novel molecular signature for early detection of cancer patients with pericardial disorder. We obtained pericardial fluids from the pericardial disorders including cancer patients. Whole genomic transcriptome scan resulted in 1,639 outlier genes as cancer-specific PE molecular signature. Further analysis showed that a cancer-specific molecular signature exists and discerns cancer patients with non-cancer patients, and that also suggested 191 gene elements as stringent classifier between cancer vs non-cancer (Welch’s t test, p < 0.05 and Ven diagrammatic analysis). Gene set enrichment analysis (GSEA) with additional computational analysis of 1,639 cancer-specific PE molecular signature identified CD24, SDC1, and ST14 as strong molecular markers for identifying cancer in PE patients. In conclusion, whole transcriptomic analysis of pericardial fluids of pericardial disorder patients suggest that etiology-specific molecular signatures can discriminate different patterns of PE, and that they provide useful information for mechanistic studies that may lead to a better understanding of the molecular basis of PE pathology.

Keywords: pericardial fluid, differential gene expression, diagnostic markers, CD24, SDC1, ST14

Citation Format: Suk Woo Nam, Sang Yean Kim, Hyung Seok Kim, Hee Doo Yang, Min Jeong Na. Characteristic molecular signature of pericardial effusion identifies malignant cancer in pericardial disorder patients [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 747.
Abstract 752: Rapid acquired resistance to alectinib in ALK-positive lung cancers with high tumor mutation burden
Authors: Ohashi Kadoaki , Makimoto Go , Tomida Shuta , Nishii Kazuya , Toyooka Shinichi , Kiura Katsuyuki
Published: August 2019

Background: Alectinib, a highly selective anaplastic lymphoma kinase (ALK) inhibitor, demonstrates an unprecedented effect in ALK-positive lung cancers. However, some tumors quickly develop resistance to alectinib. In this study, we aimed to investigate the mechanism of the rapidly acquired resistance to alectinib using clinical samples.

Methods: Autopsied specimens of lung, liver, and renal tumors were obtained from a 51-year-old male patient with advanced ALK-positive lung cancer, who had acquired resistance to alectinib in only three months. Two alectinib-resistant cell lines were established from pleural effusion (ABC-14) or liver tumors (ABC-17). A patient-derived xenograft (PDX) model was also developed from liver tumors. Next-generation sequencing (NGS), direct DNA sequencing, and quantitative real time reverse transcription-PCR were performed.

Results: ABC-14 harbored no ALK mutations and showed sensitivity to crizotinib, an ALK/MET inhibitor. It also showed mesenchymal-epithelial transition factor (MET) gene amplification and amphiregulin overexpression. The combination of crizotinib and erlotinib showed beneficial inhibitory effects on cell growth. ABC-17 and PDX tumors harbored ALK G1202R. PDX tumors metastasized to multiple organs in vivo, whereas lorlatinib, the third-generation ALK inhibitor, diminished tumor growth both in vitro and in vivo. NGS demonstrated high tumor mutation burden (TMB) and heterogeneous evolution of the patient tissues. The autopsied lung tumors harbored ALK G1202R (c. 3604 G>A) and the right renal metastasis harbored ALK G1202R (c. 3604 G>C); the mutation thus altered codon sequences.

Conclusions: High TMB and heterogeneous tumor evolution may have conferred the rapid resistance to alectinib. Timely administration of lorlatinib or combination therapies with an ALK inhibitor and other RTK inhibitors may constitute a potent therapeutic strategy.

Financial support: This research was supported by a Grant for Lung Cancer Research (K.O.), provided by the Japan Lung Cancer Society.

Citation Format: Kadoaki Ohashi, Go Makimoto, Shuta Tomida, Kazuya Nishii, Shinichi Toyooka, Katsuyuki Kiura. Rapid acquired resistance to alectinib in ALK-positive lung cancers with high tumor mutation burden [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 752.
Abstract 761: Up-regulation of miR-10a affect on prostate cancer racial disparity
Authors: Hashimoto Yutaka , Shiina Marisa , Tanaka Yuichiro , Dasgupta Pritha , Kulkarni Priyanka , Kato Taku , Wong Ryan K. , Shahryari Varahram , Maekawa Shigekatsu , Yamamura Soichiro , Bhagiratha Divya , Saini Sharanjot , Deng Guoren , Tabatabai Laura , Majid Shahana , Dahiya Rajvir , Dahiya Rajvir
Published: August 2019

Purpose: Prostate cancer (PC) is the second most diagnosed cancer in men. African-American (AfA) men have higher incidence and twice the PC mortality rates compared to Caucasian-American (CaA) men. In this study, we investigated the biochemical role of miR-10a in prostate cancer racial disparity. Methods: We used PCa cell lines from CaA (DU145, LNCaP, PC3), AfA (MDA-PCa-2b, E006AA-hT) and a normal epithelial cell line (PWR1E) to examine miR-10a expression levels. Total RNA was extracted from total 138 human prostate cancer clinical samples (AfA: n=75, CaA: n=63) and cell lines. Gene expression levels were determined by quantitative real-time PCR (qPCR). A miRNA target database (miRWalk that integrates mainly three databases, miRDB, TargetScan and miRTarBase) was used for miR-10a target prediction. Cell proliferation and apoptosis were monitored after miR-10a knockdown by using anti-miR-10a. Results: miR-10a was significantly up-regulated in AfA prostate cancer cell line, E006AA-hT compared to CaA cells. Utilizing SFVAMC and NDRI patient cohorts, we confirmed that miR-10a expression was linked to a racial difference between AfA/CaA PCa patients. Knockdown of miR-10a showed decreased growth and apoptosis, though the effect was less in CaA compared to AfA cells. We found NCOR2 which is frequently deleted in castration- resistant prostate cancer patient specimens and tumor-suppressor PTEN as miR-10a targets that overlapped in multiple miRNA target prediction databases. Conclusion: These results demonstrate that miR-10a may be a central regulator of crucial events that contribute to racial differences in prostate cancer. Regulation of miR-10a expression may be a new therapeutic strategy for AfA prostate cancer patients.

Citation Format: Yutaka Hashimoto, Marisa Shiina, Yuichiro Tanaka, Pritha Dasgupta, Priyanka Kulkarni, Taku Kato, Ryan K. Wong, Varahram Shahryari, Shigekatsu Maekawa, Soichiro Yamamura, Divya Bhagiratha, Sharanjot Saini, Guoren Deng, Laura Tabatabai, Shahana Majid, Rajvir Dahiya, Rajvir Dahiya. Up-regulation of miR-10a affect on prostate cancer racial disparity [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 761.
Abstract 772: MicroRNA regulatory networks: Concomitant in situ detection of microRNA, mRNA and protein in cancer samples
Authors: Nielsen Boye Schnack
Published: August 2019

The canonical mode of action of microRNAs involves the binding to the 3'UTR of mRNAs thereby mediating repression of protein synthesis. MicroRNAs comprise an interesting new group of cancer biomarkers and may also be therapeutic targets in cancer and other diseases using antisense oligos (ASO). The visualization of potential molecular dynamics between a microRNA, its target mRNA and the effect of protein expression can be facilitated by combined detection in tissue sections. MicroRNAs, as well as ASOs, can be detected in tissue sections or cultured cells by in situ hybridization (ISH) using LNA probes. We have developed a novel ISH assay for microRNA detection with improved sensitivity and signal-to-noise that can be used with any LNA probe on both FFPE and frozen sections. The assay is based on double-labeled LNA™ probes and detection can be either fluorescence or chromogenic staining. We have combined the fluorescence assay with immunofluorescence, for double and triple staining. We also combined the microRNA ISH assay with mRNA in situ hybridization using the highly specific RNAscope probes. We show a variety of combinations, including examples of combined staining of microRNA-17, IL-1beta mRNA or TNF-alpha mRNA, and cytokeratin proteins in automated triple staining procedures of colon cancer samples. The focal expression of IL-1beta mRNA or TNF-alpha mRNA and the loss of microRNA-17 at the invasive front involving inflammatory cells and budding cancer cells is discussed.

Citation Format: Boye Schnack Nielsen. MicroRNA regulatory networks: Concomitant in situ detection of microRNA, mRNA and protein in cancer samples [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 772.
Abstract 783: miR-375 downregulates VEGFA via YAP1-EBRR2 pathway in esophageal cancer
Authors: Ren Shuchang , Tan Xiaohui , Wu Xiaoling , Chen Tao , Latham Patricia , Fu Sidney W.
Published: August 2019

Esophageal cancer (EC), including esophageal squamous cell carcinoma (ESCC) and adenocarcinoma (EAC), is one of the deadliest cancers with extremely aggressive nature and poor survival rate. Diagnostic methods for EC mainly include endoscopy, barium swallow and ultrasound, which are either invasive or lack of sensitivity and specificity at early stages. Therefore, the development of a non-invasive and reliable diagnostic method with high sensitivity and specificity is crucial in improving early detection, diagnosis and treatment. microRNAs (miRNAs) are small RNA molecules that regulate the expression of protein-coding genes by directly binding to target mRNAs in a sequence-specific manner. The aim of this study is to identify novel miRNA biomarkers for EC diagnosis. By cross-referencing our data with others’ from NCBI, we identified a list of dysregulated miRNAs in EC, including miR-375. We assessed the expression of miR-375 in EC cell lines and tissues using real-time reverse transcriptase-polymerase chain reaction (qRT-PCR). We found that miR-375 expression is significantly decreased in human EC cell lines (KEYS-70, KEYS-180, FLO-1 and JHU-ad1) compared to the normal esophageal squamous cell line, Het-1A. Using microdissection technique, we isolated normal epithelium, dysplasia, and invasive EC components from formalin-fixed, paraffin-embedded (FFPE) tissue followed by qRT-PCR analysis. Decreased expression of miR-375 was detected in 12 of 14 EC tissue samples (86%) compared to the matched normal tissues. MTT and Matrigel invasion assay demonstrated that forced expression of miR-375 suppressed the proliferation and invasion in EC cell lines. TargetScanS and miRanda were used to identify potential target genes of miR-375, which includes epidermal growth factor receptor 2 (HER2) and YES-associated protein (YAP1), both of them act as oncogenes in EC development. Interestingly, forced expression of miR-375 resulted in significantly reduced expression of human vascular endothelial growth factor-A (VEGF-A) gene, which is a downstream target gene of YAP1/ERBB2 pathway involving in tumor angiogenesis in EC cell lines. More functional analysis is under way. These findings suggest that miR-375 may act as a tumor suppressor, and could serve as a marker for EC diagnosis and management.

Citation Format: Shuchang Ren, Xiaohui Tan, Xiaoling Wu, Tao Chen, Patricia Latham, Sidney W. Fu. miR-375 downregulates VEGFA via YAP1-EBRR2 pathway in esophageal 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 783.
Abstract 785: The inhibitory effect of compound ChlA-F onhuman bladder cancer cell invasion can be attributed to its blockage of SOX2 protein
Authors: Hua Xiaohui , Xu Jiheng , Huang Chuanshu , Li Jingxia , Tian Zhongxian , Huang Haishan
Published: August 2019

Sex-determining region Y-box 2 (SOX2), a well-known stemness biomarker, is highly expressed in a variety of cancers, including human highly invasive bladder cancer (BC). However, the role of SOX2 may vary in different kinds of malignancy. In the present study, we discovered that ChlA-F, a novel conformation derivative of isolate Cheliensisin A (Chel A), remarkably inhibits the invasive ability of human invasive BC cells through downregulation of SOX2 protein expression. We found that ChlA-F treatment dramatically decreases SOX2 protein expression in human high-grade invasive BC cells. Ectopic expression of SOX2 reversed ChlA-F inhibition of cell invasion ability in human bladder cancer cells, suggesting that SOX2 is a major target of ChlA-F during its inhibition of human BC invasion. Mechanistic studies revealed that SOX2 is downregulated by ChlA-F at both the protein degradation and protein translation levels. Further studies revealed that ChlA-F treatment induces HuR protein expression and that the increased HuR interacts with USP8 mRNA, resulting in elevation of USP8 mRNA stability and protein expression. Elevated USP8 subsequently acts as an E3 ligase to promote SOX2 ubiquitination and protein degradation. We also found that ChlA-F treatment substantially increases c-Jun phosphorylation at Ser63 and Ser73, initiating miR-200c transcription. The increased miR-200c directly binds to the 3’-UTR of SOX2 mRNA to suppress SOX2 protein translation. These results present novel mechanistic insight into understanding SOX2 inhibition upon ChlA-F treatment and provide important information for further exploration of ChlA-F as a new therapeutic compound for the treatment of highly invasive/metastatic human BC patients.

Citation Format: Xiaohui Hua, Jiheng Xu, Chuanshu Huang, Jingxia Li, Zhongxian Tian, Haishan Huang. The inhibitory effect of compound ChlA-F onhuman bladder cancer cell invasion can be attributed to its blockage of SOX2 protein [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 785.
Abstract 791: V-ATPase in glioma stem cells: V1G1 subunit expression correlates with metabolic behavior and mitochondria activity
Authors: Storaci Alessandra M. , Bertolini Irene , Caroli Manuela , Ferrero Stefano , Vaira Valentina
Published: August 2019

Background The vacuolar H+-ATPase (V-ATPase) is a multisubunit proton pump with a role in the acidification of the extra- and intra-cellular environments. Recent evidence connected V-ATPase deregulation and human diseases, including cancer. We previously demonstrated that the overexpression of the catalytic subunit V1G1 is correlated with glioma stem cells (GSC) maintenance. According to V1G1 expression, we sorted glioblastoma patients-derived neurospheres cultures (NS) into two categories: V1G1HIGH and V1G1LOW NS. These NS differ in terms of stem cell genes expression, sphere formation, invasion ability, lysosomal acidity and bioenergetics.

Methods V1G1HIGH, V1G1LOW NS treated with 10nM of BafilomycinA1 (BafA1) or vehicle for 24h (n=3 per group) were used for the functional experiments. Lactate, glucose, ATP levels were measured by commercial kits. Metabolic proteins expression was assessed by western blot. ROS production and mitochondria function were measured by flow cytometry using MitoSox and TMRE assays, respectively.

Results At basal conditions, V1G1LOW NS produce higher levels of lactate (p=0.0379) whereas V1G1HIGH NS show higher levels of extracellular glucose and intracellular ATP. Accordingly, the two NS cultures show an opposite trend in LDH subunits expression with LDHA, which pushes lactate production, being more expressed in V1G1LOW (p=0.0027). Conversely, LDHB that facilities the production of pyruvate from lactate, and the transporter MCT1 are more expressed in V1G1HIGH NS. Interestingly, using the V-ATPase specific inhibitor BafA1, metabolic characteristics of V1G1HIGH are redirected to the V1G1LOW phenotype. Moreover, in V1G1HIGH NS BafA1 induces ROS production and affects mitochondria activity, increasing their depolarization.

Conclusions Taken together these data suggest two distinct metabolic behaviors of NS according to V-ATPase expression. Indeed for energy production V1G1LOW NS could rely on a glycolytic metabolism whereas V1G1HIGH NS on oxidative phosphorylation. Moreover, when overexpressed the proton pump could also have a role in preserving mitochondria polarity. Although preliminary, this is the first evidence reporting a role for V-ATPase in the regulation of cancer stem cell metabolism.

Citation Format: Alessandra M. Storaci, Irene Bertolini, Manuela Caroli, Stefano Ferrero, Valentina Vaira. V-ATPase in glioma stem cells: V1G1 subunit expression correlates with metabolic behavior and mitochondria activity [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 791.
Abstract 799: Aberrant mitochondrial protein involvement through early PDAC initiation and progression using multiplexed DNA-PAINT and correlative histology
Authors: Rames Matthew J. , Civitci Fehmi , Zheng Ting , Wagner Josiah , Link Jason , Nan Xiaolin
Published: August 2019

Prior to pancreatic ductal adenocarcinoma (PDAC), pancreatic acini cells change their morphology through pancreatic intraepithelial neoplasia (PanIN) stages, becoming increasingly dysplastic as stroma and tissue hypoxia increase. This increasing hypoxia forms a cancer promoting microenvironment, wherein we propose metabolic changes trigger aberrant mitochondrial networks form healthy tissue. We developed tissue superresolution imaging to directly quantify structural mitochondrial response through patient histology, whereby DNA-PAINT can provide super-resolution detail decoupled from photo bleaching to visualize mitochondria through tissue layers. Expanding from preliminary data, target mitochondrial dynamics proteins’ organization will also be correlated to PanIN stages.

Introduction: Early oncogene involvement in PDAC progression links to metabolic and mitochondrial regulatory changes. Typically nutrient deprivation and hypoxia trigger cell death from increased reactive oxygen species production and organelle damage which trigger apoptosis, yet these prolonged effects can be cancer promoting when less severe. Mitochondrial dynamics and proteins related to mitochondrial fission and fusion can reduce apoptotic signaling, enhance aerobic glycolysis, and increase ROS to allow cancer progression.

Materials and Methods: Adapting from previous proof of concept, mitochondria (TOM20) within formalin-fixed paraffin embedded (FFPE) tissue sections were imaged with stochastic optical reconstruction microscopy (STORM). In brief: cadaver healthy pancreas FFPE tissue samples underwent deparafinization, antigen retrieval, indirect immuno-labeling with AlexaFluor647, TIRF illumination with a 60x objective was used for data collection, whereby data processing was conducted using the open-source FIJI and custom MATLAB software.

Results and Discussion: Preliminary data shows proof of principle that both STORM and DNA-PAINT can be correlated to histological staining of human pancreas.

Conclusions: Superresolution imaging reveals ultrastructural details of mitochondria in FFPE patient samples not resolved via conventional fluorescence imaging. Through quantitative feature analysis, we would be able to correlate aberrant mitochondria structure and abundance to PDAC progression.

Acknowledgements: Funding provided by the Cancer Early Detection Advanced Research (CEDAR) Center of OHSU Knight Cancer Institute.

Citation Format: Matthew J. Rames, Fehmi Civitci, Ting Zheng, Josiah Wagner, Jason Link, Xiaolin Nan. Aberrant mitochondrial protein involvement through early PDAC initiation and progression using multiplexed DNA-PAINT and correlative histology [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 799.
Abstract 811: Multi-targeted inhibitors of mitochondrial one-carbon metabolism and cytosolic de novo purine synthesis enzymes as anti-tumor agents
Authors: Nayeen Md Junayed , Shah Khushbu , Dekhne Aamod , Ning Changwon , O'Connor Carrie , Katinas Jade M. , Wong Jennifer , Hou Zhanjun , Dann Charles E. , Matherly Larry H. , Gangjee Aleem
Published: August 2019

Targeting cellular one-carbon (1C) metabolism in cancer cells with pyrrolo[3,2-d]pyrimidine analogs provides in vivo antitumor efficacy. We discovered a first-in-class series of 5-substituted pyrrolo[3,2-d]pyrimidine analogs (AGF291, AGF320, AGF347) with inhibition of serine hydroxymethyltransferase 2 (SHMT2), a key enzyme in mitochondrial 1C metabolism and a reported oncodriver. In addition, AGF291, AGF320 and AGF347 inhibited de novo purine nucleotide biosynthesis at glycinamide ribonucleotide formyltransferase (GARFTase) and 5-aminoimidazole-4-carboxamide (AICA) ribonucleotide formyltransferase (AICARFTase), and serine hydroxymethyltransferase 1 (SHMT1). In vivo studies demonstrated excellent antitumor efficacy for AGF347 against MIA PaCa-2 pancreatic adenocarcinoma xenografts in SCID mice with a median tumor growth delay (T-C) of >38 days for 4 mice, and 1 of 5 mice tumor-free 122 days post-treatment. AGF359, a new analog in this series, was a potent inhibitor of KB human tumor cell proliferation in vitro. AGF359 inhibition of KB human tumor cells was reversed with glycine and adenosine, establishing mitochondrial 1C metabolism and de novo purine biosynthesis as the targeted pathways; AICA plus glycine was incompletely protective, implicating AICARFTase as a direct cellular target. AGF359, like AGF291, AGF320, and AGF347, inhibited purified human SHMT2 (Ki = 0.399 ± 0.174 µM), SHMT1 (Ki = 0.70 ± 0.088 µM) and AICARFTase (Ki = 8.86 ± 2.83 µM). There was no inhibition of 5,10-methylene tetrahydrofolate dehydrogenase 2 (MTHFD2). Further structural modifications of these multi-targeted agents afforded pyrrolo[3,2-d]pyrimidine analogs AGF307 and AGF312. Cell-based glycine/nucleotide rescue experiments in KB tumor cells established that, as with AGF359, AGF307 and AGF312 were dual inhibitors of the mitochondrial 1C metabolism (AGF307) and de novo purine biosynthesis (AGF307, AGF312). These compounds are currently in further preclinical evaluation as a prelude to possible clinical development as antitumor agents.

Citation Format: Md Junayed Nayeen, Khushbu Shah, Aamod Dekhne, Changwon Ning, Carrie O'Connor, Jade M. Katinas, Jennifer Wong, Zhanjun Hou, Charles E. Dann III, Larry H. Matherly, Aleem Gangjee. Multi-targeted inhibitors of mitochondrial one-carbon metabolism and cytosolic de novo purine synthesis enzymes as anti-tumor agents [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 811.
Abstract 814: Involvement of unique endosomes in the Mieap-induced vacuoles formation during Mieap-mediated cancer cell death
Authors: Ikari Naoki , Nakamura Yasuyuki , Arakawa Hirofumi
Published: August 2019

Background: Mitochondria-eating protein (Mieap), a p53-inducible protein is known to have tumor suppressive functions by repairing or degrading unhealthy mitochondria. When Mieap degrades mitochondria, Mieap-induced vacuoles (MIVs) are formed to eat mitochondria, which subsequently induce iron-dependent cell death. Previously, we elucidated that the MIVs contain lysosomal proteins and degraded mitochondria, suggesting the MIVs represent non-canonical mitophagy. However, the process of MIV formation is still unclear.

Methods: The process of MIV formation was visualized by high spatial and temporal resolution confocal live cell imaging with the EGFP-tagged Mieap protein (EGFP-Mieap) that was ectopically expressed in A549 cells. To interpret the imaging data, we performed the Mieap-interactome analysis, and consulted the list of proteins immunoprecipitated by anti-Mieap antibody. Further, we investigated the mechanism for MIV formation using various deletion-mutant forms of EGFP-Mieap.

Results: The enforced expression of EGFP-Mieap generated characteristic endosomes whose membrane contained Mieap. The unique endosomes showed directed movement toward clusters of mitochondria, where MIVs were generated, and enlarged by eating mitochondria and fusing to each other. When we reviewed the list of Mieap-interacting proteins considering the directed movement, alpha tubulins were found to be included. The endosomes consisting of C-terminus deficient Mieap showed nondirected movement and accumulated at the edge of the cells.

Conclusions: Besides lysosomes and mitochondria, MIVs are derived from the Mieap-related endosomes via the endocytosis/pinocytosis. Possibly, the interaction between the C-terminus region of Mieap and alpha tubulins is critical for the endosome delivery to the mitochondria in MIV formation.

Citation Format: Naoki Ikari, Yasuyuki Nakamura, Hirofumi Arakawa. Involvement of unique endosomes in the Mieap-induced vacuoles formation during Mieap-mediated cancer cell death [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 814.
Abstract 819: DNA methylation genome-wide analysis in remnant gastric cancer
Authors: Ito Tomoaki , Sugimoto Kiichi , Orita Hajime , Maeda Masahiro , Moro Hiroshi , Ushijima Toshikazu , Katai Hitoshi , Wada Ryo , Sakamoto Kazuhiro , Sato Koichi , Brock Malcolm V.
Published: August 2019

Purpose: Although primary gastric cancers (PGC) and remnant gastric cancers (RGC) both originate from the same gastrointestinal organ, they have very distinct clinicopathological behaviors. Due to the importance of chronic inflammation in the carcinogenesis of gastric malignancies, we hypothesized that DNA promoter hypermethylation would play a critical role in the carcinogenesis of RGC as well as PGC.

Methods: We investigated the genome-wide DNA methylation patterns of PGC and RGC tissues from 48 patients from an academic medical center in Japan using the Infinium HumanMethylation450 BeadChip assay. The results were validated by quantitative methylation-specific PCR (qMSP) in separate, independent cohorts.

Results: We found that in our training cohort of 48 patients, genes from the gastric cancer tissues identified by the Infinium HumanMethylation 450 Beadchip clustered into high and low methylation groups on multivariate analysis (p=0.004, OR=12.33). PGCs contributed significantly to the high methylation group suggesting that the DNA promoter methylation status in PGC is higher than that in RGC. Supporting this conclusion was the finding that in a separate qMSP analysis in a test cohort, the gene A had significantly higher DNA promoter methylation in cancer tissues in the validation PGC tissues than in RGC.

Conclusion: This study demonstrated that DNA promoter methylation status in PGC is higher than in RGC. This result may reflect the effects of Helicobacter pylori on the induction of DNA methylation in the remnant stomach.

Citation Format: Tomoaki Ito, Kiichi Sugimoto, Hajime Orita, Masahiro Maeda, Hiroshi Moro, Toshikazu Ushijima, Hitoshi Katai, Ryo Wada, Kazuhiro Sakamoto, Koichi Sato, Malcolm V. Brock. DNA methylation genome-wide analysis in remnant gastric 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 819.
Abstract 82: Transforming of human osteoblasts with the combination of hTERT (T), SV40Tag (S) and c-Myc: Changes in phenotype and tumorigenesis capability
Authors: Zhang Zhongting , Wang Yifei , Zhang Wendong , Roth Michael , Gill Jonathan Benjamin , Xu Zhaohui , Tian Xiangjun , Wang Jing , Gorlick Richard
Published: August 2019

The etiology of osteosarcoma (OS), the most common primary malignant bone cancer in pediatric patients, still remains unknown. It is not clear where tat which point he osteosarcoma is originated from the osteogenic lineagein the osteogenic osteosarcoma arises., some consider Potential candidates in the literature include the human mesenchymal cells (hMSC) as the cell of origin in OS, whereas others believe theand osteoblast (OB) to be the most likely cell of origin. To understand the origin and molecular pathogenesis of osteosarcoma, we have transformed hMSCs and OBs differentiated from the same MSC with oncogenes hTERT (T), SV40Tag (S) and H-RAS (R) in series to check evaluate for spindle cell tumor formation in mice. Transformed MSC did not form osteoid, whereas OB-TSR tumor showed only scant production of osteoid, suggesting MSC may not be the origin of OS and the hTERT, SV40Tag and h-Ras may not be sufficient for osteoblast to form OS.

C-Myc is one of the genes that play a major role in oncogenic transformation of normal cells. It has been shown that c-Myc is overexpressed in OS cells and more than 10% OS patients carry c-Myc amplification mutations. We further decide to look intoThis served as the basis to examine the role of c-Myc in OS formation in the background of cells transformed by hTERT and SV40. OB transformed with hTERT and SV40Tag cells were further transfected with a retrovirus containing human c-Myc to obtain stable cell lines. Quantitative PCR and western blots assays detected both gene and protein expression patterns of c-Myc, respectively, in the transformed cell lines. Transformed cell lines were analyzed for proliferation, invasion, migration, and spheroid formation to determine the tumorigenic capacity capability. TThe tumorigenesis capability is found to be correlated with the level of c-Myc expression level based onby RT-PCR data. Furthermore, we used RNA-seq to analyze gene expression profiles of the transformed cell lines, compared with those forin comparison to primary OS cell lines and patient derived xenografts (PDXs). In -vivo tumorigenic assays, histological examination for osteoid production and chromosome analysis are still under way. Our study using an osteosarcoma model with transformed human cells may provide new insights on the cell origin of OS.

Citation Format: Zhongting Zhang, Yifei Wang, Wendong Zhang, Michael Roth, Jonathan Benjamin Gill, Zhaohui Xu, Xiangjun Tian, Jing Wang, Richard Gorlick. Transforming of human osteoblasts with the combination of hTERT (T), SV40Tag (S) and c-Myc: Changes in phenotype and tumorigenesis capability [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 82.
Abstract 825: TAPS: A bisulfite-free, base-resolution and quantitative sequencing method for cytosine modifications
Authors: Liu Yibin , Siejka Paulina , Velikova Gergana , Bi Ying , Tomkova Marketa , Schuster-Boeckler Benjamin , Song Chun-Xiao
Published: August 2019

Aberrant DNA methylation changes are powerful prognostic and predictive biomarkers in cancer diagnosis and treatment. For decades bisulfite sequencing has been the gold standard for DNA methylation analysis. However, this method relies on harsh deamination reaction of unmodified cytosine to uracil which degrades the majority of DNA and generates sequencing libraries with low complexity. Those drawbacks limit the applications on low-input samples and lead to poor sequencing quality, low mapping rates, uneven genome coverage and increased sequencing cost. Here, we present a novel bisulfite-free and base-resolution sequencing method, TET Assisted Pyridine borane Sequencing (TAPS), for direct detection of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) without affecting unmodified cytosine. We utilised TET enzymes to readily oxidize 5mC and 5hmC to 5-carboxylcytosine (5caC), which was then converted to dihydrouracil by pyridine borane via a previously unknown reductive decarboxylation/deamination reaction. This non-destructive method enables high-throughput sequencing with nanoscale input materials (genomic DNA and cell-free DNA) and preserve DNA up to 10 kbs long which could allow long read sequencing. We demonstrated that TAPS could directly replace bisulfite sequencing with higher mapping rates, more even coverage and lower sequencing costs, and enable more comprehensive, cheaper and faster methylome analyses. This would be a revolutionary tool for epigenetic study and facilitate both academic research and clinical diagnostics.

Citation Format: Yibin Liu, Paulina Siejka, Gergana Velikova, Ying Bi, Marketa Tomkova, Benjamin Schuster-Boeckler, Chun-Xiao Song. TAPS: A bisulfite-free, base-resolution and quantitative sequencing method for cytosine modifications [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 825.
Abstract 829: SOX2-mediated 5hmC dysregulation in GBM stem cells
Authors: Sudhir Sweta , Lopez-Bertoni Hernando , Lugo-Fagundo Maria , Lal Bachchu , Laterra John
Published: August 2019

Primary brain tumors are among the most devastating forms of cancer and glioblastoma (GBM) represents the most aggressive and lethal form of the disease. We now know that GBM contains small subsets of cells that display tumor-propagating stem-like phenotypes (i.e. glioma stem cells or GSCs) that act as critical determinants of resistance to current treatments and tumor recurrence for which there is no proven therapy. Altered patterns of DNA methylation are widely reported in human GBM. However, substantial knowledge gaps remain in our understanding of the molecular mechanisms responsible for this epigenetic dysregulation, its downstream consequences and its role in the GBM tumor-propagating phenotype. Understanding and ultimately targeting the epigenetic mechanisms that induce and maintain these tumor-propagating cell subsets is critical to improving GBM therapy and patient outcomes.

DNA methylation generally occurs in cytosine-guanine (CpG) sequences and is established by DNMTs, which catalyze the conversion of cytosine to 5-methylcytosine (5mC). DNA methylation is dynamically regulated and reversible by the ten-eleven translocation (TET) family of deoxygenases that catalyze the conversion of 5mC to 5-hydroxymethylcytosine (5hmC). Levels of 5hmC closely align with the differentiation state of cells; terminally differentiated cells contain high levels of 5hmC and less differentiated cells have very low levels. Multiple studies found negative correlations between 5hmC levels and glioma grade and loss of 5hmC correlates with poor prognosis of GBM patients. This strongly suggests that tumor suppressing epigenetic mechanisms are driven by TET-dependent 5hmC generation.

We now show that SOX2, a reprogramming transcription factor that induces GSCs, represses the TET2 and decreases 5hmC in multiple GSC isolates. TET2 repression using two independent shRNA hairpins efficiently decreased 5hmc levels and significantly enhanced self-renewal capacity and tumor growth capacity of low-passage GSCs. We also show that low concentrations of vitamin C, a TET enzyme cofactor, significantly increases 5hmC in low-passage GSCs. This epigenetic change induced by vitamin C associated with increased GSC death induced by temozolomide (TMZ), the main chemotherapeutic used to treat GBM. These findings support a mechanism by which SOX2 induces loss of 5hmC in GSCs by repressing TET2 and show that vitamin C can reverse this epigenetic tumor promoting pathway and potentially enhance GBM chemotherapeutics.

Citation Format: Sweta Sudhir, Hernando Lopez-Bertoni, Maria Lugo-Fagundo, Bachchu Lal, John Laterra. SOX2-mediated 5hmC dysregulation in GBM stem 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 829.
Abstract 836: From tobacco smoking to mutation signature: The role of epigenetic changes in human cancers
Authors: Chen Zhishan , Wen Wanqing , Cai Qiuyin , Long Jirong , Shu Xiao-ou , Zheng Wei , Guo Xingyi
Published: August 2019

Tobacco smoking is associated with a unique mutation signature in the human cancer genome. It is unclear whether tobacco smoking alters DNA methylations and gene expressions affect smoking-related mutation signature. In this study, we evaluated the smoking-related DNA methylation sites reported from five previous studies using peripheral blood cells to identify possible target genes. Using the mediation analysis approach, we evaluated whether the association of tobacco smoking with mutation signature was mediated through altered DNA methylation and expression of these target genes. Based on data obtained from 21,108 blood samples, we identified 374 smoking-related DNA methylation sites, annotated to 248 target genes. Using data from DNA methylations, gene expressions and smoking-related mutation signature generated from ~7,700 tumor tissue samples across 26 cancer types from The Cancer Genome Atlas (TCGA), we found 11 of the 248 target genes whose expressions were associated with smoking-related mutation signature at a Bonferroni-correction P < 0.001. This included four for head and neck cancer, and seven for lung adenocarcinoma. In lung adenocarcinoma, our results further showed that smoking increased the expression of three genes, AHRR, GPR15, and HDGF, and decreased the expression of two genes, CAPN8, and RPS6KA1, which were consequently associated with increased smoking-related mutation signature. Additional evidence showed that the elevated expression of AHRR (cg14817490) and GPR15 (cg19859270), were associated with smoking-altered hypomethylations. Our findings provide novel insight into the contributions of tobacco smoking to carcinogenesis through complex molecular mechanisms of the evaluated mutation signature by altered DNA methylation and gene expression.

Citation Format: Zhishan Chen, Wanqing Wen, Qiuyin Cai, Jirong Long, Xiao-ou Shu, Wei Zheng, Xingyi Guo. From tobacco smoking to mutation signature: The role of epigenetic changes in human cancers [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 836.
Abstract 84: Inhibition of Hedgehog signalling in ligand dependent tumours
Authors: Vaghjiani Vijesh G. , Cochrane Catherine , Watkins Neil , Mudge Stuart , Cain Jason E.
Published: August 2019

Hedgehog (Hh) signalling is evolutionarily conserved pathway that is critical during development and maintenance of stem cells in adults. Activation of the Hedgehog pathway can occur through either ligand-dependent or ligand-independent mechanisms. While mutations in key Hh signalling components (PTCH1, SUFU, SMO, GLI1) result in ligand-dependent signalling and constitutive activation in some cancers, the vast majority of cancers in which Hh signalling is implicated do not have pathway mutations and are therefore driven upstream at the level of Hh ligand. Despite this, there are no biomarkers that can predict which tumours are dependent on Hh ligand and are likely to be responsive to Hh pathway inhibition.

Using a panel of osteosarcoma cell lines derived from either radiation-induced or conditional Trp53 and Rb1 genetic inactivation murine models of osteosarcoma, we observed a dramatic increase in Gli1 mRNA, a downstream target of the Hh pathway, in response to Hh ligand stimulation in Trp53 and Rb1 deficient cells. This was accompanied by a significant increase in primary cilia frequency, a critical organelle for Hh pathway activation. Importantly, Hh response to ligand was abolished using the SMO inhibitor LDE225. Using osteosarcoma allograft models, we showed that LDE225-mediated inhibition of the Hh pathway resulted in reduced tumour growth and increased survival in ciliated Trp53 and Rb1-deficient allografts but no response in a non-ciliated Trp53 and Rb1 wildtype-allograft.

The sustained clinical use of SMO inhibitors ultimately leads to development of resistance. We next evaluated the efficacy of Itraconazole, a SMO inhibitor previously demonstrated to be effective in Hh-therapy resistant ligand-independent tumour models, in our ligand-dependent osteosarcoma models. We show that Itraconazole inhibits Hh pathway activation in response to ligand leading to reduced Gli1 mRNA in vitro, and reduced tumour growth and increased survival in vivo.

Collectively, this data implicates Trp53, Rb1 and primary cilia as a potential biomarker of Hh ligand responsiveness in osteosarcoma and response to Hh pathway inhibition by SMO inhibitors LDE225 and Itraconazole.

Citation Format: Vijesh G. Vaghjiani, Catherine Cochrane, Neil Watkins, Stuart Mudge, Jason E. Cain. Inhibition of Hedgehog signalling in ligand dependent tumours [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 84.
Abstract 840: Identification of novel colorectal tumor suppressor genes through genome-wide promoter hypermethylation analysis
Authors: Bazzocco Sarah , Dopeso Jose Higinio , Martínez-Barriocanal Águeda , Anguita Estefanía , Nieto Rocio , Sanchez Alex , Mariadason John M. , Arango Diego
Published: August 2019

Cancer initiation and progression are driven by both genetic and epigenetic changes. Although recent genome/exome sequencing efforts have significantly contributed to the thorough characterization of the genetic changes associated with the oncogenic process, further investigation is required to systematically identify the driver genes regulated by promoter hypermethylation. Using genome-wide analysis of the levels of promoter methylation (HumanMethylation27, Illumina) and the levels of mRNA expression (microarray analysis) in a panel of 30 colorectal cancer cell lines and 223 primary colorectal tumors (TCGA), we found a subset of 553 (5.6%) genes whose levels of promoter methylation showed a significant negative association with their expression. Higher overall methylation levels were associated with microsatellite instability (MSI), a CpG methylator phenotype (CIMP), faster proliferation and absence of APC mutations. Next, because genes that are epigenetically silenced could represent important drivers of the oncogenic process we investigated the role of the zinc finger transcriptional regulator ZNF238/ZBTB18, a gene silenced by promoter methylation, on the growth of colon cancer cells. Reintroduction of ZNF238 in HCT116 and HT29 colon cancer cells with low endogenous levels and promoter methylation of ZNF238, resulted in a significant reduction of cell proliferation both in vitro and in a subcutaneous xenograft NOD/SCID mouse model. Moreover, using immunohistochemical analysis with a validated antibody we found that ZNF238 is lost or reduced in the majority of the 133 primary colorectal tumors included in a tissue microarray, and that lower ZNF238 expression is associated with lymph node metastasis and shorter survival of patients with locally advanced colorectal cancer. In summary, we identified a set of 553 genes putatively silenced by promoter methylation in colorectal tumors that could significantly contribute to the oncogenic process. Moreover, as a proof of concept, we demonstrate that the epigenetically silenced gene ZNF238 has tumor suppressor activity and is associated with the survival of colorectal cancer patients.

Citation Format: Sarah Bazzocco, Jose Higinio Dopeso, Águeda Martínez-Barriocanal, Estefanía Anguita, Rocio Nieto, Alex Sanchez, John M. Mariadason, Diego Arango. Identification of novel colorectal tumor suppressor genes through genome-wide promoter hypermethylation analysis [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 840.