bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 2986: Meta-analysis of whole exome sequencing data reveals the mutational spectrum of testicular germ cell tumors
Authors: Litchfield Kevin , Houlston Richard S. , Huddart Robert , Summersgill Brenda , Shipley Janet , Turnbull Clare
Published: August 2015

Testicular germ cell tumors (TGCTs) are the most common cancer in young men. Although cure rates for TGCT are generally high, due to the sensitivity of malignant testicular germ cells to platinum-based chemotherapies, this comes at the cost of an increased risk of metabolic syndrome, infertility and secondary cancer. Furthermore, there are limited options for patients who are platinum resistant, a group for whom the long-term survival rate is less than 15%. Improved targeted therapies for TGCT are predicated on a greater understanding of mutational profile of these tumours, including identification of driver mutations and pathways disrupted. We recently published the first large-scale analysis of whole exome sequencing of TGCTs (comprising 42 tumors), which revealed a uniformly low mutation rate; some ∼ten times lower than average rates observed in other adult solid tumors, consistent with the embryological origin of TGCT. Aside from mutations in the previously known driver gene KIT, no additional novel genes with mutation frequency greater than 15% were associated with tumorigenesis. In parallel The Cancer Genome Atlas consortium is sequencing an additional series of TGCTs.

Given the apparent absence of high frequency driver genes aside from KIT, several lower frequency oncogenic pathways may exist, requiring identification through larger sample size studies and/or pooled analysis. Here we present meta-analysis across multiple datasets with variant calling re-processed through a consistent bioinformatic pipeline. We systemically searched for novel recurrent mutational events at three levels:- (i) driver genes (combined sample size was empowered to detect recurrent gene mutations of frequency >5% with 89% power), ii) pathway based associations and iii) focal copy number alterations. In addition we assessed for clinicopathological-molecular associations, with particular focus on mutational events distinguishing treatment responsive and resistant tumors. Comprehensive cataloguing of the mutational spectra, through analyses of our data in combination with other initiatives such as TCGA, is leading to increasing insights into this biology of this cancer.

Citation Format: Kevin Litchfield, Richard S. Houlston, Robert Huddart, Brenda Summersgill, Janet Shipley, Clare Turnbull. Meta-analysis of whole exome sequencing data reveals the mutational spectrum of testicular germ cell tumors. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2986. doi:10.1158/1538-7445.AM2015-2986
Abstract 2987: Next generation sequencing analysis of genetically engineered mouse models of human cancers
Authors: Chung Wei-Jen , Long Jason , Cheng Jason , Tran Chris , Dey Anwesha , Daemen Anneleen , Junttila Melissa
Published: August 2015

Genetically engineered mouse models (GEMM) of cancer are invaluable tools for oncology research. They recapitulate critical aspects of tumor etiology and allow for unique interrogation of the tumor stroma interactions during tumorigenesis. In addition, these models are poised for assessing therapeutic drug efficacy and resistance mechanisms. Although these models are widely used, the mutational landscape and transcriptional profile of GEMMs have yet to be thoroughly characterized.

In this study we sought to investigate the precise mutational landscape of four well-validated GEMMs representing three types of cancers, non-small cell lung cancer (NSCLC), pancreatic ductal adenocarcinoma (PDAC) and melanoma. Using next generation sequencing techniques, we profiled one adenovirally-induced Kras mutant NSCLC model (KrasLSL.G12D; p53frt/frt), two Kras mutant models of spontaneous PDAC (KrasLSL.G12D; p16/p19fl/fl; Pdx1.Cre and KrasLSL.G12D; p16/p19fl/wt; p53LSL.R270H/wt; Pdx1.Cre) and one Braf mutant melanoma model (BrafV600E/wt; PTENfl/fl; TyrCreER). All four GEMMs incorporate mutations that are well documented to occur in the human patient population. Additionally these models have been documented to approximate histological and phenotypic aspects of their human correlate.

We carried out whole exome sequencing for each model (NSCLC n = 120; PDAC n = 73 and n = 40, respectively; melanoma n = 17) to comprehensively characterize the baseline genomes. We also looked for additional recurrent driver mutations beyond those engineered within each model. Interestingly, both intra and intertumoral heterogeneity was evident. Furthermore, we performed RNA sequencing and identified significantly affected genes and gene sets. Finally, we compared our GEMMs to human tumors from TCGA (LUAD, LUSC, PAAD and SKCM) at the genomic and transcriptomic level. Taken together, the results we will present provide a clearer understanding of the genomic and transcriptional landscape of these GEMMs and their relationship with human patients. More importantly, these analyses will allow us to better interpret results from previous and future experiments using these GEMMs of cancer.

Citation Format: Wei-Jen Chung, Jason Long, Jason Cheng, Chris Tran, Anwesha Dey, Anneleen Daemen, Melissa Junttila. Next generation sequencing analysis of genetically engineered mouse models of human cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2987. doi:10.1158/1538-7445.AM2015-2987
Abstract 2988: International cancer genome consortium (ICGC)
Authors: Jennings Jennifer L. , Hudson Thomas J.
Published: August 2015

The International Cancer Genome Consortium (ICGC) was established to bring together researchers from around the globe to comprehensively analyze the genomic, transcriptomic, and epigenomic changes in 50 different tumour types or subtypes that are of clinical and societal importance across the globe (International network of cancer genome projects. Nature 464, 993-998 (15 April 2010)).

As of December 2014, the ICGC has received commitments from researchers and funding organizations in Asia, Australia, Europe, North America and South America for 77 project teams in 17 jurisdictions to study more than 25,000 tumour genomes.

More than 27,000 tumours that meet ICGC requirements for consent have been obtained and research pathologists have approved over 27,000 samples as meeting ICGC standards.

Processed data is available via the Data Coordination Centre (http://dcc.icgc.org) based at the Ontario Institute for Cancer Research and is updated quarterly. The data release (Version 17) in October 2014 included datasets from 42 ICGC members. The ICGC Data Portal has been improved to provide enhanced support to the research community browsing this vast dataset.

The ICGC and The Cancer Genome Atlas (TCGA) PanCancer Analysis of Whole Genomes (PCAWG) project is committing more than 2,500 tumour-normal whole genome pairs from 23 different tumour types to uniform alignment and variant calling. More than 600 researchers will contribute to the interpretation of the data and writing of papers that will focus on specific analyses covering: Identification of driver mutations outside coding regions, including those that affect cis-regulatory regions and non-coding RNAs; and identification of gene regulatory pathways altered by non-coding mutations. Because the projected size of the pan-cancer dataset for 5,000 WGS is very large, PCAWG is using a distributed compute cloud environment (at different computing centres in the USA, Europe and Asia) that meet technical requirements of the project and the bioethical framework of ICGC and its member projects. More information can be found on www.icgc.org.

Citation Format: Jennifer L. Jennings, Thomas J. Hudson. International cancer genome consortium (ICGC). [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2988. doi:10.1158/1538-7445.AM2015-2988
Abstract 2992: The landscape of long non-coding RNAs in cancer
Authors: Niknafs Yashar S. , Iyer Matthew K. , Chinnaiyan Arul M.
Published: August 2015

Long non-coding RNAs (lncRNAs) are emerging as important players in cancer. The emergence of high-throughput RNA sequencing (RNA-Seq) technology provides a revolutionary means for systematic discovery of transcribed elements. However, efforts to catalog lncRNAs have thus far failed to appreciate the transcriptional robustness and complexity in the human genome because they were confined to select cell lines or small numbers of mostly normal tissue.

In order to delineate genome-wide lncRNA expression, we curated 7,256 RNA-Seq libraries comprising over 43 terabases of sequence from 25 independent studies including tumors, normal tissues, and cell lines. The data were processed using ab initio transcriptome meta-assembly methodologies to develop a consensus of 91,013 expressed genes. Over 68% (58,648) of genes were classified as lncRNAs, of which 79% (48,952) were previously unannotated. About 1% (597) of the lncRNAs harbor ultraconserved elements and 7% (3,900) overlap disease-associated single nucleotide polymorphisms (SNPs). To prioritize lineage-specific, disease-associated lncRNA expression, we developed a non-parametric method for differential expression testing called Sample Set Enrichment Analysis (SSEA), and nominated 7,942 lineage- or cancer-associated lncRNA genes. In order to interrogate potential functionality for these lncRNAs, gene set enrichment testing was performed on genes correlated with each lncRNA.

By uncovering this expansive landscape of cancer-associated lncRNAs, we provide the scientific community a powerful starting point to begin investigating their biological relevance. We anticipate that the lncRNAs identified by this study, as well as the computational tools developed herein, will provide a foundation for lncRNA genomics, biomarker development, and the delineation of cancer disease mechanisms.

Citation Format: Yashar S. Niknafs, Matthew K. Iyer, Arul M. Chinnaiyan. The landscape of long non-coding RNAs in cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2992. doi:10.1158/1538-7445.AM2015-2992
Abstract 2994: Clinical applications of comprehensive genome analysis of papillary renal cell carcinoma, type II
Authors: Kim Ji-Yeon , Lee Se-Hoon , Cha Soojin , Moon Kyung Chul , Kim Jong-Il , Heo Dae Seog
Published: August 2015

Purpose

Papillary renal cell carcinoma (PRCC) is the most common type of non-clear cell renal cell carcinoma (NCCRCC). Of PRCC, PRCC,type2(PRCC2) is known to have highly aggressiveness and relative drug resistance. In contrast of clear cell renal cell carcinoma (CCRCC), we poorly understand genetic alterations to drive PRCC2. Moreover, though many tyrosine kinase inhibitors (TKIs) are now available to treat PRCC, the predictive marker of TKIs is not in existence. Therefore, we aimed to analyze genetic alteration of PRCC2.

Patients and Methods

We performed whole-exome sequencing (WES) of DNA from 9 PRCC2 samples and matched normal tissue. In addition, whole-transcriptome sequencing (WTS) was conducted for 7 of these samples. We analyzed somatic mutations, copy number alterations, germ line mutations using WES data, pathway alterations and gene fusions through RNA expression data. We investigated the relationship between clinical prognosis and genetic alterations.

Results

Of nine samples, average non-silent mutation rate is 1.13 mutations per Mb (range, 0.49 to 2.14 mutations per Mbs). Forty-seven somatic mutations including SRRD(four), PKD1L2(three) and MX1(three) are discovered in more than 2 specimens. The genes associated to histone modification and ubiqutination are frequently altered. We found FH somatic mutation, a potent target of VEGFR TKI, in young female PRCC2 patient. Her tumor is extremely shrunk after 6cycles treatment and after that she received pazopanib for 52weeks maintaining effective response.

Conclusion

We suggest the possibility of translational research from sequencing data to real medical treatment of PRCC2. This pivotal study demonstrates that novel somatic FH mutation is the therapeutic target of VEGFR TKI. Therefore, further large scaled comprehensive genetic analysis of PRCC2 is required to propose personalized medicine for conquering papillary renal cell carcinoma, type 2.

Note: This abstract was not presented at the meeting.

Citation Format: Ji-Yeon Kim, Se-Hoon Lee, Soojin Cha, Kyung Chul Moon, Jong-Il Kim, Dae Seog Heo. Clinical applications of comprehensive genome analysis of papillary renal cell carcinoma, type II. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2994. doi:10.1158/1538-7445.AM2015-2994
Abstract 2999: DEC1 and DEC2 crosstalk between circadian rhythm and tumor progression
Authors: Sato Fuyuki , Muragaki Yasuteru
Published: August 2015

Circadian rhythms are tightly regulated by clock genes. It has been reported that clock genes plays important roles in biological function of normal cells. However, it is not well understood the function of clock genes in tumor cells. We showed that basic helix-loop-helix (bHLH) transcription factors, differentiated embryonic chondrocyte gene 1 (DEC1/BHLHE40/Sharp2/Stra13) and DEC2 (BHLHE41/Sharp1), are clock genes and play important roles in circadian rhythm, cell proliferation, apoptosis, hypoxia response, various stresses and epithelial-to-mesenchymal transition (EMT) in tumor cells. Various stresses, such as exposure to transforming growth factor-beta (TGF-β), hypoxia, cytokines, serum-free, and anti-tumor drugs affect DEC expression. The increased or decreased DEC expression by such stresses is involved in tumor progression. The evidence that DEC2 has circadian expression in implanted mouse sarcoma cells and negatively regulates VEGF expression under hypoxia. DEC1 has pro-apoptotic effects in human breast cancer cells, whereas DEC2 has anti-apoptotic effects. DEC1 induced EMT by TGF-β in human pancreatic cancer cells, whereas DEC2 suppressed EMT. Immunohistochemical analysis showed that DEC1 is highly expressed in tumor cells and the expression is involved in malignancy. On the other hand, DEC2 is little expressed in tumor cells compared with adjacent non-tumor cells. These findings suggest that DEC1 and DEC2 have different roles in tumor progression under circadian rhythm. DEC2 regulates the circadian rhythm and VEGF expression by negative feed-back system, and DEC1 and DEC2 regulates EMT by positive and negative feed-back system in tumor cells. So, DEC acts as “accelerator” and “brake” in tumor progression. In this study, we show current progress of the DEC genes in tumor progression.

Citation Format: Fuyuki Sato, Yasuteru Muragaki. DEC1 and DEC2 crosstalk between circadian rhythm and tumor progression. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2999. doi:10.1158/1538-7445.AM2015-2999
Abstract 300: increasing radiation dose enhances immunotherapy efficacy and together prolongs tumor dormancy in a subgroup of mice treated for advanced intracerebral melanoma
Authors: Smilowitz Henry M. , Micca Peggy , Sasso Daniel , Wu Qian , Dyment Nathanial , Kuo Lynn
Published: August 2015

Previously we developed a clinically relevant therapy model for advanced intracerebral B16 melanomas in syngeneic mice combining radiation and immunotherapies. Here seven days after B16F10Luc2 melanoma cells were implanted intracerebrally (D7), syngeneic mice with bioluminescent tumors that had formed (1E105 to 7E106 photons/minute (>1E106, large (L); <1E106, small (S)) were segregated into L/S-balanced subgroups. Then, mice received either radiation therapy alone (RT) or radiation therapy plus immunotherapy (RT plus IT). Radiation dose was varied (15, 18.75 or 22.5 Gray (Gy), given on D8), while immunotherapy was provided similarly to all mice. The data support the hypothesis that increasing radiation dose increases the efficacy of immunotherapy in a subgroup of mice. The tumors that were greatly delayed in beginning their progressive growth were bioluminescent in vivo–some for many months, indicating prolonged tumor “dormancy,” in some cases presaging long-term cures. Mice bearing such tumors far more likely had received radiation plus immunotherapy than radiation alone. Radiotherapy is a very important adjunct to immunotherapy; the greater the tumor debulking by radiation therapy, the greater should be the benefit to tumor immunotherapy.

Citation Format: Henry M. Smilowitz, Peggy Micca, Daniel Sasso, Qian Wu, Nathanial Dyment, Lynn Kuo. increasing radiation dose enhances immunotherapy efficacy and together prolongs tumor dormancy in a subgroup of mice treated for advanced intracerebral melanoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 300. doi:10.1158/1538-7445.AM2015-300
Abstract 3004: Contextual RNAi screen identifies ACLY and ACC1 as mediators of hypoxia-induced apoptosis through metabolic and transcriptional mechanisms
Authors: Keenan Melissa M. , Liu Beiyu , Tang Xiaohu , Wu Jianli , Cyr Derek , Stevens Robert D. , Ilkayeva Olga , Lucas Joseph , Muoio Deborah M. , Kim So Young , Chi Jen-Tsan
Published: August 2015

To become established as a solid tumor, cancer cells must adapt to and survive under various tumor microenvironment (TME) stresses, such as hypoxia or lactic acidosis. While many stress-signaling mechanisms have been well-studied, much remains unknown about how tumor cells survive these stresses during tumor progression. To identify genes that modulate cellular survival under stresses, we performed genome-wide shRNA screens under hypoxia or lactic acidosis. We discovered that the genetic depletion of acetyl-CoA carboxylase (ACACA or ACC1) or ATP citrate lyase (ACLY) protected cancer cells from hypoxia-induced apoptosis through both metabolic and transcriptional mechanisms. First, while depleting α-ketoglutarate in control cells, hypoxia unexpectedly increased levels of α-ketoglutarate in the cells depleted of ACC1 or ACLY. Supplementation with α-ketoglutarate rescued the hypoxia-induced apoptosis in a mitochondria-dependent manner. Second, loss of ACLY or ACC1 reduced protein levels and activity of the oncogenic transcription factor ETV4. Silencing of ETV4 protected cells from hypoxia-induced apoptosis and triggered remarkably similar global transcriptional responses as with silenced ACLY or ACC1. Importantly, within tumor expression datasets, ETV4 transcriptional activity was highly correlated with ACLY or ACC1 gene expression signatures. Therefore, ETV4 acted as a key regulator of the transcriptional output of lipogenic activity via ACLY or ACC1. These results reveal a novel interconnectedness between cellular metabolic and transcriptional responses for life or death decisions under stress. Since lipogenic inhibitors are under investigation as cancer therapeutics, our findings suggest that the use of these inhibitors will need to be carefully considered with respect to tumor hypoxia, progression and dormancy. More broadly, our screen provides a framework for studying additional tumor cell stress-adaption mechanisms in the future.

Citation Format: Melissa M. Keenan, Beiyu Liu, Xiaohu Tang, Jianli Wu, Derek Cyr, Robert D. Stevens, Olga Ilkayeva, Joseph Lucas, Deborah M. Muoio, So Young Kim, Jen-Tsan Chi. Contextual RNAi screen identifies ACLY and ACC1 as mediators of hypoxia-induced apoptosis through metabolic and transcriptional mechanisms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3004. doi:10.1158/1538-7445.AM2015-3004
Abstract 3005: Investigating the impact of hypoxia-induced changes in splicing on tumor microenvironment
Authors: Brady Lauren , Popov Vladimir , Ivan Mircea , Radovich MIlan , Koumenis Constantinos
Published: August 2015

Hypoxia is a cellular stress that influences both normal and tumor development. For many types of cancer, including head and neck carcinomas, tumor hypoxia shows a strong positive correlation with poor patient outcome. In order to gain a deeper understanding of the biology of the tumor microenvironment, we are investigating how transcription is differentially regulated in hypoxic compared to normoxic regions of tumors. Previously, we reported that the expression of over 500 genes differs between hypoxic and normoxic tumors. However, there has not yet been a global sequencing analysis of tumors to characterize hypoxia-induced differences in transcriptional regulation at base-pair resolution. To achieve this, we developed a method to isolate RNA from hypoxic regions of tumors in vivo and used this method to carry out deep RNA-sequencing of normoxic and hypoxic regions of human head and neck tumors grown in mice. Corresponding in vitro samples from cells cultured in 0.5% oxygen conditions (in which the only stress was hypoxia) were also sequenced. We used these data to examine which genes showed changes in isoform-level expression and to define hypoxia-mediated changes in splicing. The data revealed more than 1,000 hypoxia-responsive mRNA isoforms. Using a combined informatics approach to analyze both differential expression of alternatively spliced exons and entire isoforms, we observed that many hypoxia-induced isoforms showed changes in alternate last exons, retained introns and 3′ untranslated regions compared to the isoforms expressed in normoxic cells. Moreover, we observed global repression of genes involved in RNA processing and splicing, which may represent one mechanism to shift patterns in splicing and lead to expression of stress-induced isoforms. Differentially expressed isoforms include genes that play critical roles in fundamental processes involved in hypoxic adaptation, such as the translation, glycolysis and cell cycle. Therefore, we propose that alternative splicing plays a role in mediating tumor growth and survival in hypoxic conditions by regulating stress-responsive pathways.

Citation Format: Lauren Brady, Vladimir Popov, Mircea Ivan, MIlan Radovich, Constantinos Koumenis. Investigating the impact of hypoxia-induced changes in splicing on tumor microenvironment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3005. doi:10.1158/1538-7445.AM2015-3005
Abstract 3007: Intracellular detection of hypoxia in live cells
Authors: Mandavilli Bhaskar S. , O'Grady Michael
Published: August 2015

Hypoxia is an important phenomenon in many physiological processes and involved in many human diseases including cancer. Inflammation can lead to significant hypoxia in tissues. The study of hypoxia has been complicated with the lack of proper instrumentation to induce hypoxia in cells and image cells under hypoxic conditions. Here, we describe a live cell-based method to conveniently measure hypoxia using a new Image-iT® Hypoxia Probe and a specialized microscope incubator which can control oxygen concentrations down to 1%, The Image-iT® Hypoxia Probe is an oxygen sensing fluorescent probe, is quenched with increasing oxygen concentrations, and has excitation and emission peaks of 483 and 616 nm respectively. The probe is sensitive to varying concentrations of oxygen and can detect as low as 1% O2 concentrations in cells. Imaging of cells with the Image-iT® Hypoxia Probe in the incubator prevents re-oxygenation of cells and gives more precise measurement of hypoxia in cells, allowing for reversible and dynamic measurements of hypoxia in cells. Using this system, we measured hypoxia in several cell lines including A549, HeLa and U-2 OS. The Image-iT® Hypoxia Probe is also very good at detecting hypoxia in 3D tumor spheroids generated using different methods. The new hypoxia probe gives good signal to noise with more than 3-fold changes at 5% O2 levels with robust statistics. The Image-iT® Hypoxia Probe provides a good system for precise, robust and reproducible measurements of hypoxia in cells.

Citation Format: Bhaskar S. Mandavilli, Michael O'Grady. Intracellular detection of hypoxia in live cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3007. doi:10.1158/1538-7445.AM2015-3007
Abstract 301: RealTVac, a novel strategy to treat advanced, late-stage tumors with real-time tumor vaccination
Authors: Jachimczak Piotr , Mitsch Andreas , Aigner Achim
Published: August 2015

Novel anti-cancer immune therapeutic strategies, like various new antibody formats and/or tumor vaccines, show promising results in patients. However, due to the “immune-escape phenomenon” driven by tumor-secreted Transforming Growth Factor-beta (TGF-beta), the host immune system of cancer bearing patients frequently fails to control tumor re-growth.

The RealTVac® approach aims at avoiding this “immune-escape phenomenon” by intratumoral inhibition of active TGF-beta isotypes while simultanously and synergistically inducing an efficient immune response by a highly potent combination of immune stimulating factors.

The synergistic effects of the proposed combination of a TGF-beta inhibitor with immunostimulating cytokines upon human immune cell activation are demonstrated in in-vitro experiments. Both, immune cell proliferation and tumor cell cytotoxicity were significantly enhanced. Initial experiments in a syngenic B16 melanoma xenograft model in immunocompetent mice indicate that the RealTVac® therapy of established subcutaneous B16 tumor xenografts reduced local tumor growth compared to untreated controls.

To summarize, the goal of the local application of RealTVac® is to allow the host immune system to scan in real-time all Tumor Associated Antigens (TAAs) being currently expressed by those tumor cells that are exposed to sufficient TGF-beta inhibition. The repetitive administration of the RealTVac® approach is furthermore expected to provide a continuous update of the immune response, reflecting the changing pattern of TAAs related to the dynamic intratumoral heterogeneity in malignant tumors.

Citation Format: Piotr Jachimczak, Andreas Mitsch, Achim Aigner. RealTVac, a novel strategy to treat advanced, late-stage tumors with real-time tumor vaccination. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 301. doi:10.1158/1538-7445.AM2015-301
Abstract 3011: Two-sided story of DNA repair proteins MRE11 and RAD51 in breast cancer
Authors: Yuan Shyng-Shiou F. , Hung Amos , Hou Ming-Feng
Published: August 2015

Background

DNA repair proteins are required for the maintenance of genome integrity in normal cells. However previous studies also showed that altered expression of MRE11 and RAD51, proteins crucial for DNA double-strand break repair, DNA replication, and telomere maintenance, are associated with cancer outcomes. In this study, we investigated the role of MRE11 and RAD51 in breast cancer in both clinical and in vitro settings.

Methods

The expression of MRE11 and RAD51 in breast tumor tissues were determined by immunohistochemistry, and associations with clinicopathological characteristics and overall survival were assessed using Cox proportional hazards regression models and Kaplan-Meier survival curves. The effect of altered expression of MRE11 and RAD51 on cell proliferation, invasion, and treatment resistance was assessed in vitro using breast cancer cell lines, with the underlying mechanisms explored. The effect of MRE11 and RAD51 overexpression on tumor growth was assessed in an orthotopic xenograft model. All statistical tests were two-sided.

Results

High MRE11 expression was statistically significantly associated with malignant cancer behaviors including increased tumor size and lymph node metastasis as well as resistance to radiotherapy and chemotherapy. MRE11 overexpression in breast cancer cell lines promoted cell proliferation through STAT3, cell cycle entry, invasion and migration, and radioresistance via enhanced DNA repair activity, and also inhibited apoptosis; knockdown of MRE11 had the opposite effect. In xenograft tumor-bearing mice, tumor growth was increased in MRE11-overexpressing group.

RAD51 was expressed both in the cytoplasm and nucleus of all the breast cancer and non-cancerous cell lines. Expression levels of cytoplasmic RAD51 were positively correlated with cancer stage, tumor size, lymph node metastasis in breast cancer patients. High expression of cytoplasmic RAD51 predicted a poor patient survival as well as resistance to radiotherapy and chemotherapy.

Conclusion

High MRE11 and RAD51 expression was associated with a more malignant behavior in breast cancer. MRE11 and RAD51 may serve as potential targets for therapeutic intervention against breast cancer.

Note: This abstract was not presented at the meeting.

Citation Format: Shyng-Shiou F. Yuan, Amos Hung, Ming-Feng Hou. Two-sided story of DNA repair proteins MRE11 and RAD51 in breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3011. doi:10.1158/1538-7445.AM2015-3011
Abstract 302: High throughput drug screening with engineered tumor spheroids
Authors: Lemmo Stephanie , Aefi Ehsan , Luker Gary , Tavana Hossein
Published: August 2015

Physiologically relevant, in vitro cell-based models are important tools to identify effective anticancer drugs. Three-dimensional (3D) clusters of cancer cells, called tumor spheroids, mimic key characteristics of avascular tumors such as diffusion limitations and hypoxia, and hence serve as a relevant model for compound screening. Nevertheless, current spheroid formation techniques face difficulties to generate large number of uniform sized spheroids, conveniently maintain them in culture and biochemically analyze them using commercially available tools. To address this need, we developed a robotic, high throughput technology for three-dimensional culture of cancer cells that is simple to implement, produces consistent size spheroids in standard microwell plates, and allows convenient robotic media exchange and drug addition, and analysis of cellular responses using microplate readers. The technology is based on the use of an aqueous two-phase system. Cancer cells confined in a drop of the denser aqueous phase are robotically dispensed into a microwell containing the immersion aqueous phase. Cells remain within the drop at the bottom of the microwell and form a viable spheroid.

We demonstrate the robust performance of this technology by evaluating the effect of two clinically used drugs against biologically aggressive skin cancer and triple negative breast cancer (TNBC) cells. Skin cancer spheroids treated with paclitaxel and cisplatin show dose-dependent decrease in cell viability, but with a greater resistance compared to the monolayer culture (2D) of cells (paclitaxel: LD50,3D = 178.5 nM, LD50,2D = 22.1 nM and cisplatin: LD50,3D = 131.7 μM and LD50,2D = 29.2 μM. TNBC cell monolayer and spheroids treated with cisplatin displayed a similar LD50 value of ∼13.1 μM. However, unlike the monolayer culture of TNBC cells that showed a dose-response to paclitaxel with LD50,2D = 8.0 nM, TNBC spheroids showed resistance to this drug, even at micromolar-scale concentrations, and maintained a high viability.

In conclusion, our tumor spheroid technology enables quick and efficient generation of relevant tumor models in standard microplates to conveniently treat with chemical compounds and analyze. This technology allows investigating the efficacy of drug compounds against cancer cells and reveals cellular responses not captured with widely-used monolayer cultures. The ease of conducting and analyzing high throughput experiments will substantially reduce labor and cost, and expedite discovery of effective anti-cancer compounds with a physiologic tumor model.

Citation Format: Stephanie Lemmo, Ehsan Aefi, Gary Luker, Hossein Tavana. High throughput drug screening with engineered tumor spheroids. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 302. doi:10.1158/1538-7445.AM2015-302
Abstract 3020: Homology-directed repair in mouse mammary tissue
Authors: Kass Elizabeth , Moynahan Mary Ellen , Jasin Maria
Published: August 2015

Numerous factors are linked to breast cancer risk including reproductive history, early exposure to radiation, age and genetic background. Defects in homology-directed repair (HDR)_considered the most accurate of the three major pathways for repairing double-strand breaks (DSBs) in DNA in mammalian cells_can lead to genomic instability and are associated with tumor predisposition. Mutations in multiple genes involved in HDR are linked to breast cancer susceptibility including BRCA1 and BRCA2. The mammary gland is highly influenced by its hormonal environment and undergoes significant changes during pregnancy, lactation and involution, but many questions remain about how the DNA damage response in mammary tissue, and specifically DNA DSB repair, is impacted by these developmental changes.

A widely used reporter for studying DSB repair by HDR is DR-GFP in which direct repeats of two defective GFP genes are induced to recombine by I-SceI endonuclease cleavage of one of the repeats, resulting in a functional GFP gene. We have recently generated DR-GFP containing mice that express I-SceI under the control of a tetracycline-inducible promoter. Induction of I-SceI by doxycycline administration ex vivo results in substantially increased HDR in primary cultures of mammary epithelial cells compared to transient transfection experiments.

To gain insight into the effects of breast cancer predisposing mutations on HDR in mammary epithelial cells, the I-SceI/DR-GFP mice were crossed to mice carrying hypomorphic mutations in Brca1 (Brca1tr/tr) or Brca2 (Brca2ex27/ex27). An approximately 3-fold reduction in HDR was observed in primary mammary epithelial cells from virgin Brca1tr/tr I-SceI DR-GFP mice compared to littermate controls, while an 8-fold reduction was observed in primary mammary epithelial cells from virgin Brca2ex27/ex27 mice. Primary ear fibroblasts from these animals showed similarly reduced HDR levels.

In order to understand how DSB repair processes in the mammary gland are affected by developmental changes we are assessing HDR at different stages of mammary gland development in vivo. We observe a significant increase in HDR relative to overall repair in mammary tissue from mice at mid-pregnancy compared to virgin littermates. We are now attempting to determine if there is a relationship between repair proficiency, lineage specificity of mammary epithelial cells and fate after involution in mammary tissue from normal and BRCA mutant mice.

Citation Format: Elizabeth Kass, Mary Ellen Moynahan, Maria Jasin. Homology-directed repair in mouse mammary tissue. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3020. doi:10.1158/1538-7445.AM2015-3020
Abstract 3021: FoxP1 increases nucleotide excision repair capacity through activating xeroderma pigmentosum group C in human breast cancer
Authors: Kim Haesung , Kim Lee-Su , Lee Jae-yong , Kim Jeong-Hyeon , Lee Jeong-Min , Kim Chea-Ha
Published: August 2015

Breast cancer is the most common cancer in the women. Recent study revealed that nucleotide excision repair (NER) deficiency might play an important role in the cause of sporadic breast cancer, showing significant reduction of five NER gene products including xeroderma pigmentosum group C (XPC) in human breast cancer tissue. Thus, XPC inactivation might be related to occurrence and progression of breast cancer.

FoxP1 is a member of the forkhead O family of transcription factors which have a broad range of functions. FoxP1 is considered as a putative tumor suppressor in breast cancer because of its ability to cause apoptosis. The relationship between FoxP1 and XPC has been yet to be known in breast cancer.

In our data, Western blot analysis of 8 human primary tumors showed that the high expression of FoxP1 was correlated with the high level of XPC. In three established mammary epithelial cell lines (MCF-7, MDA-MB-231, 4T1), FoxP1 expression in MCF-7 was higher than in MDA-MB-231 and 4T1. Similar to the result of primary tumors, the expression of XPC was also high in MCF-7 and low in MDA-MB-231 and 4T1. Next, we found that the level of XPC decreased with the knockdown of FoxP1 compared to control in MCF-7. On the other hand, the ectopic expression of FoxP1 in MDA-MB-231 and 4T1 elevated XPC expression for protein level. In real time RT-PCR, the level of XPC mRNA was the same as in Western blot analysis. We tested whether NER activity is dependent on FoxP1. In vitro NER activity increased by overexpression of FoxP1 in MB-231. Sh-FoxP1 transfected MCF-7 cell shows reduced in vitro NER activity. In addition, immunostaining assay showed that XPC expression was much accumulated in the nucleus of MDA-MB-231 with overexpression of FoxP1 than in control.Taken together, these data suggested FoxP1 could up-regulate NER activity by activating XPC in breast cancer. However, the way how FoxP1 can regulate XPC remained to be cleared. To clarify the mechanism, we are performing biological interaction between FoxP1 and XPC in both breast cancer cell lines and human primary breast tumors.

Citation Format: Haesung Kim, Lee-Su Kim, Jae-yong Lee, Jeong-Hyeon Kim, Jeong-Min Lee, Chea-Ha Kim. FoxP1 increases nucleotide excision repair capacity through activating xeroderma pigmentosum group C in human breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3021. doi:10.1158/1538-7445.AM2015-3021
Abstract 3023: Chromatin accessibility underlies the tumor suppressor role of BAF (mSWI/SNF) complexes in many human cancers
Authors: Hodges Courtney , Hargreaves Diana C. , Miller Erik L. , Crabtree Gerald R.
Published: August 2015

Recent exome sequencing studies of human cancer indicate that ∼20% of malignancies have mutations in the subunits of the BAF (mSWI/SNF) chromatin remodeling complex, implicating the complex as a major tumor suppressor. However, the mechanisms of tumor suppression remain uncertain. To understand how defective BAF subunits contribute to cancer, we analyzed the genomic states of 5,659 patient samples derived from 22 tumor types, and compared samples containing genetic defects of the BAF complex against samples containing no such defects. We show that in the majority of cancer types, tumors with BAF subunit defects have significantly higher mutation counts than cancers of the same tissue lacking BAF defects. By reconstructing the mutational processes active in each patient sample, we find that excess mutations in BAF-deficient cancers represent the signatures of defective mismatch repair, cytidine deamination, UV-damage response, DNA end joining, Pol epsilon function, and other enzymatic processes acting on DNA. In four cancer types where ChIP-seq tracks were available for the healthy cell type of origin, we tracked the locations of over 280,000 mutations, and find that excess mutations in BAF-deficient tumors are biased towards regions of elevated H3K36me3 and low H3K27me3 in their corresponding non-cancerous tissues. We hypothesized that these mutations resulted from inaccessibility for repair and maintenance factors when a BAF subunit is defective. We tested this hypothesis using topoisomerase IIa binding as model for processes that require enzymatic access to DNA, and found that rapid conditional deletion of the ATPase Brg led to an increase in genome-wide deposition of H3K27me3 at Brg-dependent topoisomerase IIa sites from an initial low state. Our data indicate that the tumor-suppressive functions of BAF complexes arise largely from the requirement for BAF complexes to maintain genomic stability by providing access to DNA for repair and maintenance mechanisms.

Note: This abstract was not presented at the meeting.

Citation Format: Courtney Hodges, Diana C. Hargreaves, Erik L. Miller, Gerald R. Crabtree. Chromatin accessibility underlies the tumor suppressor role of BAF (mSWI/SNF) complexes in many human cancers. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3023. doi:10.1158/1538-7445.AM2015-3023
Abstract 3026: Gastric cancer associated variant of DNA polymerase Beta (Leu22Pro) induce genomic instability and cellular transformation
Authors: Kidane Dawit , Rozacky Jenna , B. Sweasy Joann
Published: August 2015

DNA polymerase beta (Pol β) is a key enzyme for the protection of oxidative DNA lesions via its role in base excision repair (BER). Approximately 1/3 of tumors studied to date express Pol β variant proteins, and several tumors overexpress Pol β. Pol β possesses DNA polymerase and dRP lyase activities, both of which are known to be important for efficient BER. The dRP lyase activity resides within the 8kDa amino terminal domain of pol β, is responsible for removal of the 5′ phosphate group and the polymerase domain at subsequent steps fills the gaps. Previously, we demonstrated that gastric cancer-associated variant of pol β (Leu22Pro (L22P)) lacks dRP lyase function in-vitro. Here, we have employed DNA fiber labeling to quantify, for the first time, the extent to which the normal function of dRP lyase of DNA polymerase beta is critical to maintain global replication fork rates in normal gastric epithelial cells. We report that replication fork rates in L22P expressing cells are on average half of those observed with wild-type cells. In addition, we show that L22P variant promotes replication dependent double strand breaks that may likely responsible for chromosomal aberrations. These data may implicate normal function of dRP lyase is critical to stabilize replication fork stall and prevent replication fork collapse to DNA double strand breaks.

Citation Format: Dawit Kidane, Jenna Rozacky, Joann B. Sweasy. Gastric cancer associated variant of DNA polymerase Beta (Leu22Pro) induce genomic instability and cellular transformation. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3026. doi:10.1158/1538-7445.AM2015-3026
Abstract 3028: Tolerance and intolerance of proximal thymine glycol in DNA double-strand break repair by nonhomologous end joining
Authors: Bafail Duaa , Chalasani Sri Lakshmi , Al mohaini Mohammed , Akopiants Konstantin , Povirk Lawrence F.
Published: August 2015

Double-strand breaks (DSBs) induced by ionizing radiation are often accompanied by ancillary oxidative base damage that could prevent or delay their repair. In order to better define the features that make some DSBs repair-resistant, plasmid DSB substrates were constructed with two blunt ends, one having the oxidatively modified nonplanar base thymine glycol at the first, second or third positions from the 3′ terminus (Tg1, Tg2 and Tg3, respectively). End joining was examined in extracts of Bustel fibroblasts, wherein end joining is completely dependent on addition of exogenous recombinant XLF, and therefore reflects classical nonhomologous end joining. Tg at the third position had almost no effect on end-joining even when present on both ends of the break. However, the Tg1 and Tg2 substrates yielded tenfold to twentyfold less head-to-tail joins and no detectable head-to-head joins, suggesting that Tg as the terminal or penultimate base is a serious barrier to NHEJ and an absolute barrier when present at both ends. For all substrates, Tg-containing ligation products predominated at early times, and remarkably, a 3′ terminal Tg was in some cases directly ligated to an undamaged blunt end. Dideoxy trapping of base excision repair intermediates indicated that Tg was excised in the extracts, but largely if not exclusively after DSB ligation. For all substrates, accurate blunt end ligation without end trimming predominated. However addition of excess Artemis nuclease to the extracts resulted in a slightly shorter ligation product, and a slight increase in yield, for the Tg2 substrate only. For the Tg1 substrate, there was a distinct 2-hr delay between addition of substrate and the detection of ligated products, while the unmodified and Tg3 products showed a much shorter delay of ∼30 min. Overall, the results suggest that Tg can be a major impediment to DSB repair when present as the terminal or penultimate base, being relatively resistant to both trimming and ligation.

Citation Format: Duaa Bafail, Sri Lakshmi Chalasani, Mohammed Al mohaini, Konstantin Akopiants, Lawrence F. Povirk. Tolerance and intolerance of proximal thymine glycol in DNA double-strand break repair by nonhomologous end joining. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3028. doi:10.1158/1538-7445.AM2015-3028
Abstract 3029: Role of eIF3a expression in cellular sensitivity to radiation treatment
Authors: Tumia Rima A.
Published: August 2015

Translation initiation involving eukaryotic translation initiation factors (eIFs) is a crucial mechanism in controlling gene expression. eIF3a has been shown previously to regulate protein synthesis and cellular response to cisplatin treatment. Its expression has also been shown to negatively associate with prognosis. In this study, we tested a hypothesis that eIF3a regulates synthesis of DNA repair proteins, which in turn regulates cellular response to ionizing radiation (IR) treatments. We found that eIF3a overexpression sensitizes cellular response to IR while its knockdown causes resistance to IR. We also found that eIF3a over-expression increases IR-induced DNA damage and decreases Non-Homologous End Joining (NHEJ) repair activity by suppressing expression level of NHEJ repair proteins such as DNAPKcs. Consistently, eIF3a knockdown decreases IR-induced DNA damage and increases Non-Homologous End Joining (NHEJ) repair activity by enhancing expression level of NHEJ repair proteins. Together, we conclude that eIF3a plays an important role in cellular response to DNA-damaging treatments by regulating synthesis of DNA repair proteins and, thus, eIIF3a likely plays an important role in the outcome of cancer patients treated with DNA-damaging strategies.

Note: This abstract was not presented at the meeting.

Citation Format: Rima A. Tumia. Role of eIF3a expression in cellular sensitivity to radiation treatment. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3029. doi:10.1158/1538-7445.AM2015-3029