bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2442: Assessing how macrophages attain distinct iron handling phenotypes in a tumor via a mathematical model
Authors: Vieira Luis Sordo , Masison Joseph , Adams Jacqueline , Torti Suzy , Laubenbacher Reinhard , Chifman Julia
Published: August 2019

Tumor-associated macrophages are critical regulators of iron levels. It is well established that macrophages display distinct iron handling phenotypes based on the several signals they receive from other cells in the tumor microenvironment. In order to shed light on how macrophages respond to these different signals, we are building a knowledge-based mathematical model for how macrophages attain distinct, yet related, iron handling capabilities.

Citation Format: Luis Sordo Vieira, Joseph Masison, Jacqueline Adams, Suzy Torti, Reinhard Laubenbacher, Julia Chifman. Assessing how macrophages attain distinct iron handling phenotypes in a tumor via a mathematical model [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 2442.
Abstract 2456: NetZen: A comprehensive network-based pathway and target discovery platform
Authors: Le Son , Riva Alberto , Deng Changwang , Tran David D.
Published: August 2019

BACKGROUND: Success in precision medicine depends on the ability to accurately and rapidly identify master regulators of critical gene subnetworks. To achieve this goal requires a seamless computational target discovery platform that integrates global network visualization with identification of master regulatory subnetworks (MRS) and their key influencers.

METHODS: We have created NetZen - a computer cluster-capable computational suite allowing for 1) robust network generation with million edges; 2) network-based MRS identification; 3) hierarchical analysis with automated annotations; and 4) whole-network 3D visualization of all 20,000 genes. To identify cancer MRS, we applied NetZen to expression datasets of tumors and normal tissues, tumor-initiating cells (TICs), non-TIC cancer cells, and normal stem and non-stem cells from TCGA, GEO and internal sources. For validation, we introduced these subnetworks in normal cells to recapitulate TIC phenotypes in vivo or disrupted them in patients-derived TICs and mouse xenografts (PDX). To identify MRS of mortality, we applied NetZen to survival data in TCGA.

RESULTS: Networks of 28 human cancers were generated, integrating transcriptomics, microRNAs, proteomics, and clinical data. For cancers with TIC datasets, we identified TIC-specific MRS with clear-cut hierarchy controlling immune surveillance, EMT and developmental functions. Interestingly, in some cancers, the immune subnetwork including immune checkpoints was entirely encompassed in the developmental subnetwork. We present representative validation on glioblastoma TIC (GIC) MRS with features common in other cancers’ TICs. Exogenous introduction of this subnetwork in normal astrocytes forced them to become GICs and form tumors in mice. Disrupting this subnetwork in 10 independent PDXs profoundly blunted tumor growth and prolonged survival. Lastly, we present detailed MRS of mortality analysis of multiple cancer types and identify common pathways directly influencing outcomes.

DISCUSSION: NetZen provides a complete solution to big data analysis by effortlessly processing million-edge hierarchical networks with biological insights through automated annotations. The findings that the immune and developmental subnetworks were intimately linked suggest that immune escape by TICs is intrinsic of cellular transformation rather than a product of selection. Lastly, detailed network hierarchy provides a logical roadmap for cancer therapeutic development.

Citation Format: Son Le, Alberto Riva, Changwang Deng, David D. Tran. NetZen: A comprehensive network-based pathway and target discovery platform [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 2456.
Abstract 2479: A survey of molecular factors of DNA methylation and demethylation in a broad range of cancer cell lines
Authors: Vural Suleyman , Krushkal Julia
Published: August 2019

DNA methylation has a powerful influence on cellular processes. It plays important roles in cancer initiation and progression. Cancer cells exhibit frequent epigenomic alterations that affect expression of genes involved in apoptosis, cell proliferation, and DNA repair. We used expression information of known methylating and demethylating genes in cancer cell lines to analyze their association with methylation levels of individual probes and median epigenome-wide methylation levels. We utilized RNA-seq gene expression measures and Illumina Infinium HumanMethylation450 BeadChip DNA methylation data of 645 cancer cell lines from 23 cancer types from the Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer resources. We analyzed 73 genes, products of which are involved in methylation and demethylation processes, and examined correlations between expression levels of these genes and methylation status of individual probes and epigenome-wide. In a combined analysis across all cancer types, we observed statistically significant correlations between expression of global DNA methyltransferase genes, DNMT1, DNMT3A, and DNMT3B, and probe methylation levels in 566, 909, and 230 genes, respectively. In particular, DNMT3A expression was associated with methylation status of growth factor and receptor genes FGF1, FGFR4, and EGFR. Expression of hydroxymethylating genes TET1, TET2, and TET3 was correlated with methylation status of probes in 363, 1, and 1055 genes, respectively, including associations of TET3 expression with methylation of probes in the FGF1, KIT, FHIT, IGF1R, IGF2BP2, and PARP3 genes, which may affect growth, proliferation, and DNA repair of tumor cells. APOBEC1, CBX2, UHRF1 and ZBTB38, products of which are involved in various molecular steps of DNA methylation or demethylation, had the largest numbers of associations in different cancer types. For example, in chronic lymphocytic leukemia cell lines we observed a strong negative correlation between ZBTB38 expression, the product of which binds methylated DNA, and methylation levels of probes in 204 genes. We also found a strong correlation between UHRF1 gene expression and methylation levels of probes in 150 genes in chronic lymphocytic leukemia cell lines, in agreement with reported roles of the product of this gene in binding DNA methyltransferases and affecting DNMT1 methylation activity. These results provide a better understanding of molecular mechanisms of epigenetic dysregulation in cancer cells and may provide strategies for future analysis of molecular targets of drug response that may be affected via epigenetic processes.

Citation Format: Suleyman Vural, Julia Krushkal. A survey of molecular factors of DNA methylation and demethylation in a broad range of cancer cell lines [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 2479.
Abstract 2482: A portable bioinformatics pipeline for the FDA authorized IMPACT DNAseq assay
Authors: Tang Zuojian , Kumar Nikhil , Bolipata Allan , Song Timothy , Kandoth Cyriac , Liu Feng , Mcmanamon David , Hampton Oliver , Socci Nicholas , Solit David
Published: August 2019

On November 2017, the US Food and Drug Administration (FDA) authorized Memorial Sloan Kettering Cancer Center's (MSKCC) IMPACT (Integrated Mutation Profiling of Actionable Cancer Targets) tumor profiling assay and bioinformatics pipeline for clinical decisions in treating all solid tumors. The IMPACT test uses next-generation sequencing (NGS) to identify the presence of mutations in 468 unique genes, fusions at known sites, copy-number alterations, and microsatellite instability (MSI). A version freeze of the bioinformatics pipeline was made public in May 2017 in Zehir et al. "Mutational Landscape of Metastatic Cancer Revealed from Prospective Clinical Sequencing of 10,000 Patients", Nat Med. 23(6): 703-713. Active development of the pipeline has continued in its CLIA-compliant computing environment, and the resulting improvements have been incorporated into an open-source portable research-grade bioinformatics pipeline. Here, we present results from Roslin, our research bioinformatics pipeline built to be functionally equivalent to the IMPACT clinical reporting workflow that includes manual filtering/annotation and signout by molecular oncologists. Roslin can be installed and executed on laptops, workstations, local compute clusters, or cloud compute servers. As a research-grade pipeline it is flexible enough to handle new assays including exomes/genomes, and extensible enough to add or replace components like reference genomes, sequence aligners, variant callers, false-positive filters, and functional/clinical annotation. Tracking all components and parameters used in each pipeline run allows researchers to easily generate method sections for manuscripts that are sufficiently detailed for peer-review and reproducibility of published results. Roslin has been deployed and tested on multiple high-performance clusters and cloud computing resources. It offers end users GUI driven workflow logging, run reporting, and real-time tracking (https://github.com/mskcc/roslin). Evaluated on NGS data from 128 tumors with signed out IMPACT clinical reports, Roslin achieves a recall rate of 98% for both somatic substitutions and indels, and 60% for fusions. Precision was 96% for substitutions, and 86% for indels. We present our work in maximizing precision and recall rates while retaining the portability, modularity, and reproducibility of Roslin.

Citation Format: Zuojian Tang, Nikhil Kumar, Allan Bolipata, Timothy Song, Cyriac Kandoth, Feng Liu, David Mcmanamon, Oliver Hampton, Nicholas Socci, David Solit. A portable bioinformatics pipeline for the FDA authorized IMPACT DNAseq assay [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 2482.
Abstract 2495: Glucosamine enhances TRAIL-induces apoptosis in DU145 and Hela cancer cell lines
Authors: Sun Chao , Chesnokov Viktor , Itakura Keiichi
Published: August 2019

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) has attracted wide attention and extensive research because of selective apoptosis of tumor cells but not of normal cells. However, the clinical trials using TRAIL alone were disappointed due to the resistance of cancer cells to the TRAIL-treatment. Several resistant mechanisms, such as low expression of death receptors, and overexpression of cFLIP, BCL-2 antiapoptotic family members and XIAP were proposed. Tunicamycin was reported to enhance the expression of death receptor 5 through the activation of CHOP. Similar to tunicamycin, glucosamine is known to increase the expression of CHOP by inducing ER stress. We thus investigated whether glucosamine could increase the expression of death receptor 5 to overcome TRAIL-resistance. We found that glucosamine treatment of DU145 cells did not increase the expression of death receptor 5 on the cell surface, even though the overall expression was increased. However, glucosamine significantly increased TRAIL-induced apoptosis in DU145 and Hela cells. Our investigation revealed that glucosamine sensitized cancer cells to TRAIL-induced apoptosis through both intrinsic and extrinsic apoptosis pathways, as evidenced by the cleavage of both caspase 9 and caspase 8. Our data further showed that the combination of TRAIL with glucosamine suppressed the expression of cFLIP, BCL-2 and XIAP, translocated BAK to the mitochondrial outer membrane, and released cytochrome C and SMAC from mitochondrial. Interestingly, in addition to the activation of apoptotic pathways, TRAIL-mediated inflammatory responses were attenuated by glucosamine treatment. Pretreatment of glucosamine reduced the TRAIL-induced nuclear NF-kB level and the expression of IL-6 and IL-8. Together, these results demonstrated that the combination treatment, TRAIL with glucosamine, may provide a promising novel strategy for treating different human cancers by overcoming TRAIL-resistance and inhibiting TRAIL-induced inflammation.

Citation Format: Chao Sun, Viktor Chesnokov, Keiichi Itakura. Glucosamine enhances TRAIL-induces apoptosis in DU145 and Hela cancer cell lines [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 2495.
Abstract 25: Identifying new inhibitors of ABCG2 by high-throughput screening
Authors: Kokubo Shoji , Ohnuma Shinobu , Karasawa Hideaki , Yamamura Akihiro , Suzuki Hideyuki , Murakami Megumi , Sugisawa Norihiko , Kanehara Keigo , Sato Keisuke , Ishida Takanori , Kamei Takashi , Naitoh Takeshi , Unno Michiaki
Published: August 2019

Background: ABCG2, also known as breast cancer resistance protein (BCRP), is a member of the ATP-binding cassette (ABC)-family of membrane transport proteins associated with multidrug resistance (MDR). No ABCG2 inhibitor has shown clinical significance yet.

Aim: To identify novel inhibitors of ABCG2 by high-throughput screening.

Material & Methods: After higher expression of ABCG2 in HCT-116/BCRP cells was confirmed by flow cytometry (FACSVerse) using 5D3 antibody, high-throughput fluorescent cell-based assay was performed with the original chemical library of Tohoku University containing about 6000 compounds using ABCG2-specific fluorescent substrate, Pheophorbide a (PhA) in HCT-116/BCRP cells overexpressing ABCG2. Ko143, a potent ABCG2 inhibitor, was used as positive control. Fluorescence intensity in the cells was read by a fluorescence plate reader (SpectraMax M2e) in bottom read mode, with 395 nm excitation, 670 nm emission. To validate the results, flow cytometry assay was performed with 10 µM PhA in HCT-116/BCRP cells. The fluorescence intensity was analyzed by flow cytometry (FACSVerse).

Results: Fluorescenct cell-based assay with high-throughput screening could detect 23 candidate compounds, which showed higher fluorescence intensity as positive control Ko143 showed. Then, these 23 compounds were validated with flow cytometry assays. The flow cytometry assays revealed that 16 compounds increased fluorescence intensity of PhA in HCT-116/BCRP cells in dose dependent manner, suggesting that these candidate compounds inhibited transport function of ABCG2. Among 16 compounds, 6 compounds showed further inhibitory effect for transport function of ABCG2 as equivalent to that of Ko143.

Conclusion: These 6 compounds may provide a basis for further investigation of ABCG2 function.

Citation Format: Shoji Kokubo, Shinobu Ohnuma, Hideaki Karasawa, Akihiro Yamamura, Hideyuki Suzuki, Megumi Murakami, Norihiko Sugisawa, Keigo Kanehara, Keisuke Sato, Takanori Ishida, Takashi Kamei, Takeshi Naitoh, Michiaki Unno. Identifying new inhibitors of ABCG2 by high-throughput screening [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 25.
Abstract 2536: O-GlcNAcylation of Ewing sarcoma EWSR1/FLI1 protein impairs relocation of Aurora B to the midzone during mitosis
Authors: Azuma Mizuki
Published: August 2019

Ewing sarcoma is a pediatric bone cancer expressing chimeric EWSR1/FLI1 protein derived from a t(22;11)(q24;12) translocation. Our previous study demonstrated that EWSR1/FLI1 impairs the relocation of Aurora B kinase from the centromere to the midzone during the metaphase-anaphase transition (referred to as “Aurora B-relocation”). Aurora B-relocation to the midzone is essential for faithful cell division, whereas compromising this process will lead to formation of a binucleated cell or chromosomal mis-segregation. This cellular activity might play a role in the pathogenesis of Ewing sarcoma; however, how EWSR1/FLI1 impairs Aurora B-relocation remains unclear. In the present study, we tested the hypothesis that O-GlcNAcylation of EWSR1/FLI1 impairs Aurora B-relocation. The results showed that the increase in cellular O-GlcNAc levels in Ewing sarcoma A673 cells induced by treatment with the O-GlcNAcase inhibitor Thiamet-G (TMG) increased the level of aberrant Aurora B-relocation, and the O-GlcNAcylation level of endogenously expressed EWSR1/FLI1. To map the amino acid(s) required for O-GlcNAcylation of EWSR1/FLI1, HeLa cells were used because they do not express EWSR1/FLI1 endogenously. Among the putative point mutant constructs of EWSR1/FLI1, the substitution of Thr79 of EWSR1/FLI1 by Asp (phosphor mimic mutant) significantly reduced the O-GlcNAcylation level of EWSR1/FLI1, suggesting that Thr79 is the site of O-GlcNAcylation. Finally, the EWSR1/FLI1-T79D and EWSR1/FLI1-T79A mutant constructs failed to induce aberrant Aurora B-relocation in HeLa and A673 cells. Therefore, we propose that O-GlcNAcylated EWSR1/FLI1-T79 is required for the induction of aberrant Aurora B-relocation. In addition, EWSR1/FLI1-dependent disruption of Aurora B-relocation leads to defects in chromosome instability (CIN). Here, we demonstrate a unique role of EWSR1/FLI1 that may be a part of the pathogenesis of Ewing sarcoma.

Citation Format: Mizuki Azuma. O-GlcNAcylation of Ewing sarcoma EWSR1/FLI1 protein impairs relocation of Aurora B to the midzone during mitosis [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 2536.
Abstract 2543: Cyclin-dependent kinase 1(CDK1)-mediated phosphorylation of SET in mitosis inhibits its oncogenic activity
Authors: Yin Ling , Zeng Yongji , Dong Jixin
Published: August 2019

SET is a protein phosphatase 2A (PP2A) inhibitor and functions as an oncogene in multiple human cancer types. Here we revealed a novel phosphorylation site of SET in mitosis and its biological significance in tumorigenesis. We found that the mitotic kinase cyclin-dependent kinase 1 (CDK1) phosphorylates SET in vitroand in vivo at serine 7 during antimitotic drug-induced G2/M phase arrest and in normal mitosis. SET deletion resulted in massive mitotic defects including centrosome amplification, multipolar spindles, and chromosome missregregation. Moreover, we revealed that SET mitotic phosphorylation is required for proper mitotic progression and its oncogenic function in vitro and in xenograft animal models. Furthermore, we found STAT3 and p53 signaling are regulated by SET. Collectively, our findings suggest a novel layer of regulation for SET in mitosis and its role in tumorigenesis. (This is supported by Fred and Pamela Buffett Cancer Center Support Grant P30 CA036727)

Citation Format: Ling Yin, Yongji Zeng, Jixin Dong. Cyclin-dependent kinase 1(CDK1)-mediated phosphorylation of SET in mitosis inhibits its oncogenic 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 2543.
Abstract 2549: Allostatic conditions in human glioblastoma stem cells are maintained with the contribution of CLIC1 membrane protein functional expression
Authors: Verduci Ivan , Carlini Valentina , Barbieri Federica , Daga Antonio , Florio Tullio , Mazzanti Michele
Published: August 2019

Allostasis is a cellular condition physiologically occurring during transient hyper-activated state of the cell. When stress is persistent, cells are unable to restore a basal homeostatic state and allostasis become chronic. Most of the cells are not able to cope with these prolonged conditions and die. Conversely, solid tumors reveal a persistent stress state strengthen by chronic overexpression of several elements and hyper-activation of intracellular pathways. In glioblastoma stem cells (GSCs), the most evident allostatic outcome is a high rate of cell division. Several proteins present in the allostatic GSC plasma membrane are deregulated in either expression or function. Altering any of these proteins disrupts the allostatic equilibrium, causing drastic functional changes and, in some cases, cell death. Several of these proteins, potentially used as valuable pharmacological targets, are also crucial for the physiological activity of the cells. Consequently, modifying their functions would affect the survival of both cancer and healthy cell population. On the contrary, chloride intracellular protein 1 (CLIC1) is active as a membrane charge carrier only during periods of chronic stress, while during homeostatic conditions it is essentially irrelevant. Inhibition of NADPH oxidase or the NHE1 proton pump, both overexpressed in CSCs, causes death of both GSCs and normal mesenchymal stem cells. Conversely, impairing CLIC1 activity delays GSC cycle progression but leaves healthy cells functions unaltered. Our work suggests that CLIC1 protein and its associated chloride permeability are a crucial element involved in the stabilization of GB CSC allostasis. (This work is supported by a GRANT from AIRC to MM)

Citation Format: Ivan Verduci, Valentina Carlini, Federica Barbieri, Antonio Daga, Tullio Florio, Michele Mazzanti. Allostatic conditions in human glioblastoma stem cells are maintained with the contribution of CLIC1 membrane protein functional expression [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 2549.
Abstract 2555: Modulation of protein interaction states in the cell cycle and during cancer drug action
Authors: Nordlund Pär
Published: August 2019

The cellular thermal shift assay (CETSA) is a biophysical technique allowing direct studies of ligand binding to proteins in cells and tissues. The proteome-wide implementation of CETSA with mass spectrometry detection (MS-CETSA) is an efficient method to discover targets for orphan clinical drugs and hits from phenotypic screens, as well as to identify off-targets, explain poly-pharmacology and toxicity. Recently implemented highly sensitive multidimensional MS-CETSA techniques can now also access binding of physiological ligands to proteins, such as metabolites, nucleic acids and other proteins. MS-CETSA can thereby provide comprehensive information on modulations of protein interaction states in cellular processes, including downstream effects of drugs and transitions between different physiological cell states (Dai, Cell 2018, Tan, Science 2018). Such horizontal information on ligand modulation in cells is largely orthogonal to vertical information on the levels of different proteins, and therefore opens novel opportunities to understand operational aspects of cellular proteomes in more detail. We will discuss recent application of MS-CETSA to understand the cell cycle as well as cancer drug action and resistance.

References:

- Modulation of Protein-Interaction States through the Cell Cycle. Dai L, Zhao T, Bisteau X, Sun W, Prabhu N, Lim YT, Sobota RM, Kaldis P, Nordlund P. Cell. 2018 173(6):1481-1494.

- Thermal proximity coaggregation for system-wide profiling of protein complex dynamics in cells. Tan CSH, Go KD, Bisteau X, Dai L, Yong CH, Prabhu N, Ozturk MB, Lim YT, Sreekumar L, Lengqvist J, Tergaonkar V, Kaldis P, Sobota RM, Nordlund P. Science. 2018 359(6380):1170-1177.

Citation Format: Pär Nordlund. Modulation of protein interaction states in the cell cycle and during cancer drug action [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 2555.
Abstract 2557: X-ray repair cross complementing 1 (XRCC1) is a critical factor in response to DNA damaging agents in triple negative breast cancer
Authors: Lee Kevin J. , Andrews Joel , Gassman Natalie R.
Published: August 2019

Triple negative breast cancer (TNBC) is a breast cancer subtype that is characterized by the lack of expression of estrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor 2 (HER2). The lack of these targetable molecules makes cytotoxic chemotherapies a first-line choice, thus there is a pressing need to better understand how TNBC will respond to alternative or emerging treatments. Base excision repair (BER) proteins play an essential role in DNA damage response and repair, yet they have been overlooked in the analysis of chemotherapeutic treatment response. XRCC1 is a critical scaffold protein that mediates the protein-protein and protein-DNA interactions involved in BER and single-strand break repair. Several studies have linked single nucleotide polymorphisms (SNPs) within XRCC1 to increases in breast cancer incidence, yet XRCC1’s role in modulating treatment response is unknown. Here we show XRCC1 expression in a panel of TNBC cell lines correlates to an increase in basal DNA damage levels. DNA damage was analyzed using the Repair Assisted Damage Detection (RADD) assay which measures the broadest spectrum of DNA damage in vitro. We show MDA-MB-157 with low levels of XRCC1 correlate to low levels of basal DNA damage, MDA-MB-231 and HCC1806 have moderate levels of XRCC1 and DNA damage, and MDA-MB-468 contain high expression levels of XRCC1 that correlate with high levels of DNA damage. XRCC1 localization is also variable across the panel of TNBC cell lines with XRCC1 being mislocalized to the cytoplasm in the HCC1806 cells and highly localized to the nuclear compartment in the MDA-MB-468. These expression and localization changes in XRCC1 alter the ability of cells to repair single strand break (SSB) lesions induced by 355 nM laser microirradiation and DNA damaging chemicals. Here we show that TNBC cell lines respond differently to alkylating damage (methyl methanesulfonate) and PARP inhibition (olaparib) based on XRCC1 expression and localization in combination with levels of basal DNA damage. Taken together it is important to consider XRCC1 in context with DNA damage levels and expression of other BER proteins in TNBC in order to effectively design experiments utilizing DNA damaging agents or chemotherapeutics targeting DNA repair pathways.

Citation Format: Kevin J. Lee, Joel Andrews, Natalie R. Gassman. X-ray repair cross complementing 1 (XRCC1) is a critical factor in response to DNA damaging agents in triple negative 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 2557.
Abstract 2561: DNA-PKCS deacetylation by SIRT2 promotes DNA double-strand break repair by non-homologous end joining
Authors: Head PamelaSara E. , Ganji Nagaraju P. , Wang Shi-Ya , Duong Duc , Zhang Hui , Daddacha Waaqo , Li Shuyi , Seyfried Nicholas T. , Smalley David M. , Wang Ya , Deng Xingming , Dynan William S. , El-Rayes Bassel , Davis Anthony J. , Yu David S.
Published: August 2019

DNA-dependent protein kinase (DNA-PK) plays a critical role in non-homologous end joining (NHEJ), the predominant pathway that repairs DNA double-strand breaks (DSB) in response to ionizing radiation (IR) to govern genome integrity. How DNA-PK is activated in response to DSBs has remained elusive. Here, we show that the SIRT2 sirtuin deacetylase and tumor suppressor directs the activation of DNA-PK through deacetylation of its catalytic subunit (DNA-PKcs). SIRT2 deacetylase activity governs cellular resistance to DSB-inducing agents and promotes NHEJ. SIRT2 furthermore interacts with and deacetylates DNA-PKcs in response to IR, which facilitates its interaction with Ku and recruitment to DSBs, thereby leading to DNA-PKcs autophosphorylation and DNA-PK signaling to downstream NHEJ substrates. Moreover, SIRT2 inhibitor sensitizes resistant cancer cells and tumors to IR. Our findings define a mechanism for DNA-PK activation by SIRT2-mediated deacetylation, elucidating a critical upstream signaling event initiating the repair of DSBs by NHEJ to promote genome integrity and govern IR resistance, which can be exploited for improvements in cancer therapy.

Citation Format: PamelaSara E. Head, Nagaraju P. Ganji, Shi-Ya Wang, Duc Duong, Hui Zhang, Waaqo Daddacha, Shuyi Li, Nicholas T. Seyfried, David M. Smalley, Ya Wang, Xingming Deng, William S. Dynan, Bassel El-Rayes, Anthony J. Davis, David S. Yu. DNA-PKCS deacetylation by SIRT2 promotes DNA double-strand break repair by non-homologous end joining [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 2561.
Abstract 2563: A comparison of microsatellite instability analysis methods for detection of mismatch repair deficiency
Authors: Lewis Samantha R. , Peterson Shaun , Oostdik Kathryn , Tomlinson Heather
Published: August 2019

Use of biomarkers for patient treatment stratification is an increasingly important topic with the recent FDA approval of Pembrolizumab and Nivolumab for microsatellite instability (MSI) high/mismatch repair (MMR) deficient cancers. This ground-breaking approval allows physicians to make patient treatment decisions based on molecular biomarker status. Thus, performance of detection methods for these biomarkers is of great importance. The gold standard for microsatellite instability (MSI) analysis is PCR followed by fragment analysis using capillary electrophoresis to resolve DNA fragment size. Recently, PCR followed by melt curve analysis and Next Generation Sequencing (NGS) have been presented as alternative approaches. In this set of experiments, these methods will be compared using a subset of human cancer samples.

A cohort of matched cancer and adjacent normal FFPE samples were used for this comparison. From this cohort, individuals were selected that contained subtle MSI shifts, low tumor volume, or heterozygosity at microsatellite loci. Samples from these individuals were tested for MSI by either fragment or melt curve analysis or by sequencing. Samples were then classified as MSI-High, MSI-Low or microsatellite stable (MSS) according to manufacturer specifications for melt curve analysis or by an interpretive software for fragment analysis and NGS. Preliminary results indicate that there may be differences in sensitivity when using challenging samples with melt curve analysis compared to the gold standard method, fragment analysis.

The standard method for determining MSI status utilizes PCR and fragment analysis. Recently, melt curve analysis and NGS have been proposed as alternative methods. We present preliminary findings of decreased sensitivity with melt curve compared to fragment analysis. Additional studies with a larger, more diverse sample set will be required to define this relationship further.

Citation Format: Samantha R. Lewis, Shaun Peterson, Kathryn Oostdik, Heather Tomlinson. A comparison of microsatellite instability analysis methods for detection of mismatch repair deficiency [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 2563.
Abstract 2564: Tracing extrachromosomal DNA inheritance patterns in glioblastoma using CRISPR
Authors: Yi Eunhee , Verhaak Roel
Published: August 2019

Glioblastoma (GBM) is the most lethal brain tumor; it is characterized by poor response to standard post-resection radiation and cytotoxic therapy, resulting in a dismal prognosis with a five-year survival rate of 10%. Recurrence after therapy for GBM is unavoidable. There are substantial differences among the cells of GBM tumors in the abundance and types of genetic material. This heterogeneity likely is the major cause of therapy failure, the development of treatment resistance, and ultimately recurrence. A recent study has suggested that the majority of GBM activate oncogenes through amplification extrachromosomal DNA (ecDNA). Despite the speculation that ecDNA is a key factor of tumor heterogeneity, how ecDNA is propagated and distributed among—and how it behaves within—cancer cells is completely unknown. To address this gap in knowledge, this study focused on developing a novel cytogenetic tool that enables visualization and tracking ecDNA behavior in live GBM cells. We found breakpoint sequences resulting from genome rearrangements during ecDNA formation by performing computational analysis whole genome sequencing data from a pair of primary/recurrent GBM neurospheres. Each breakpoint represents a unique target sequence for ecDNA-specific labeling. Breakpoint were validated by breakpoint-PCR, focusing on breakpoints shared by the primary and recurrent neurosphere. We determined location and quantity of all ecDNA breakpoints through breakpoint-FISH (BP-FISH) analysis, which showed that all signal was found outside of the conventional chromosomes in the metaphase spread, validating the extrachromosomal nature of the ecDNA breakpoints. The breakpoint quantities, reflecting the number of ecDNA elements per cell, varied between primary and recurrent neurosphere suggesting that ecDNAs quantitatively changed during GBM evolution. These results emphasize the necessity of tracking ecDNA dynamics to answer the question of how ecDNA is distributed into the daughter cells. Our findings will be strong evidence to make ecDNA-specific CRISPR system in further research. Tracing ecDNA dynamics will provide new insight into the impact of ecDNA on cancer evolution.

Citation Format: Eunhee Yi, Roel Verhaak. Tracing extrachromosomal DNA inheritance patterns in glioblastoma using CRISPR [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 2564.
Abstract 2567: TRACERx: Intra-tumor subclonal driver mutation results in defective DNA damage response (DDR) and genome instability
Authors: Lu Wei-ting , Zalmas Lykourgos-Panagiotis , Webber Thomas , Kanu Nnennaya , Swanton Charles
Published: August 2019

TRACERx is a longitudinal clinical study, which explores the evolutionary trajectories of NSCLC by temporally dissecting genomic aberrations through multi-region sequencing. Analysis of the first 100 patients revealed extensive intra-tumor heterogeneity (ITH) and chromosomal instability (CIN), manifesting as subclonal somatic mutations and copy number aberrations. Although mutations in “trunk” clonal drivers such as Ras, C-Myc, p53 or MAPK pathway have been thoroughly characterized after the herculean efforts of TCGA consortium, the roles of sub-clonal drivers remain largely obscure as their identification is only made possible by multi-region sequencing.

Harnessing the power of the TracerX consortium, we investigate the molecular mechanisms that underpins the role of sub-clonal drivers in ITH. By means of high content imaging of DNA damage markers and tracking human artificial chromosome loss, we aim to elucidate the link between subclonal drivers and CIN events (Zalmas et al, in press). Importantly, we found the ablation of a large proportion of subclonal drivers result in genomic instability. This suggests that genome instability is not merely a standalone hallmarks of cancer, but also a major step that drivers sub-clonal expansion and functionally connect genome instability to tumor evolution. However, the molecular pathways directly linking genome maintenance, chromosomal instability events and cancer evolution is unclear. More intriguingly, only a fraction of these subclonal drivers has been previously reported to be involved in canonical DNA damage response (DDR). This suggests the error-prone DNA repair is favored during tumor clonal evolution.

Here, we present previously uncharacterized subclonal driver that are important for error-free DNA repair pathway. The ablation of these genes causes mis-localization of key homologous repair (HR) proteins, such as BRCA1 and Rad51, to DNA damage sites after IR irradiation. Strikingly, early signaling events such as ATM phosphorylation, phosphorylation and ubiquitylation of histone H2A.X, and 53BP1 localization remain unaffected. The exact molecular mechanism the leads to DNA repair pathway selection will be discussed.

Citation Format: Wei-ting Lu, Lykourgos-Panagiotis Zalmas, Thomas Webber, Nnennaya Kanu, Charles Swanton. TRACERx: Intra-tumor subclonal driver mutation results in defective DNA damage response (DDR) and genome instability [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 2567.
Abstract 2569: Synergism between RAD51AP1 and RAD54 during late stages of homologous recombination DNA repair
Authors: Selemenakis Platon , Maranon David , Wiese Claudia
Published: August 2019

Genomic instability is one of the enabling hallmarks of cancer and can arise by exposure to several factors: endogenous challenges such as stress to DNA replication forks or exogenous compounds such as exposure to ionizing radiation (IR) and other environmental mutagens. These insults can lead to the induction and propagation of mutations through the generation of multiple DNA lesions, the most toxic one of which is a DNA double-strand break (DSB). DSBs can be repaired by several different repair pathways. One of these pathways is homologous recombination (HR), a relatively faithful DSB repair pathway that relies on the availability of a homologous template for DNA repair synthesis.

DNA strand exchange in HR is mediated by the RAD51 recombinase which forms a nucleoprotein filament on single-stranded DNA for strand invasion. In human cells, RAD51-mediated strand invasion is supported by the DNA motor protein RAD54 and by the RAD51-Associated Protein 1 (RAD51AP1). While RAD54 translocates on double-stranded DNA and promotes DNA strand separation, RAD51AP1 enhances the assembly of the synaptic complex. However, if RAD54 and RAD51AP1 work independently or together in the HR reaction is unclear. Since functional loss of either RAD54 or RAD51AP1 each leads to a moderate defect in HR, we speculate that RAD54 and RAD51AP1 may act in parallel in the promotion of RAD51 activity. We hypothesize that simultaneous inactivation of both RAD54 and RAD51AP1 may lead to a synthetic phenotype in HR impairment.

The main focus of this study is to investigate the potential synergism between RAD51AP1 and RAD54 during late stages of the HR reaction to improve targeted cancer therapy. We are investigating if loss of RAD54 in human cells exacerbates the phenotype of RAD51AP1-deficiency and renders cells more sensitive to DNA damaging agents. We have generated RAD51AP1 knockout HeLa cells using CRISPR/Cas9 technology. In these cells, we have also inactivated the RAD54 gene using a very similar approach. Moreover, we have generated RAD54 knockout (KO) cells in a wild type RAD51AP1 background. All cell lines are tested for their response to chemotherapeutic agents in cell survival and DNA replication assays. Our first results show that the double KO cells are more sensitive to mitomycin C exposure than single KO cells, suggesting that RAD51AP1 and RAD54 function largely independently of each other in the HR reaction.

The results from our investigation are expected to lead to the potential development of new cancer drugs targeted at ancillary factors of RAD51 in late stages of the HR reaction.

Citation Format: Platon Selemenakis, David Maranon, Claudia Wiese. Synergism between RAD51AP1 and RAD54 during late stages of homologous recombination DNA repair [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 2569.
Abstract 2571: Role of XRCC4 downregulation in bioflavonoid-induced chromosomal translocations
Authors: Ghosh Anindita , Richardson Christine A.
Published: August 2019

The faithful repair of DNA double strand breaks (DSBs) is crucial for genome integrity, and aberrant repair can result in chromosomal translocations, including those associated with infant leukemia. Evidence from our lab and others suggests that environmental and dietary compounds such as bioflavonoids have the potential to promote chromosomal translocations in utero. DSBs can be repaired by either classical non-homologous end joining (C-NHEJ) that is mostly accurate or by microhomology-mediated alternative end joining (Alt-EJ) that is more error prone and can cause chromosomal translocations. Ku70/80, Ligase 4 (LIG4), X-Ray Repair Cross-Complementing factor 4 (XRCC4), and XRCC4 like factor (XLF) are central to C-NHEJ. Poly ADP Ribose 1 (PARP 1) and C terminal binding protein interacting protein (CtIP) participate in Alt-EJ. CtIP favors Alt-EJ pathway by promoting the resection of DNA ends to uncover microhomologies supporting the formation of large deletions and chromosomal translocations. Studies suggest that loss of efficient activity in the C-NHEJ repair pathway may promote the use of the other more error-prone pathway such as Alt-EJ. The purpose of the study is to understand the relationship between bioflavonoid exposure, induction of DNA repair proteins, and repair pathway choice to understand the underlying mechanisms of bioflavonoid-induced infant leukemias. Embryonic stem (ES) cells were exposed to genistein 1 hr at doses 0μM-100μM based on determined LD50 values. Following exposure, cells were recovered 4hr, 24hr and 48 hr, followed by protein extraction and Western blotting. Protein levels of γH2AX, Ku70, XRCC4, XLF, CtIP and PARP-1 were measured by densitometry and analyzed using one way ANOVA. CRISPR-Cas9 was used to downregulate C-NHEJ DNA repair protein XRCC4. XRCC4-/- cells will subsequently be exposed to genistein, recovered, followed by protein extraction and Western blotting. Protein levels of γH2AX, Ku70, XRCC4, XLF, CtIP and PARP-1 will measured by densitometry. Protein levels before and after gene editing of XRCC4 will be compared and analyzed using two way ANOVA. We have downregulated the XRCC4 gene in ES cells and anticipate that following exposure to genistein in the absence of XRCC4, we will detect elevated chromosomal translocation frequency in those stem cells by stimulating the Alt-EJ pathway. This research will be highly impactful in understanding how bioflavonoid-induced DNA damage is repaired by cells and whether these compounds promote genome instability analogous to that observed in infant leukemia.

Citation Format: Anindita Ghosh, Christine A. Richardson. Role of XRCC4 downregulation in bioflavonoid-induced chromosomal translocations [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 2571.
Abstract 2572: Integrated viral sequences in esophageal cancer tissue
Authors: Patten Victoria A. , Mwaphaga Lamech , Mathew Christopher G. , Hendricks Denver T. , Parker M. Iqbal
Published: August 2019

The human genome exhibits a significant degree of invasion by viral genomes. During the course of evolution, several RNA and DNA viruses have become integrated into the vertebrate genome and there is no reason to believe that the process has ended. Integrated viral DNA may account for as much as 10% of the human genome and it is very likely that they may act as carcinogens by altering normal cellular gene expression.

In this study we have compared the viral DNA sequences present in normal and tumor tissues from esophageal squamous cell carcinoma (ESCC) samples. DNA was extracted from paired normal and tumor biopsies and subjected to whole genome sequencing. Raw data run folders were converted to FASTQ files and progressed through a QC pipeline. Normal and tumor data sets were then blasted and aligned against an extensive viral sequence database using specialized DRAGEN software, so that all integrated viral DNA fragment sequences were identified. The identified viral sequences were then compared between the paired normal and tumor samples.

Using this approach, a large number of viral DNA sequences were identified; these included human papilloma viruses, Herpes simplex viruses, adenoviruses and Hepatitis C viruses. Furthermore, some unexpected viruses of interest were also identified. These include the Autographa Californica Nucleopolyhedrovirus, the Emiliana Huxleyi virus 86, the Gryllus bimaculatus nudivirus and the Trichodysplasia spinulosa-associated polyomavirus, present in different tumour DNA samples.

Amplification and rearrangements of endogenous retroviruses such as the Human Endogenous Retrovirus K113 were also observed. Endogenous retroviruses (ERV) can serve as long-lasting viral reservoirs and provide evidence for the coexistence between retroviruses and their hosts over many generations. Defective ERVs can also recombine to affect the expression of adjacent genes.

It has been proposed that integrated viral sequences may play a possible role in the etiology and promotion of diseases such as cancer, thus targeted studies of specific viral sequences may elucidate the mechanisms of gene disruption and may ultimately lead to the development of more effective therapeutic interventions.

Note: This abstract was not presented at the meeting.

Citation Format: Victoria A. Patten, Lamech Mwaphaga, Christopher G. Mathew, Denver T. Hendricks, M. Iqbal Parker. Integrated viral sequences in esophageal cancer tissue [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 2572.