bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5338: Transcriptional elongation mediates human papillomavirus type 16 late promoter activation
Authors: Songock William , Bodily Jason
Published: July 2017

Human papillomaviruses (HPVs) are small DNA viruses that infect keratinocytes of squamous epithelia and cause benign lesions that can progress to several malignancies, including cervical cancer. Upregulation of viral transcripts by the HPV16 late promoter is dependent on host cellular differentiation. We sought to investigate the main cellular transcription regulators that correlate with differentiation-dependent increase in the late viral transcripts. Here, we show evidence that the step in transcription cycle that is responsible for late promoter activation is transcriptional elongation.

We began by establishing that the increase in viral transcripts originating from the late promoter is primarily regulated at the transcript synthesis level rather than post-transcription level. Based on this observation, we investigated whether there is a correlation between late promoter activation and activities that occur during the initiation, pausing or elongation steps of transcription cycle. Using chromatin immunoprecipitation (ChIP), we did not observed a differentiation-dependent increase of RNA Polymerase II (RNAP II) in the late promoter region. However, there was a significant increase of RNAP II at downstream sites of late promoter suggesting elongation is regulated by differentiation.

We then sought to investigate the cyclin dependent kinases (CDKs) that are known to regulate transcription elongation. ChIP did not show a significant increase of the elongation factor CDK9 in the late promoter; however, the enrichment of CDK9 at downstream sites of the viral genome significantly increased upon differentiation, suggesting that differentiation induces recruitment of CDK9 to facilitate elongation. Additionally, CDK8, a component of the Mediator complex that recruits CDK9 to the RNAP II, was increased upon differentiation. Kinase inhibitors against CDK8 did not affect late viral transcripts; however, knocking down CDK8 resulted in significant reduction of viral transcripts, suggesting that CDK8 could be involved in protein-protein interactions that are required for the synthesis of late viral transcripts.

These findings suggest that HPV has evolved to utilize cellular transcriptional elongation machinery as a way of regulating its late gene expression. This mechanism allows the virus to persistently infect host cells and cause HPV-associated malignancies.

Citation Format: William Songock, Jason Bodily. Transcriptional elongation mediates human papillomavirus type 16 late promoter activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5338. doi:10.1158/1538-7445.AM2017-5338
Abstract 5342: Epigenetic polymorphisms and human papillomavirus E6/E7 oncoproteins transfection: Induce ATM gene deletion11q22-23 and chromosomal instability
Authors: McGhee Eva , Zarate Liliana , Opara Chiamaka , Alugbue Antoinette , Tyler Meidrah , Wissa Bileko , Ballard Billy , Pattillo Roland
Published: July 2017

Persistent infections involving high-risk types 16 and 18 of Human Papillomavirus (HPV) have been established as the etiological agent of cervical cancer. Recent data indicate that several polymorphisms of key regulators from the DNA damage repair pathways are associated with cancer development susceptibility. The long arm of chromosome 11 has received much attention as a high frequency of epigenetic deletions of various sites, observed in HPV-long term infection that lead to cervical cancer, indicating the presence of putative tumor suppressor genes and its association with disease progression. To better understand the importance of chromosome 11q22-23 region in the development of cervical cancer, we investigated genetic and epigenetic alterations of the tumor suppressor ATM gene in human keratinocytes transfected with HPV E6/E7 oncoproteins (16-MT). In the present study we examined 16-MT cells for epigenetic changes and chromosome imbalances, using FISH, and arrayCGH. We used several FISH probes, mapping to region 11q22-23 where the ATM gene is located. Our study revealed that the ATM gene located on chromosome 11q is deleted, relating to chromosome deletions, that are recurrent abnormalities in cervical cancer, implicating the loss of tumor suppressor genes as a significant mechanism that drive cells to become cancerous. This study was designed to investigate genetic instability using HPV16 E6/E7 oncoproteins to better understand the importance of chromosome 11q epigenetic changes. Deletion of 11q22-23 was observed in all metaphases scored. With restricted areas of deletions, two chromosomal regions of possible importance in cervical carcinomas could be distinguished. The first region is D11S1325 and the second region is D11S2179. The second region may contain part of the ATM gene. The results indicate that a tumor suppressor gene on chromosome 11q22-23 may be involved in the carcinogenesis of the cervix and the involvement of the ATM gene, as the driving factor remains a possibility.

Citation Format: Eva McGhee, Liliana Zarate, Chiamaka Opara, Antoinette Alugbue, Meidrah Tyler, Bileko Wissa, Billy Ballard, Roland Pattillo. Epigenetic polymorphisms and human papillomavirus E6/E7 oncoproteins transfection: Induce ATM gene deletion11q22-23 and chromosomal instability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5342. doi:10.1158/1538-7445.AM2017-5342
Abstract 5346: Combining droplet tagged short read sequencing with optical DNA mapping technology for improved assembly of cancer genomes
Authors: Jiang Yuan , Bilke Sven , Meltzer Paul S.
Published: July 2017

Kataegis, chromothripsis, chromoplexy and forms of structural hyper-mutation generically described as chromoanagenesis are found in many cancer types. Structural rearrangements can result in deletions, translocations, inversions and insertions, and gene amplification with important functional consequenes as cancer drivers and clonal diversity. Whole genome sequencing is increasingly used to sensitively detect strucutural rearrangements. Yet, it remains difficult to detect these events reliably without an excessive number of false positives. This is largely a consequence of the still relatively short read-length of commonly used sequencing platforms. As a result, in larger cohorts, PCR based methods are generally not feasible for validating all predicted structural rearrangements. Accordingly, new approaches for robust mapping of both simple and complex rearrangements are needed. Here we used a hybrid of two technologies, 10X genomics droplet tagged Illumina sequencing (10X Genomics ) and DNA fiber molecular imaging (BioNano Irys) for high throughput validation. First, utilizing a sequencing-assisted synthetic genome: for each rearrangement predicted by NGS, we build a “pseudo-chromosome” offering this structure as one alternative to the BioNano aligner. This combination compensates for the weaknesses of each platform resulting in a more robust result. In this way, even short reads can validate structural rearrangements, as the requirement for split reads is obviated. Provided that sufficient coverage is achieved, statistical analysis can be used to confidently recognize NGS false positive predictions. This allows the cataloging of complex repetitive regions and large-scale rearrangements. In our study we have evaluated this combined approach to large scale genome mapping to identify hallmark fusions and other rearrangements from the highly complex genomes of osteosarcoma cancer cell lines.

Citation Format: Yuan Jiang, Sven Bilke, Paul S. Meltzer. Combining droplet tagged short read sequencing with optical DNA mapping technology for improved assembly of cancer genomes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5346. doi:10.1158/1538-7445.AM2017-5346
Abstract 5350: A comprehensive and integrated approach to genomic and proteomic analysis of FFPE NSCLC tumor specimens
Authors: Hinerfield Douglas , Mellen Jennifer , Geiss Gary , Webster Philippa , Merritt Chris , Barker Kristi , Demirkan Gokhan , Filanoski Brian , Polakiewicz Roberto , Crosby Katherine
Published: July 2017

This poster describes a novel fully integrated method for the multiplexed DNA, RNA and Protein analysis of formalin fixed paraffin embedded tumor specimens. Cancer progression is typically a result of aberrations in the molecular pathways that regulate cell growth. Identifying and understanding these changes is necessary for the detection of drug targets as well as the identification of novel biomarkers that can predict response to such drugs. Deep molecular profiling of clinical tumor samples is challenged by limited material and the compromised quality resulting from variable handling and the fixation processes that are commonly employed in the clinical laboratories. While multiplexed gene expression and mutational analysis of such samples has become well established, we describe a new method that enables multiplexed DNA, RNA and protein analysis from a single formalin fixed paraffin embedded (FFPE) tumor specimen on the NanoString nCounter system. We have adapted the sample preparation methods to enable the multi-analyte analysis of FFPE samples and have demonstrated concordance with the clinical gold standard immunohistochemistry (IHC) methodology. As a proof-of-concept, five FFPE non-small cell lung cancer (NSCLC) specimens were simultaneously stained with a panel of antibodies specific to a variety of cancer-relevant proteins, five of which were validated on serial sections by IHC using antibodies from Cell Signaling Technology. In addition to the protein analysis, from the same specimen, expression of 770 cancer-related genes and >100 single nucleotide variants (SNVs) were also analyzed on the nCounter platform. By integrating the analysis of DNA SNV’s, as well as RNA and protein expression from single FFPE specimens, it is now possible to reveal the molecular mechanisms of tumor progression, uncover novel drug targets and identify biomarkers from precious specimens.

Citation Format: Douglas Hinerfield, Jennifer Mellen, Gary Geiss, Philippa Webster, Chris Merritt, Kristi Barker, Gokhan Demirkan, Brian Filanoski, Roberto Polakiewicz, Katherine Crosby. A comprehensive and integrated approach to genomic and proteomic analysis of FFPE NSCLC tumor specimens [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5350. doi:10.1158/1538-7445.AM2017-5350
Abstract 5357: OncoGxSelectTM - a beneficial supplement to comprehensive cancer genetic test
Authors: Han Yang , Yu Pengfei , Song Qingxuan , Wei Min , Hu Guanghui
Published: July 2017

Genetic profiling of tumor has been widely accepted both in oncology research and clinical field. However, clinical doctors are sometimes overwhelmed by tremendous and complicated information provided by comprehensive genetic tests, although they are welcomed by research-oriented oncologists. In order to provide accurate key findings to aid diagnosis and treatment in clinical practice, a careful tailored NGS based test - OncoGxSelectTM was developed and validated. Targeting 7 popular cancer types (NSCLC, Colorectal cancer, Breast cancer, Melanoma, Thyroid cancer, Esophageal and GIST cancer), OncoGxSelectTM detects all 4 types of genomic alterations including single nucleotide mutation, insertion/deletion, copy number variation and gene rearrangement in tumor tissue for 12 well-characterized clinical actionable genes strictly following NCCN (National Comprehensive Cancer Network) guidelines. OncoGxSelectTM offers high sensitivity and specificity greater than 99%, low cost and fast turnaround time using both DNA and RNA as starting material from FFPE samples with as low as 10% tumor cell content. This CLIA certified and CAP accredited genetic test panel is a valuable supplement to clinical oncologists to further facilitate diagnosis and treatment choosing.

Citation Format: Yang Han, Pengfei Yu, Qingxuan Song, Min Wei, Guanghui Hu. OncoGxSelectTM - a beneficial supplement to comprehensive cancer genetic test [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5357. doi:10.1158/1538-7445.AM2017-5357
Abstract 5359: An ultrasensitive and highly reproducible hybridization capture-based Next-Generation Sequencing clinical assay to enable precision oncology in patients with solid tumors
Authors: Hernandez Moises , King Sara , Raghunath Sharanya , Johnson Christopher , Loughmiller David , Nadauld Lincoln , Mishra Pravin J.
Published: July 2017

Sequencing whole cancer exome and whole genome provides useful information to study cancer evolution. However, cost and amount of data may become overwhelming. Enrichment capture-based methods to design custom targeted gene panel have rapidly evolved recently in cancer genomics arena. Custom targeted gene sequencing entails several advantages (i.e., better quality, ethical, affordable, technically suitable, personalized and reimbursable) for cancer patients over whole exome and whole genome sequencing. With this in mind, and to enable precision oncology in patients with solid tumors, we developed ICG100 2.0 panel, a hybridization capture-based next-generation sequencing assay for targeted sequencing of all exons and flanking introns of 162 commonly mutated cancer genes in formalin-fixed, paraffin-embedded (FFPE) tumors. To establish ICG100 2.0 panel, we compared three commercially available capture based technologies and evaluated reproducibility, sensitivity, specificity and the detection limit for low-frequency variants using internally developed bioinformatics pipeline. Cell lines, reference standards/synthetic DNA and solid tumor samples with the known genetic information was utilized in this analysis. Results were derived from MiSeq and NextSeq platforms and cross-compared on other platforms, including MassArray and ddPCR for establishing concordance and uniformity. Intra and interrun replicates were utilized to assess the quality, precision and reproducibility of variant calling. Mean depth of coverage was observed at >300X with >99% sensitivity and specificity. These findings and observations will guide other clinical laboratories to establish new assay that require less DNA input, enzyme based fragmentation and reduced preparation time. While we show that the three capture based methods (after comparison) had an overall accuracy in SNP and CNV detection similar to each other with minor differences, we describe an approach to assess and establish the best assay from the clinical standpoint to guide treatment decisions and match cancer patients to the most appropriate clinical trials.

Citation Format: Moises Hernandez, Sara King, Sharanya Raghunath, Christopher Johnson, David Loughmiller, Lincoln Nadauld, Pravin J. Mishra. An ultrasensitive and highly reproducible hybridization capture-based Next-Generation Sequencing clinical assay to enable precision oncology in patients with solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5359. doi:10.1158/1538-7445.AM2017-5359
Abstract 5368: Comparative analysis of multiple copy number alteration tools in the detection of amplifications and deletions on both whole-exome and targeted NGS panel platforms
Authors: Virk Selene M. , Boyle Sean Michael , Alla Ravi , Yen Jennifer , Chen Richard
Published: July 2017

Somatic copy number alterations, or CNAs, are frequent occurrences in the tumor landscape and can present in the form of focal alterations or as chromosome level events. Reliable detection of CNAs is crucial to understanding the impact of these events on a wide range of factors including tumor progression and treatment outcomes. The clinical use of NGS panels, and more recently exomes, continues to expand in cancer but the reliable detection of CNAs remains a challenge. A major factor contributing to the challenge is the availability of reliable tools that have been well tested and validated.

We sought to characterize the sensitivity and specificity of multiple CNA detection tools with a focus on clinically relevant genes in NGS panel and whole-exome. Our approach utilized a variety of publicly available CNA tools to identify whole gene amplifications and deletions in a set clinically relevant genes identified in the Catalogue of Somatic Mutations in Cancer (COSMIC) database. The tested CNA tools included two that supported both whole exome and panel NGS data, allowing cross platform in addition to cross tool comparisons. This work involved more than 20 well characterized cell lines derived from a broad spectrum of tumor types, including breast, lung, melanoma, and prostate cancers. The cell lines were analyzed in both tumor/normal and tumor only (which utilizes a proxy normal) modes. Analyses of patient-matched and proxy-normal data were performed with both assays.

Using our approach, we were able to identify clinically relevant CNAs on both platforms. The performance of the tools tested varied, with some tools performing best on either amplification or deletion events. These results represent a comprehensive comparison of recent copy number alteration tools and provides data that can be utilized to make the tools even more robust and reliable.

Citation Format: Selene M. Virk, Sean Michael Boyle, Ravi Alla, Jennifer Yen, Richard Chen. Comparative analysis of multiple copy number alteration tools in the detection of amplifications and deletions on both whole-exome and targeted NGS panel platforms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5368. doi:10.1158/1538-7445.AM2017-5368
Abstract 537: TRAF3 serves as a tumor suppressor in HPV-associated head and neck squamous cell carcinomas
Authors: Chen Tony W. , Zhang Jialing , Yang Xinping , Chen Zhong , Waes Carter Van
Published: July 2017

The molecular mechanisms that control transformation and tumorigenicity of human papillomavirus-associated (HPV+) head and neck squamous cell carcinomas (HNSCCs) are currently being investigated. The number of HPV+ HNSCC cases has increased sharply in recent years, especially in tonsil and oropharyngeal cancers. These HPV+ HNSCCs often respond differently to treatments. Recently, The Cancer Genome Atlas (TCGA, 2015) identified novel loss-of-function genomic alterations of TNF receptor-associated factor 3 (TRAF3) in HPV+ HNSCCs. TRAF3 is a ring-finger E3 Ubiquitin Ligase which inhibits downstream alternative NF-κB signaling and promotes anti-viral immunity by promoting degradation of ubiquitinated proteins. To assess TRAF3’s role in HPV+ HNSCCs, we identified cell lines with lower level of TRAF3 protein, consistent with deficient TRAF3 expression identified in TCGA data. Functional studies showed that TRAF3 expression led to decreased steady-state protein levels of the alternative NF-κB pathway components RELB and NF-κB2/p52, as assessed by Western blot, reporter assays and immunofluorescence. Additionally, TRAF3 expression led to decreased cell proliferation, tumorigenic activity and migration, and increased sensitivity to chemotherapy agent cisplatin. Interestingly, TRAF3 increases the steady state protein level of the classical tumor suppressors RB and p53 in HPV+ HNSCC cell lines as assessed by Western blot. Further in vitro characterization of TRAF3’s function in HPV+ HNSCCs was assessed using clinically identified TRAF3 loss-of-function mutagenic isoforms, mimicking both TCGA data and HNSCC cell line data for TRAF3 defects. In contrast to wtTRAF3, these mutant forms of TRAF3 do not as strongly restore p53 or inhibit RELB and NF-κB2/p52 protein levels. One specific mutation which causes a frameshift at residue 210 greatly inhibits TRAF3 function as assessed by Western blot. To further assess effects of wt or TRAF3 loss-of-function mutants in vitro and in vivo, an inducible HPV+ HNSCC line expressing TRAF3 using the TET-ON system, and TRAF3 knockout in HPV+ HNSCC line and Human Oral Keratinocyte (HOK) line using CRISPR-Cas9 genomic editing are generated. In conclusion, the ring-finger E3 ubiquitin ligase TRAF3 inhibits the pro-survival alternative NF-κB signaling pathway and restores TP53 and RB, thereby serving as a tumor suppressor in HPV+ HNSCCs.

(Supported by NIDCD intramural project ZIA-DC-000016, 73 and 74)

Citation Format: Tony W. Chen, Jialing Zhang, Xinping Yang, Zhong Chen, Carter Van Waes. TRAF3 serves as a tumor suppressor in HPV-associated head and neck squamous cell carcinomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 537. doi:10.1158/1538-7445.AM2017-537
Abstract 5370: Examination of DNA looping near oncogenes reveals variable patterns of epigenetic landscapes in cancer
Authors: Perez Monika , Johannes Ty
Published: July 2017

INTRODUCTION:

The compaction of chromatin in the nucleus into hierarchical three-dimensional (3D) structures plays a key role in the regulation of gene expression. Advancements in high-throughput chromatin conformation capture assays have enabled the determination of this hierarchical structure in a variety of cell states, including cancer. Of note, recent studies have implicated pathogenic alterations in 3D topology of the genome with the activation of proto-oncogenes in a multitude of cancers.

METHODS:

To characterize the topological changes associated with different cancer phenotypes, we examine publically available epigenetic profiles from the ENCODE Project, including 3D data from Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET) from breast cancer and leukemia cell lines. Integrating the chromatin topology data with transcription factor binding, DNase accessibility, and histone modification data, we uncover variable epigenetic landscapes associated with differentially active oncogenes in these cancer cell lines.

RESULTS:

We provide evidence of topological variability influencing differentially expressed oncogenes and tie these observations to other epigenetic changes. In particular, we show insulated-neighborhoods mediated by CTCF looping partition segments of the genome containing active oncogenes away from repressive markings like H3K4me3. Additionally, we show that variation in topology differentially localizes H3K27ac in distal regulatory regions to promoters of oncogenes, providing a means for gene activation in non-coding regions.

CONCLUSIONS:

Variable patterns of chromatin topology provide a unique signature and mechanism of oncogenes for cancerous phenotypes. We use publically available data to show mechanisms of activity associated with chromatin topology variation.

Citation Format: Monika Perez, Ty Johannes. Examination of DNA looping near oncogenes reveals variable patterns of epigenetic landscapes in cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5370. doi:10.1158/1538-7445.AM2017-5370
Abstract 5372: Alterations in the chromatin accessibility in nonalcoholic steatohepatitis-associated hepatocellular carcinoma
Authors: Dechassa Mekonnen Lemma
Published: July 2017

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Accumulated evidence in the past decade has stablished that NASH, in addition to other well-identified risk factors, is becoming a major cause of HCC in United States. In the present study, we investigated cancer-associated chromatin alterations in the Stelic Animal Model (STAM) of NASH-related HCC. Using Assay for Transposase-Accessible Chromatin with high throughput sequencing (ATAC-seq), we identified 1666 differentially opened chromatin regions in HCC tumor tissue as compared to normal livers. Annotation of these regions showed that they were associated with 1773 differentially expressed genes, which were enriched in lipid and steroid metabolism, obesity, and cancer. Among the differentially opened chromatin regions, 10% resided within 5 kb from the transcription start site (TSS), while the remaining 90% were located distal to the TSS (> 5 kb). By combining ATAC-seq and transcriptomic profiles, we demonstrated that 182 of the up-regulated genes were associated with open chromatin regions in tumor tissue. Furthermore, 24 of these up-regulated genes, including Apoa4, Nupr1, Anxa2, and Igfbp1, had accessible chromatin regions located proximal to the TSS. Importantly, analysis of histone modifications at the open chromatin regions of the over-expressed cancer-related Apoa4 and Anxa2 genes demonstrated enrichment by H3K4 mono-methylation (H3H4me1) and H3K27 acetylation (H3K27ac), histone marks that associated with the active gene promoters (H3K27ac) and enhancers (H3Kme1 and H3K27ac). In conclusion, these data provide evidence for alterations in the chromatin accessibility in tumor tissue and potential role of open chromatin regions in the over-expression of critical cancer-related genes in HCC.

Citation Format: Mekonnen Lemma Dechassa. Alterations in the chromatin accessibility in nonalcoholic steatohepatitis-associated hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5372. doi:10.1158/1538-7445.AM2017-5372
Abstract 538: The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) anti-apoptosis ability
Authors: Hou Hsin-Han , Yu Chong-Jen
Published: July 2017

Emerging evidence has indicated that proteins of a disintergrin and metalloprotease (ADAM) family contribute to cancer progression and metastasis. One member of this family, ADAM15, has been shown to be upregulated in multiple cancers, including gastric, lung, breast, and prostate cancers, and the enzymatic activities of its extracellular metalloprotrease domain promote breast cancer proliferation and migration through mediating ErbB signaling pathway. The patients with ADAM15 high-expressing lung tumors have shorter survival time and ADAM15 has been proved to enhance synovial fibroblasts anti-apoptosis ability via focal adhesion kinase signaling pathway. We firstly demonstrated other than extracellular enzymetic activity, the longest isoform of ADAM15 (ADAM15 i6), which contains the most cytoplasmic Src homology 3 (SH3) binding motifs, significantly upregulated in primary lung cancer tissues and promoted NSCLC proliferation via growth factor receptor-bound protein 2 (Grb2) and Src homolog 2 domain containing (Shc) association. In this study, we further explore the roles of ADAM15 cytosolic domain in NSCLC apoptosis resistance. Overexpression of ADAM15 i6 promoted CL1-0 cell anti-apoptosis ability according to the trypan blue inclusion assay. Ablation of nephrocystin (NPHP1) attenuated the ADAM15 i6-promoted anti-apoptosis ability. Thus, we identified a novel mechanism of the ADAM15 cytoplasmic domain in NSCLC tumor progression, which will shed light on the molecular mechanisms of ADAM proteins, and facilitate development of novel therapy in NSCLC.

Note: This abstract was not presented at the meeting.

Citation Format: Hsin-Han Hou, Chong-Jen Yu. The cytosolic domain of a disintergrin and metalloprotease (ADAM) 15 promotes non-small cell lung cancer (NSCLC) anti-apoptosis ability [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 538. doi:10.1158/1538-7445.AM2017-538
Abstract 5386: Does chemotherapeutics contribute to DNMT1 expression level in colorectal cancer cells
Authors: VAROL NURAY , TERZİ SUNA ARIKAN , SOYLEMEZ ZAFER , YILDIZ HANDAN , OZDEMİR MUJGAN , SOLAK MUSTAFA
Published: July 2017

Epigenetic modifications, particularly DNA methylation in selected gene promoters is a pivotal role in the development of colorectal cancer. DNA methylation is considered as one of the most important epigenetic mechanisms and it is catalyzed by DNA methyltransferases (DNMTs). DNMT1 abundance has been frequently seen in colorectal cancers but the reasons are not well understood. We investigated to the effect of chemotherapeutics used in treatment of colorectal cancer on expression of DNMT1 and this effect is achieved over which signalling pathway. In this study, GSK3β; IWP2 for β-catenin) and chemotherapeutics (oxaliplatin, fluorouracil, irinotecan) were detected by WST1. DNMT1 expression level was determinated by real-time PCR; and protein levels of GSK3β, pGSK3β(Ser9), Akt1, pAkt1(Ser473), β-catenin, pβ-catenin(Ser675) and DNMT1 by western blot. Our results indicated taht Akt1 increased the protein level of DNMT1 expression with coordinate transcriptional change via β-catenin pathway. Fluorouracil and irinotecan decreased DNMT1 expression at both transcriptional and translational levels but not oxaliplatin. Oxaliplatin increased DNMT1 expression at mRNA and protein levels. This effect is achieved by specific phosphorylation of β-catenin protein. The results revealed that use of some chemotherapeutic, particularly oxaliplatin, with spesific inhibitors (such as β-catenin inhibitor) in combination led to a reduced DNMT1 expression. Our findings may offer a new approach for determining the molecular effects of β-catenin signal pathway on DNMT1. This may allow us to identify new molecular targets for the treatment of colorectal cancers. However, the results revealed that some chemotherapeutics may contribute the aberration of DNA methylation.

Citation Format: NURAY VAROL, SUNA ARIKAN TERZİ, ZAFER SOYLEMEZ, HANDAN YILDIZ, MUJGAN OZDEMİR, MUSTAFA SOLAK. Does chemotherapeutics contribute to DNMT1 expression level in colorectal cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5386. doi:10.1158/1538-7445.AM2017-5386
Abstract 5387: Droplet digital PCR for sensitive quantification of DNA methylation in non-invasive material: Development of a robust control
Authors: Pharo Heidi D. , Andresen Kim , Jeanmougin Marine , Lind Guro E.
Published: July 2017

Background: Droplet digital PCR (ddPCR) is a highly sensitive method for quantification of nucleic acids, with great potential for detection of biomarkers present in low quantities. As for DNA methylation analyses using ddPCR, there is no consensus on whether or not a control should be included. The aim of the present study was to; 1) demonstrate that use of a control is essential in such analyses, and 2) develop a robust control with better performance than the existing alternatives.

Results and methods: A 4-gene control panel was developed in our lab, and its performance compared to the existing alternatives ACTB and C-LESS-C1. This was done through analyzing the methylation level of four target genes in DNA from 34 colon cancer cell lines. All ddPCRs were performed using the QX200™ PCR platform (BioRad). Details will be presented.

Conclusions: In summary, use of a control is essential for robust and consistent ddPCR based DNA methylation, and can prevent erroneous interpretation of technical artifacts.

Citation Format: Heidi D. Pharo, Kim Andresen, Marine Jeanmougin, Guro E. Lind. Droplet digital PCR for sensitive quantification of DNA methylation in non-invasive material: Development of a robust control [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5387. doi:10.1158/1538-7445.AM2017-5387
Abstract 5389: A personalized medicine approach to identifying dysregulated epigenetic enzyme activity in the development of castrate-resistant prostate cancer
Authors: Lee Jin-Hee , Boersma Melissa , Yang Bing , Damaschke Nathan , Corey Eva , Denu John , Jarrard David F.
Published: July 2017

Purpose: Recent advances in proteomic and chromatin immunoprecipitation tools have been vital in studying cancer epigenetics, but the ability to measure directly the enzyme activities of dysregulated histone-modifiers understudied. We utilize a novel approach to identify altered histone-modifying enzymes in the progression from hormone sensitive (HS) to castrate-resistant prostate cancer (CRPC) progression.

Methods: We developed, validated and utilized a high-throughput peptide microarray assay to identify altered histone lysine (de)acetylation activity in tumor lysates. Functional assays, novel HS and CRPC human tumor arrays and xenografts were utilized to confirm these findings.

Results: This microarray-based activity assay revealed up-regulated histone acetyltransferase (HAT) activity against specific histone H3 sites in a castrate-resistant (CR) PCa cell line compared to its hormone-sensitive (HS) isogenic counterpart. NAD+-dependent deacetylation assays revealed down-regulated Sirtuin activity in validated CR lines. Levels of acetyltransferases GCN5, PCAF, CBP and p300 were unchanged between matched HS and CR cell lines. However, auto-acetylation of p300 at K1499, a modification known to enhance HAT activity and a target of deacetylation by SIRT2, was highly elevated in CR cells, while SIRT2 protein level was reduced in CR cells. Interrogation of HS and matched CR xenograft lines reveals that H3K18 hyperacetylation, increased p300 activity, and decreased SIRT2 expression are associated with progression to CR in 8/12 (66%). Tissue microarray analysis revealed that hyperacetylation of H3K18 is a feature of CRPC. Inhibition of p300 results in lower H3K18ac levels and increased expression of androgen receptor.

Conclusions: This novel microarray approach provides a method to identify commonly dysregulated chromatin enzymes during progression providing a personalized therapeutic strategy to direct available drugs to target enzymes. Reduced SIRT2 expression and increased p300 activity lead to a concerted mechanism of hyperacetylation at specific histone lysine sites (H3K9, H3K14, and H3K18).Support: DOD PCRP (Jarrard)

Citation Format: Jin-Hee Lee, Melissa Boersma, Bing Yang, Nathan Damaschke, Eva Corey, John Denu, David F. Jarrard. A personalized medicine approach to identifying dysregulated epigenetic enzyme activity in the development of castrate-resistant prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5389. doi:10.1158/1538-7445.AM2017-5389
Abstract 5401: Assessment of whole genome amplification for whole exome sequencing in detecting genetic mutation
Authors: Xue Crystal , Gardner Laura , Jiang Guanglong , Shen Fei , Schneider Bryan
Published: July 2017

Whole exome sequencing (WES) has been widely used for studying genetic mutations in DNA coding regions to elucidate cancer etiology and for identifying biomarkers to optimize chemotherapy. However, these studies can be limited when only small amount of DNA is available. Whole genome amplification (WGA) technology can be combined with WES to make these studies technically possible. Here, we evaluated WGA using a phi 29 polymerase prior to library preparation for WES in samples with various DNA concentrations. WES was performed by targeted exon amplification followed by massively parallel sequencing. We compared the base calls of single nucleotide variants for individual same samples with or without WGA prior to library construction to determine the concordance rate of variant calls. We also assessed genetic variant call rate in the same samples with or without WGA. The number of variants obtained ranged from 159,851,999 to 390,784 in the non-amplified samples and from 353,215 to 384,118 in the same amplified samples. The average concordance rate of identical variants in the samples with or without WGA was 96.5%. Our results indicate that WGA in prior to WES can increase detection efficiency of single nucleotide variants in samples with relatively small amount of DNA and/or low DNA concentration.

Citation Format: Crystal Xue, Laura Gardner, Guanglong Jiang, Fei Shen, Bryan Schneider. Assessment of whole genome amplification for whole exome sequencing in detecting genetic mutation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5401. doi:10.1158/1538-7445.AM2017-5401
Abstract 5402: Single cell sequencing of high grade serous ovarian cancer
Authors: Starr Timothy K. , Winterhoff Boris , Maile Makayla , Beckman Kenneth , Daniel Jerry , Geller Melissa , Bazzaro Martina , Klein Molly , Hellweg Raffaele , Abrahante Juan , Mitra Amit K. , Sebe Atilla , Mullany Sally A.
Published: July 2017

We performed RNA sequencing of single cells derived from a high grade serous ovarian cancer (HGSOC) specimen to determine the extent of heterogeneity and to determine if it was feasible to identify cancer stem cells or gene expression signatures of chemo resistance. To perform RNA sequencing we enzymatically digested a fresh specimen from an HGSOC derived from the ovary. Immune cells were depleted by flow cytometry and single cell sequencing was performed using the Fluidigm C1 chip in tandem with Illumina HiSeq 2500 sequencing. Multiple bioinformatics tools were used to identify subgroups and activated pathways. Immunohistochemistry was performed on an adjacent tumor section to analyze markers of epithelium, stroma and stem cells. We found that gene expression patterns in single cells could be used to separate cells into stroma-like and epithelial-like groups. Gene set enrichment analysis identified proliferative genesets (oxidative phosphorylation and MYC targets) associated with the epithelial-like cells while epithelial-to-mesenchymal-transition (EMT) genes associated with the stroma-like cells. Neither group was significantly associated with genesets derived from chemo-resistant cells. Using known marker analysis, we could identify a small percentage of cells that expressed ovarian cancer stem cell markers and we could group cells into functional categories. Using four molecular subtypes established from large-scale bulk sequencing studies we show that single cells from a single patient are heterogeneous and each molecular subtype is represented. In conclusion, we show the feasibility of performing single cell sequencing on an epithelial ovarian cancer and reveal a heterogeneous population of cells. Expanding these findings to a larger cohort of patients could allow for identification of targetable sub-populations of cells that were previously undetectable in studies that use bulk samples to interrogate the transcriptome and genome of ovarian cancer patients.

Citation Format: Timothy K. Starr, Boris Winterhoff, Makayla Maile, Kenneth Beckman, Jerry Daniel, Melissa Geller, Martina Bazzaro, Molly Klein, Raffaele Hellweg, Juan Abrahante, Amit K. Mitra, Atilla Sebe, Sally A. Mullany. Single cell sequencing of high grade serous ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5402. doi:10.1158/1538-7445.AM2017-5402
Abstract 541: A comprehensive analysis delineating the immunotherapeutic terrain of cancer-related clinical trials
Authors: Statz Cara M. , Patterson Sara E. , Yin Taofei , Mockus Susan M.
Published: July 2017

Technological innovations have facilitated a greater understanding of how the tumor microenvironment contributes to cancer, leading to rapid FDA approval of four immunotherapies. To assess how these therapies are being further investigated in combination with other therapies and in tumor types outside of the current FDA approval, we performed a comprehensive analysis of the curated clinical trials in the JAX Clinical Knowledgebase (JAX-CKB). In brief, clinical trials investigating Atezolizumab, Nivolumab, Pembrolizumab, and Ipilimumab, curated from clinicaltrials.gov, were queried in the JAX-CKB and then analyzed for comparison. Further analyses were executed to illustrate possible unmet needs within the field of cancer therapeutics. Of the four immunotherapies, Pembrolizumab was identified with the greatest number of clinical trials overall, with 305 compared to Atezolizumab, 79, Nivolumab, 183, and Ipilimumab, 126. Of these trials the number of trials investigating Pembrolizumab, Atezolizumab, Nivolumab, or Ipilimumab in combination with another therapy was higher than those investigating one of the four immunotherapies as a monotherapy. Phase II trials for both single therapy and combinatorial therapies were greater than both Phase I and Phase III for the same groups, regardless of therapy. On average, 12% ± 3.8% of the combined trials for all four drugs included any advanced solid tumor. Nivolumab combined with Ipilimumab demonstrated the greatest number of trials (61) investigating an immunotherapy in combination with another immunotherapy. Among those, 30 were Phase II trials, 16 were Phase I, and 14 were Phase III. Across five cancer indications (lung, pancreatic, ovarian, prostate, and colon), lung cancer was most commonly indicated in the trials, among all four drugs. Prostate was indicated in the least number of trials, with Ipilimumab ranking the highest (10). The recent success with immunotherapies has garnered significant interest in understanding how these therapies will perform in different tumor types and whether specific combinations will have a greater impact. Interrogation of the clinical trial terrain in the JAX-CKB provides a basis for determining additional investigations that might be warranted.

Citation Format: Cara M. Statz, Sara E. Patterson, Taofei Yin, Susan M. Mockus. A comprehensive analysis delineating the immunotherapeutic terrain of cancer-related clinical trials [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 541. doi:10.1158/1538-7445.AM2017-541
Abstract 5419: The relationship between glutamatergic signaling and altered glutamine metabolism in melanoma
Authors: Shah Raj , Boreland Andrew , Chen Suzie
Published: July 2017

Aberrant glutamatergic signaling has been implicated in many cancer types, and is associated with dysregulated growth leading to cellular transformation and tumorigenesis. Our laboratory has previously illustrated the oncogenic properties of a neuronal receptor, metabotropic glutamate receptor 1 (GRM1) in melanocytes. Glutamate is the natural ligand of GRM1 and the major excitatory neurotransmitter in the central nervous system. Our group has demonstrated that glutamate production/release is upregulated in GRM1 expressing melanoma cells, resulting in constitutive activation of GRM1 and GRM1-associated downstream signaling pathways. We hypothesize that this activation of GRM1 in melanoma cells is associated with higher expression of c-Myc and increased enzymatic activity of glutaminase (GLS) converting glutamine to glutamate. We showed that reducing the extracellular glutamate levels by an inhibitor of glutamate release, Riluzole, led to significantly reduce melanoma cell proliferation in vitro and tumor progression in vivo. We also demonstrated that inclusion of CB-839, a potent, selective, and orally bioavailable GLS inhibitor, resulted in a significant inhibition of GRM1-expressing melanoma cell proliferation compared to GRM1 negative melanoma cell lines. Interestingly, we found that simultaneous targeting of glutamate production (CB-839) and release (Riluzole) in GRM1 expressing melanoma cells led to enhanced suppression of cell proliferation in vitro. Additionally, we found that wild type BRAF melanoma cells are more sensitive to CB-839 than mutated BRAF cells while responses to Riluzole were independent of BRAF genotypes. Furthermore, our data indicate that CB-839 acts by reducing the function, not the expression, of GLS. Currently, we are investigating the relationship between GRM1, c-Myc and GLS by genetic and pharmacological approaches, and if this cascade of events is linked with altered glutamine metabolisms described for many human cancers including melanoma.

Citation Format: Raj Shah, Andrew Boreland, Suzie Chen. The relationship between glutamatergic signaling and altered glutamine metabolism in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5419. doi:10.1158/1538-7445.AM2017-5419
Abstract 5420: PGAM1-REDD1 pathway promotes paclitaxel resistance through enhancing aerobic glycolysis in ovarian cancer cells
Authors: Feng Yan , Lv Weiguo , Xie Xing , Yu Yan
Published: July 2017

Aerobic glycolysis is a dominant pathway for energy production in cancer cells, but the association of this phenomenon with chemoresistance remains still uncertain. Here, we found that the expression of phosphoglycerate mutase 1 (PGAM1), a key catalytic enzyme in aerobic glycolysis, was significantly higher in paclitaxel-resistant ovarian cancer cell line SKOV3. Up-regulated PGAM1 expression by gene transfection and down-regulated PGAM1 expression by siRNA knockdown altered the process of aerobic glycolysis, paclitaxel sensitivity and DNA damage response 1 (REDD1) expression in ovarian cancer cells (SKOV3 and A2780). Further, the change of the concentration of pyruvic acid or lactic acid in culture medium and REDD1 expression by gene transfection or siRNA knockdown also altered the sensitivity to paclitaxel. REDD1 knockdown of by siRNA reversed partially paclitaxel resistance induced by PGAM1 over-expression in cancer cells, vice versa. Structural interaction between PGAM1 and REDD1 was predicted by molecular docking. Our results suggested that PGAM1-REDD1 pathway promotes paclitaxel resistance through enhancing aerobic glycolysis in ovarian cancer cells.

Citation Format: Yan Feng, Weiguo Lv, Xing Xie, Yan Yu. PGAM1-REDD1 pathway promotes paclitaxel resistance through enhancing aerobic glycolysis in ovarian cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5420. doi:10.1158/1538-7445.AM2017-5420