bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 4713: Targeting the mitochondria for the treatment of MLH1-deficient disease
Authors: Rashid Sukaina , Bridge Gemma , Yao Zhi , Szabadkai Gyorgy , Martin Sarah A.
Published: August 2015

The DNA Mismatch repair (MMR) pathway is responsible for the repair of base-base mismatches and insertion/deletion loops that arise during DNA replication. MMR deficiency is currently estimated to be present in 15-17% of colorectal cancer cases and 30% of endometrial cancers. MLH1 is one of the key proteins involved in the MMR pathway. MMR deficient tumours are often resistant to standard chemotherapies, therefore there is a critical need to identify new therapeutic strategies to treat MMR deficient disease.

We have performed synthetic lethal compound and RNAi screens to identify potential therapeutic targets for the treatment of MLH1 deficient tumours. Strikingly, upon analysis, a number of the hit compounds have been shown to target mitochondrial function. Our previous work has shown that MLH1 can localize to the mitochondria and silencing of the mitochondrial genes, POLG and PINK1 are synthetically lethal with MLH1 deficiency.

We demonstrate that MLH1 deficient tumours are synthetically lethal with mitochondrial targeted agents (Menadione, Parthenolide) which are known to induce reactive oxygen species and cause oxidative stress as one of their main mechanisms of action. Upon functional analysis we show that loss of MLH1 is associated with a reduction in Complex I activity, reduced basal oxygen consumption rate, reduced spare respiratory capacity and reactive mitochondrial biogenesis (upregulation of pgc1β), suggesting that mitochondrial function is deregulated upon loss of MLH1.

Taken together, our results thus far suggest that targeting the mitochondria may be a potential therapeutic strategy for the treatment of MLH1 deficient disease.

Citation Format: Sukaina Rashid, Gemma Bridge, Zhi Yao, Gyorgy Szabadkai, Sarah A. Martin. Targeting the mitochondria for the treatment of MLH1-deficient disease. [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 4713. doi:10.1158/1538-7445.AM2015-4713
Abstract 4718: Inositol polyphosphate 4-phosphatase II activates PI3K/SGK3 signaling to promote proliferation of human melanoma cells
Authors: Jiang Chen Chen , Chi Meng Na , Guo Su Tang , Wilmott James S. , Guo Xiang Yun , Yan Xu Guang , Wang Chun Yan , Liu Xiao Ying , Jin Lei , Tseng Hsin-Yi , Croft Amanda , Hondermarck Hubert , Liu Tao , Scolyer Richard A. , Zhang Xu Dong
Published: August 2015

Inositol polyphosphate 4-phosphatase type II (INPP4B) inhibits phosphatidylinositol 3-kinase (PI3K)/Akt signaling and is emerging as a tumor suppressor in a variety of tissues. Here we report that, conversely, it functions as an oncogenic regulator in human melanoma through activating PI3K/SGK3 signaling. While it was upregulated in a subset of melanomas, knockdown of INPP4B inhibited melanoma cell proliferation in vitro, and retarded melanoma growth in a xenograft model. In contrast, overexpression of INPP4B resulted in increased proliferation and anchorage-independent growth of melanocytes. Strikingly, INPP4B did not impinge on activation of Akt in melanocytic cells. Instead, it promoted PI3K/SGK3 signaling, in that INPP4B knockdown inhibited, whereas overexpression of INPP4B enhanced, activation of SGK3. Indeed, the effect of INPP4B on melanocytic cell proliferation was due to enhanced activation of SGK3, as co-introduction of an active form of SGK3 rescued melanocytes and melanoma cells from inhibition of proliferation triggered by INPP4B knockdown, and knockdown of SGK3 abolished enhancement in cell proliferation resulting from INPP4B overexpression. Upregulation of INPP4B appeared largely due to downregulation of microRNA-494 (miR-494) and/or miR-599 as a result of gene copy number reduction in melanoma cells. Collectively, these results reveal that INPP4B upregulation mediated by loss of miR-494 and/or miR-599 promotes melanoma cell proliferation through activation of PI3K/SGK3 signaling, and suggest that the role of INPP4B in the pathogenesis of cancers of different origins needs to be defined discretely.

Citation Format: Chen Chen Jiang, Meng Na Chi, Su Tang Guo, James S. Wilmott, Xiang Yun Guo, Xu Guang Yan, Chun Yan Wang, Xiao Ying Liu, Lei Jin, Hsin-Yi Tseng, Amanda Croft, Hubert Hondermarck, Tao Liu, Richard A. Scolyer, Xu Dong Zhang. Inositol polyphosphate 4-phosphatase II activates PI3K/SGK3 signaling to promote proliferation of human melanoma 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 4718. doi:10.1158/1538-7445.AM2015-4718
Abstract 473: Functional polarization of macrophage triggered by tumor-conditioned medium
Authors: Zhang Weina
Published: August 2015

Purpose: Macrophages are important components of leukocyte infiltrates in various tumor stroma. Macrophages that infiltrate into tumor tissues are called tumor-associated macrophages (TAMs). TAMs show anti-tumorigenesis(M1) or pro-tumorigenesis (M2) functions depending on the cytokine milieu of the tumor microenvironment. TAMs perform some key homeostatic functions that allow tumor maintenance and growth. However, the signals involved in communication between tumor cells and macrophages are poorly defined. The aim of this study is to explore the macrophage polarization treated with the tumor-conditioned medium.

Methods: Ana-1 macrophages, human THP-1 monocytes, and bone-marrow-derived macrophages (BMDMs) from C57BL/6J mice were incubated with HCT116-conditioned medium, respectively. Then we detected M1/M2 specific molecular markers by flow cytometry, Western blot and Real-time PCR. Flow cytometry was used to quantitatively assess the change of macrophage populations in macrophages treated with conditioned medium or normal medium. CD68(human) and CD11b(human), F4/80(mouse) and CD11b(mouse) were used as total-macrophage markers, CD206 as a marker of M2 macrophage. Secretory cytokine profile of the polarized macrophages was measured by cytokine array. Then, HCT116 cells were treated with conditioned medium of M2-like macrophage and applied to cell growth and colony formation assay.

Results: By Western blot, we found that HCT116-conditioned medium could induce BMDMs and Ana-1 cells to overexpress arginase1(Arg1), but Ana-1 cells to express low inducible nitric oxide synthase(iNOS). Flow cytometry showed high expression level of CD68(human) and CD11b(human), F4/80(mouse) and CD11b(mouse), CD206 in HCT116-conditioned medium induced macrophages. Additionally, mRNA level of Arg1 was higher and M2-related cytokines IL-10,IL13 were also significantly upregulated, while mRNA level of iNOS was lower and M1-related cytokines IL-12,IFN-γwere downregulated in HCT116-conditioned medium induced macrophages. Conditioned medium of M2-like macrophages promoted the HCT116 cell growth and proliferation.

Conclusions: HCT116 can secrete the soluble factors into conditioned medium, which would induce TAM polarization. Subsequently, conditioned medium of M2-like macrophages can promote tumor cells growth.

Citation Format: Weina Zhang. Functional polarization of macrophage triggered by tumor-conditioned medium. [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 473. doi:10.1158/1538-7445.AM2015-473
Abstract 4743: A population-based approach to address clinical cancer care: The national genomics platform
Authors: Abaan Ogan , Basu Amrita , Brown Noah , Light Bret , Deal David , Hultner Michael
Published: August 2015

Our understanding of the genetic basis for cancer is advancing at a rapid rate due to the application of next generation sequencing (NGS) technologies. It is becoming common to sequence tumors and patients in an attempt to find actionable mutations that could offer the patient a more targeted and effective treatment. However, the genomics of cancer is very complex and only a handful of actionable mutations have been characterized. Larger-scale studies are needed to understand the pathogenic spectrum of cancer variants and deliver reliable clinical decision-making support to providers.

Several large-scale sequencing projects are underway to collect NGS data on tumors (e.g. TCGA, ICGC, and TARGET) but a national platform for analysis, interpretation, and reporting does not exist. We propose that a consolidated informatics platform for the collection of outcomes data with genomics and clinical data would accelerate research and provide patients with the opportunity for personalized cancer treatment. In addition, with 1,665,540 new cancer cases predicted in the US for 2014, a national-scale genomics platform is needed, capable of sequencing 3 million genomes per year, storing exabytes of data, while supporting over 20,000 oncologists, researchers, and analysts.

At Lockheed Martin, we deliver highly scalable and reliable information systems for a variety of missions and citizen services. Here, we will present our vision for a national cancer genomics platform to include NGS data collection, cost-efficient storage, scalable and modular processing pipelines, and collaborative analytics and data sharing capabilities, within a compliant privacy and security framework. In conclusion, by leveraging the scale of clinical cancer sequencing and capturing these data into a case management system for translational research, this platform provides data at-scale needed for finding actionable mutations, designing effective treatments and implementing prevention strategies, faster.

Citation Format: Ogan Abaan, Amrita Basu, Noah Brown, Bret Light, David Deal, Michael Hultner. A population-based approach to address clinical cancer care: The national genomics platform. [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 4743. doi:10.1158/1538-7445.AM2015-4743
Abstract 4752: Genomic cytometry: Single cell mutation analysis combining PCR with flow cytometry
Authors: Hyun William C. , Sciambi Adam , Pellegrino Maurizio , Eastburn Dennis
Published: August 2015

Genomic cytometry is a new technology field combining the high-throughput capabilities of flow cytometry and cell sorting with single-cell multiplex genomic analysis in microfluidic drops. The genomic cytometry method we will describe is PCR-activated cell sorting (PACS). PACS is analogous to Fluorescence-Activated Cell Sorting (FACS) in throughput and the ability to analyze single cells; however, unlike conventional flow cytometric immunophenotyping, PACS uses extremely sensitive microdroplet TaqMan PCR reactions to identify and, if needed, sort cells of interest for additional downstream molecular characterization. This unique approach enables PACS to identify cells based on the presence of transcripts, genomic DNA sequences, mRNA splice variants, SNPs, non-coding RNAs, and other biomarkers not amenable to antibody-based probe detection. PACS can be used to sort and enrich cells targeting single-copy genomic DNA for the analysis of circulating tumor cells (CTCs), intra-tumor heterogeneity, rare stem cells, viral or pathogen infections and genomic characterization of cancer immune system response and disorder. By analyzing individual cells within a population, it is possible to identify rare CTC populations or transient cell states, critical to human health and cancer progression, that are otherwise unobservable by ensemble measurements. With PACS, we identified and sorted prostate cancer cells from a heterogeneous population by performing >132,000 simultaneous single-cell TaqMan RT-PCR reactions targeting vimentin mRNA. Following vimentin-positive droplet sorting and downstream analysis of recovered nucleic acids, we found that cancer-specific genomes and transcripts were significantly enriched.

Citation Format: William C. Hyun, Adam Sciambi, Maurizio Pellegrino, Dennis Eastburn. Genomic cytometry: Single cell mutation analysis combining PCR with flow cytometry. [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 4752. doi:10.1158/1538-7445.AM2015-4752
Abstract 4754: Development of fluorescent probes for detection of DNA base excision repair in human cancer cells
Authors: Condie Allison G. , Yan Yan , Gerson Stanton L. , Wang Yanming
Published: August 2015

Many cancer chemotherapeutics target DNA, which makes them vulnerable to repair by innate mechanisms the cell has evolved to ward off endogenous and exogenous DNA damage. In the course of chemotherapy, these repair pathways lead to drug resistance and reduced efficacy of cancer treatment. To address these issues, our goal was to develop imaging agents to visualize and quantify DNA repair in cancer. Thus, we have developed small molecule, fluorescent probes that bind to the abasic (or AP for apurinic/apyrimidinic) site in DNA, a key intermediate in the base excision repair (BER) pathway.

Taking advantage of the biochemistry of BER, we have developed an assay based on a fluorescently tagged DNA oligomer to evaluate the ability of these probes to bind to AP sites and stall further repair. This assay relies on blocking the DNA single strand breaking activity of the enzyme, AP endonuclease (APE), when its natural substrate, the AP site, is obstructed by probe binding. Using this assay, we have screened our novel probes for their AP site binding potential. Our cyanine-(Cy7) based probe outperformed other, commercially available AP site binding probes including methoxyamine (MX) and aldehyde reactive probe (ARP).

In addition, we have developed a method for analyzing a genomic DNA sample that demonstrates the potential to evaluate physiologically relevant quantities of AP sites. 5-Fluoro-2′-deoxyuridine (FUDR) is indicated for treatment of many types of cancer; however, uracil DNA glycosylase (UDG) can repair the lesions in DNA introduced by FUDR. Therefore, we prepared a UDG knockdown of the DLD1 colon cancer cell line and treated both wild type and knockdown cells with FUDR for 24, 48, and 72 h. Following extraction of the DNA and in vitro treatment with purified UDG, we observed a nearly 9-fold increase in fluorescence in treated versus untreated cells. This confirmed that our probe could report of AP site quantity in cancer cells in vitro.

Our cyanine-based probes have several benefits over existing probes including red and NIR fluorescence for application in animal imaging. Our studies indicate that these probes bind to AP sites more efficiently than existing probes. Further, these fluorescent AP site-binding agents could have application in drug development and discovery or be used in assays to monitor patient response to therapy.

Citation Format: Allison G. Condie, Yan Yan, Stanton L. Gerson, Yanming Wang. Development of fluorescent probes for detection of DNA base excision repair in human cancer 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 4754. doi:10.1158/1538-7445.AM2015-4754
Abstract 4756: Genomic profile of AML-ETO rearranged acute myeloid leukemia
Authors: Koh Youngil , Kim Dae-Yoon , Kim Jong-Kwang , Chung Yeun-June , Yoon Sung-Soo , Kim Hyung-Lae
Published: August 2015

Introduction

Although it is well known that core-binding factor(CBF) acute myeloid leukemia(AML) harbors good prognosis, some patients with CBF-AML have dismal outcome. In this study, we tried to find out genomic alterations in CBF-AML so as to find genetic changes that determine prognosis of CBF-AML.

Materials and methods

We performed whole genome sequencing (WGS) of 9 AML-ETO rearranged AML patients. For whole genome sequencing, we used Hiseq 2000 (Illumina, SanDiego, CA, USA). Single nucleotide variant (SNV) calling was performed using 4 different callers. Structural variant calling was performed using three different callers.

Results

Among 9 patients, two patients had excellent prognosis, while 3 patients showed unfavorable outcome with conventional chemotherapy. 4 patients had undetermined prognosis. As for SNV, the most frequent common non-synonymous SNV was KIT mutation, while ADAMTS17 and CFAP46 mutation was found in 2/9 patients respectively. Noticeable non-synonymous SNV found only in patients with poor prognosis included NRAS, ADAM29, AGO1, and ALAS2 mutation. For structural variation, LOC44166 inversion, ANKRD26P1 deletion, EMBP1 translocation were noticeable structural variants other than AML-ETO rearrangement in these patients.

Conclusion

Not a single genetic factor was attributable to the prognosis in CBF-AML. Further validation of genetic alterations found study in this study is ongoing.

Citation Format: Youngil Koh, Dae-Yoon Kim, Jong-Kwang Kim, Yeun-June Chung, Sung-Soo Yoon, Hyung-Lae Kim. Genomic profile of AML-ETO rearranged acute myeloid leukemia. [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 4756. doi:10.1158/1538-7445.AM2015-4756
Abstract 4759: Whole genome sequencing of sequentially acquired lung and lymph node metastatic sites from a never smoker lung adenocarcinoma patient revealed extensive genomic heterogeneity
Authors: Gao Shaojian , Cultraro Constance , BIswas Romi , Carter Corey A. , Maity Tapan K. , Thomas Anish , Rajan Arun , Meltzer Paul , Schrump David , Giaccone Giuseppe , Khan Javed , Guha Udayan
Published: August 2015

Recent large-scale cancer genome sequencing studies have uncovered extensive diverse mutational landscapes in lung cancer patients. Furthermore, tumor heterogeneity has been widely recognized and it has significant clinical implications in selection of targeted treatment strategies as well as treatment response. Using whole genome sequencing, we demonstrate an unprecedented genomic heterogeneity between sequentially acquired lung and lymph node metastatic sites from an African American never-smoker lung adenocarcinoma patient who has survived with metastatic disease for over seven years while being treated with single or combination HER2-directed therapies. We determined that less than 1% of somatic variants were common between the two tumor sites. Copy number variations were more intense in the lung tumor than in the metastatic lymph node. We identified several novel somatic mutations in key cancer genes in both sites. Interestingly, one novel translocation, PLAG1-ACTA2 was highly expressed in both the lung and lymph node metastases resulting in overexpression of ACTA2, which has been suggested to increase the metastatic potential in lung adenocarcinoma. Using ultra deep targeted re-sequencing, we validated all non-synonymous variants, and approximately 80% of those identified in the metastatic lymph node were also present in a second lymph node biopsied two years after the first one. Although this degree of tumor heterogeneity was surprising, somatic variants affected key hallmarks of tumorigenesis in both sites. These findings suggest a model of early metastatic spread and parallel clonal evolution in disparate metastatic sites.

Citation Format: Shaojian Gao, Constance Cultraro, Romi BIswas, Corey A. Carter, Tapan K. Maity, Anish Thomas, Arun Rajan, Paul Meltzer, David Schrump, Giuseppe Giaccone, Javed Khan, Udayan Guha. Whole genome sequencing of sequentially acquired lung and lymph node metastatic sites from a never smoker lung adenocarcinoma patient revealed extensive genomic heterogeneity. [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 4759. doi:10.1158/1538-7445.AM2015-4759
Abstract 4764: The Bromodomain inhibitors regulate long noncoding RNAs controlling glioblastoma progression
Authors: Pastori Chiara , Kapranov Philip , Penas Clara , Laurent Georges St. , Ayad Nagi , Wahlestedt Claes
Published: August 2015

Bromodomain and extra-terminal domain (BET) proteins have emerged as promising therapeutic targets in glioblastoma and many other cancers. Small molecule mimics of acetylated histones inhibit BET protein function and reduce expression of several oncogenes required for glioblastoma (GBM) progression. However, the mechanism through which BET protein inhibition reduces GBM growth is not completely understood. Long noncoding RNAs (lncRNAs) are important epigenetic regulators with critical roles in cancer initiation and malignant progression but mechanistic insight into their expression and regulation by bromodomain inhibitors remains elusive. In this study we utilized single molecule sequencing (SMS) to comprehensively profile lncRNAs differentially expressed in GBM and we identified a subset of GBM-specific lncRNAs whose expression is regulated by BET proteins. Treatment of GBM cells with the BET protein inhibitor I-BET151 reduced levels of the tumor-promoting lncRNA HOTAIR and restored the expression of several other GBM-down-regulated lncRNAs. Conversely, overexpression of HOTAIR in conjunction with I-BET treatment rescues the antiproliferative activity of I-BET151. Moreover, chromatin immunoprecipitation analysis demonstrated binding of Brd4 to the HOTAIR promoter suggesting that BET proteins can directly regulate lncRNA expression. Our data unravel a previously unappreciated mechanism through which BET proteins control tumor growth of glioblastoma cells and suggest that lncRNA networks may, in part, mediate the anti-proliferative effects of many epigenetic inhibitors currently in clinical trials for cancer and other diseases.

Note: This abstract was not presented at the meeting.

Citation Format: Chiara Pastori, Philip Kapranov, Clara Penas, Georges St.Laurent,Nagi Ayad, Claes Wahlestedt. The Bromodomain inhibitors regulate long noncoding RNAs controlling glioblastoma 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 4764. doi:10.1158/1538-7445.AM2015-4764
Abstract 4767: DNA sequences differentially associated with Sirt1 and Dnmt3b during melanoma progression
Authors: Marcelino Meliso Fabiana , da Silva Camila Tainah , Coetzee Simon , Sarraf Sabedot Thaís , Noushmehr Houtan , Schneider-Stock Regine , Galvonas Jasiulionis Miriam
Published: August 2015

Melanoma is one of the most worrying tumors due to its high capacity to cause metastasis. Sirt1, a class III histone deacetylase, was shown to be attracted to sites of double strand breaks in DNA, favoring, by recruiting components of the epigenetic machinery, aberrant epigenetic silencing in that region (O’HAGAN et al., 2008). Beyond that, subsequent studies have attributed important roles of Sirt1 and other components of the epigenetic machinery, such as DNA methyltransferases (such as Dnmt3b), in tumor initiation and progression. In this sense, using a murine model of malignant transformation of melanocytes, associated with sustained stress conditions, our goal was to identify genomic sequences differentially associated to Sirt1 and/or Dnmt3B before and after stress condition, represented in our model by anchorage blockade, and analyze if these sequences are targeted by aberrant methylation in cell lines obtained by this process that represent different steps in melanoma progression. We have showed increased DNA damage after 24 hours of anchorage impediment followed by increased Sirt1 expression. In addition, we have showed Sirt1/Dnmt3b association just after this stress condition that leads to malignant transformation. It was identified several DNA sequences differentially associated to Sirt1 and also to Dnmt3b after stress condition which are also associated to genes related to different metabolic processes. Some of these sequences remain associated to Sirt1 and Dnmt3b during melanoma progression, indicating that Dnmt3b remains associated to Sirt1 during malignant transformation. Among the sequences, Tiam1 and Mxd1 are potential targets of Sirt1 and Dnmt3b that have important roles in tumor progression. We have also identified motifs where Sirt1 and Dnmt3b are probably bound before and after stress condition. The main motif related to Sirt1 was coincident with Boris motif. Subsequent analysis are been developed to elucidate the importance of these interesting discoveries. Supported by FAPESP and DAAD.

Citation Format: Fabiana Marcelino Meliso, Camila Tainah da Silva, Simon Coetzee, Thaís Sarraf Sabedot, Houtan Noushmehr, Regine Schneider-Stock, Miriam Galvonas Jasiulionis. DNA sequences differentially associated with Sirt1 and Dnmt3b during melanoma 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 4767. doi:10.1158/1538-7445.AM2015-4767
Abstract 4774: Histone H2AX is a novel regulator of epithelial to mesenchymal transition
Authors: WEYEMI SOSSOU U. , Redon Christophe E. , Choudhuri Rohini , Maeda Daisuke , Kasoji Manjula , Abrams Natalie , Bonner William M.
Published: August 2015

The vast majority of deaths from cancer are due to its progression from primary tumor to metastatic disease. Understanding how some cells can migrate from the primary tumor to seed new tumors throughout the body is a longstanding challenge in developing novel cancer treatments. Current models propose that these migratory cells acquire an invasive phenotype through a process known as the epithelial to mesenchymal transition (EMT), characterized by the loss of cell polarity and cell-to-cell adhesion, and the acquisition of migratory and invasive properties. Such changes allow the cells to invade the extracellular matrix and migrate throughout the body, aided by the formation of lamellipodia, filopodia and invadopodia. In a converse transition, the MET, mesenchymal cells may revert back to the epithelial phenotype and establish tumors at distant sites.We have preliminary observations showing that when one member of the H2A family of histones, H2AX, is silenced or inactivated in HCT116 cells, these cells exhibit mesenchymal-like characteristics including increased invasiveness. Furthermore, genome-wide expression analysis implicates the critical EMT transcription factors, SLUG and ZEB1, as mediators of H2AX loss-induced EMT. To make these studies more rigorous, we generated a novel HCT116 cell line with H2AX destroyed. Our findings show that ectopic expression of H2AX in H2AX-null cells reverses the invasiveness induced by H2AX inactivation. Moreover, in 233 human colon cancer samples, a strong correlation is found between the levels of H2AX and SLUG/ZEB1 expression. These observations lead us to hypothesize that H2AX is a key regulator of EMT and plays a critical role in the downstream events leading to increased metastasis.

Note: This abstract was not presented at the meeting.

Citation Format: SOSSOU U. WEYEMI, Christophe E. Redon, Rohini Choudhuri, Daisuke Maeda, Manjula Kasoji, Natalie Abrams, William M. Bonner. Histone H2AX is a novel regulator of epithelial to mesenchymal transition. [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 4774. doi:10.1158/1538-7445.AM2015-4774
Abstract 4777: Triptolide inhibits Wnt signaling due to DNA methylation alteration that is determined by dynamic histone 3 K79 lysine methylation in NSCLC
Authors: Li Youqiang , Shen Binghui , Kim Jae , Raz Dan
Published: August 2015

Epigenetic alterations including DNA methylation and histone modifications have been associated with virtually every step of tumor development and progression. This realization has triggered an impressive quest for the development of “epigenetic drugs” and epigenetic therapies. Here we present data demonstrating that triptolide, with a unique mechanism, has anticancer effect in lung cancer development and progression. With the use of our unique non-small cell lung cancer (NSCLC) mouse model (ED160, Fen1 mutation), data shows triptolide decreases lung cancer incidence from 70% to 10%. In addition, triptolide effectively inhibits lung cancer growth and metastasis in NSG mouse Xenograft. Triptolide inactivates Wnt signaling by restoring WIF1 expression which is due to WIF1 gene demethylation. Furthermore, we revealed histone 3 lysine methylation alteration determines DNA methylation status by the communication between DNMT1 and histone 3. Together, these evidences indicated that sequential histone and DNA methylation active Wnt signaling; further it contributes to NSCLC development and progression. This discovery potentially is yielding epigenetic therapeutic opportunities for NSCLC.

Note: This abstract was not presented at the meeting.

Citation Format: Youqiang Li, Binghui Shen, Jae Kim, Dan Raz. Triptolide inhibits Wnt signaling due to DNA methylation alteration that is determined by dynamic histone 3 K79 lysine methylation in NSCLC. [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 4777. doi:10.1158/1538-7445.AM2015-4777
Abstract 478: EWS/FLI1 transcription is modulated by the PI3K pathway via SP1 in Ewing sarcoma
Authors: Giorgi Chiara , Boro Alexander , Lopez-Garcia Laura A. , Schaefer Beat W. , Niggli Felix K.
Published: August 2015

Ewing sarcoma (ES) is the second most frequent bone cancer in childhood and it is characterized by the presence of the balanced t(11;22)(q24;q12) translocation in more than 85% of cases, generating a dysregulated transcription factor EWS/FLI1. ES belongs to small-round-blue-cell tumors and it is a very aggressive osteolytic cancer with early tendency for development of metastasis. Mostly it affects bones such as pelvis, femour and ribs but can also arise in soft tissues, mainly in adults. EWS/FLI1 is an essential oncogenic component of ES development which is necessary for tumor cell maintenance, through inappropriate regulation of target genes that are crucial for the fully malignant phenotype. Therefore, EWS/FLI1 represents an attractive therapeutic target. Screening of a small library of 153 targeted compounds has identified inhibitors of the PI3K pathway as main modulators of EWS/FLI1 activity and surprisingly this was due to an effect of the compound on EWS/FLI1 transcription. Indeed, treatment of four different ES cell lines with BEZ235 (PI3K-mTOR inhibitor) resulted in downregulation of EWS/FLI1 by 50% expression with subsequent modulation of target gene expression. Analysis of the EWS/FLI1 promoter region (-2239/+67) using various deletion constructs identified two 14bp minimal elements, named Del2 and Del23 region, as being important for EWS/FLI1 transcription. Based on in silico prediction and on in vivo data, we were able to predict 4 transcription factor candidates to bind this region and being responsive to PI3K regulation. Among these, SP1 was identified as modulator of EWS/FLI1 gene expression by siRNA. Various approches such as qRT-PCR, Western Blot and Immunofluorescence confirmed a reduction of EWS/FLI1 in Ewing cells when PI3Kαγδ and SP1 were depleted; Electrophoretic Mobility Shift Assay (EMSA) and Chromatine Immuno Precipitation (ChIP) both confirmed that the transcription factor SP1 is indeed binding to Del23 region of the EWS/FLI1 promoter. In summary, our results provide the very first insights on the transcriptional regulation of EWS/FLI1, an area that has not been investigated so far, and offer a molecular explanation for the known sensitivity of ES cell lines to PI3K inhibition.

Citation Format: Chiara Giorgi, Alexander Boro, Laura A. Lopez-Garcia, Beat W. Schaefer, Felix K. Niggli. EWS/FLI1 transcription is modulated by the PI3K pathway via SP1 in Ewing sarcoma. [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 478. doi:10.1158/1538-7445.AM2015-478
Abstract 4780: Nucleosome organization and accessibility of p53 response elements in the chromatin context
Authors: LoVerso Peter R. , Zhurkin Victor B. , Cui Feng
Published: August 2015

p53 is one of best known human proteins that induces or represses hundreds of genes and plays a pivotal role in preventing tumor development. Recent studies established unambiguously that p53 is a nucleosome-binding protein. The rotational positioning of a nucleosome is critical to determine the accessibility of a p53 site. In particular, the p53 site in a nucleosome is accessible if it is bent in the direction similar to that found in the p53-DNA co-crystals; the site becomes inaccessible if the orientation is changed by ∼180°. Importantly, we have shown that p53 response elements (REs) associated with cell cycle arrest are often exposed on the nucleosomal surface, suggesting a new role of nucleosomes in mediating the p53 binding and subsequent gene induction.

The present study aims to make a detailed analysis of nucleosome organization of all p53 functional REs in normal and cancer cells. Published genome-wide nucleosome maps from lymphoblastoid (normal) and K562 (cancer) cell lines are used. In general, the p53 REs are located in nucleosome-enriched regions, in accordance with previous findings that p53 can interact with nucleosomal DNA. At the level of individual sites, the p21 5′ RE resides in a genomic region with high nucleosome occupancy, consistent with earlier studies. The GADD45 RE that was believed to be in nucleosome-free region, is, however, characterized with medium-to-high nucleosome occupancy.

To evaluate the accessibility of p53 REs in different functional groups such as cell cycle arrest, apoptosis and DNA repair, we used a more rigorous set of nucleosomes, containing 134 million 147-bp fragments (about 7x genome coverage). The REs in the cell cycle arrest group (such as p21) and the DNA repair group (such as GADD45) are often accessible in the chromatin context, consistent with fast induction of these genes after p53 activation. Interestingly, apoptotic REs are divided - the REs associated with the mitochondrial pathway (BAX and p53AIP1) are accessible, while the REs associated with the death receptor pathway (FAS and KILLER) are inaccessible, suggesting different mechanisms for induction of apoptotic genes in vivo.

Finally, we observe a clear correlation between the nucleosome occupancy and accessibility of p53 REs. That is, the REs with the higher nucleosome occupancy tend to be accessible, while the REs with lower nucleosome occupancy are likely to be inaccessible to p53.

Citation Format: Peter R. LoVerso, Victor B. Zhurkin, Feng Cui. Nucleosome organization and accessibility of p53 response elements in the chromatin context. [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 4780. doi:10.1158/1538-7445.AM2015-4780
Abstract 4781: A comprehensive screen for protein kinases involved in histone phosphorylation using a biochemical in-vitro approach
Authors: Mueller Daniel , Totzke Frank , Ketterer Constance , Beisenherz-Huss Christian , Krämer Diane , Weber Thomas , Heidemann-Dinger Carolin , Kubbutat Michael H.G.
Published: August 2015

Gene expression is regulated by multiple mechanisms. The structure of the chromatin has been identified as one major regulator of gene expression. Histone proteins constitute the core of the nucleosomal particles, which are essential for structuring the DNA into chromatin. Various post-translational modifications of histones mainly in the N-terminal region of these proteins will compact or loosen the chromatin which in turn changes the accessibility of the DNA.

Such histone modifications like methylation, acetylation and phosphorylation can therefore result either in activation or in repression of the transcription of the respective genes, depending on the specific amino acid residues modified. While histone-acetylases and histone-methyl-transferases as well as their corresponding counterparts, histone-deacetylases and histone-demethylases, are currently in the focus of epigenetic research, it is also well documented that histones are extensively modified and regulated by phosphorylation.

In order to identify protein kinases able to phosphorylate histones we have generated a series of N-terminal peptides of human histone proteins H1, H2A, H2B, H3 and H4 as well as recombinant histone proteins. In a biochemical in-vitro activity assay (“KinaseFinder”) we tested these peptides and proteins as potential substrates against a panel of 339 recombinant human serine-threonine- and tyrosine-kinases. The results of the screens will be presented showing different protein kinases able to phosphorylate various histones in vitro. Additional data of approaches to verify and to characterize hit kinases, e.g. with respect to the specific target amino acid sequences, will be shown.

Citation Format: Daniel Mueller, Frank Totzke, Constance Ketterer, Christian Beisenherz-Huss, Diane Krämer, Thomas Weber, Carolin Heidemann-Dinger, Michael H.G. Kubbutat. A comprehensive screen for protein kinases involved in histone phosphorylation using a biochemical in-vitro approach. [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 4781. doi:10.1158/1538-7445.AM2015-4781
Abstract 4782: Genome-wide chromatin profiling in bladder and prostate cancers
Authors: Kim Sohyoung , Varticovski Lyuba , Lao Qizong , Baek Songjoon , Sung Myong-Hee , Grøntved Lars , Nickerson Michael L. , Thompson Bethtrice , Theodorescu Dan , Dean Michael , Hager Gordon H.
Published: August 2015

Mutations in a few driver genes can cause cancer, and a main goal of cancer genome analysis has been the identification of these cancer genes. Recent findings, however, indicate that deregulation of enhancers can play a major role in carcinogenesis. In this study, we aim to understand how enhancer networks evolve during cancer initiation and progression and to identify epigenetic states that are predictive of cancer stage and drug response. We surveyed 15 bladder cell lines, including the T24 lineage (T24, T24T, FL, and SL) and the UMUC3 lineage (UMUC3 and LUL2), and 10 prostate cancer cell lines including BPH1, DU145 and LNCap. These cell lines display different tumorigenic and metastatic potentials, and thus present a model for cancer progression and development of castration-resistance. DNase-seq was performed to map genome-wide regulatory profiles in these cells. The enhancers detected as DNase I hypersensitivity sites (DHSs) showed large-scale changes in the chromatin accessibility landscape during the acquisition of tumorigenic and metastatic phenotypes. Selective sets of gained or lost DHSs were identified. Among the changes during the T24 to T24T transition, the loss of DHSs was remarkable. Some of these DHSs were found near genes that are in the downstream response to HDACs. Induction of expression by the HDAC inhibitor, Na butyrate, was correlated with DNase-seq signal for these genes. Gene Ontology (GO) enrichment analysis also indentified a set of genes neighboring lost DHSs that belong to the focal adhesion class in GO category. This study identified a set of DHSs that were associated with cancer progression in bladder and prostate cancer, and the potential clinical application of the identified DHSs will be discussed.

Citation Format: Sohyoung Kim, Lyuba Varticovski, Qizong Lao, Songjoon Baek, Myong-Hee Sung, Lars Grøntved, Michael L. Nickerson, Bethtrice Thompson, Dan Theodorescu, Michael Dean, Gordon H. Hager. Genome-wide chromatin profiling in bladder and prostate 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 4782. doi:10.1158/1538-7445.AM2015-4782
Abstract 4784: Effect of epigenetic drugs on chromatin remodeling complexes disrupted in cancer
Authors: Pérez-Salvia Montserrat , Simó-Riudalbas Laia , Esteller Manel
Published: August 2015

Chromatin remodeling complexes undergo dynamic assembly of chromatin to facilitate the access to DNA packed in nucleosomes during DNA-template cellular processes. They present genetic alterations in multiple of its components in a broad range of hematological and solid malignancies. The SWI/SNF ATP-dependent chromatin remodeling complex subunits BRG1 and ARID1A were reported to act as tumor suppressors and they are mutated in a variety of human cancers including breast cancer, prostate cancer, lung cancer and pancreatic cancer.

In contrast with the irreversible genomic mutations, epigenetic modifications can be reversed by therapeutic treatment. New generation of epigenetic drugs consists on specific inhibitors against chromatin associated proteins which present abnormal expression in tumors. Bromodomain and BET inhibitors block the assembly of a protein complex at a particular gene locus by impairing the interaction of the bromodomain with the acetylated lysine of a histone tail. To date, some BET inhibitors have been tested for the treatment of the rare NUT midline carcinoma disease, acute myeloid leukemia and multiple myeloma.

Our aim was to find new potential biomarkers for a personalized cancer treatment with new generation epigenetic drugs. Thus, we tested whether cell lines with a component of the SWI/SNF complex disrupted (BRG1 or ARID1A) present more sensitivity to bromodomain inhibitors.

Citation Format: Montserrat Pérez-Salvia, Laia Simó-Riudalbas, Manel Esteller. Effect of epigenetic drugs on chromatin remodeling complexes disrupted 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 4784. doi:10.1158/1538-7445.AM2015-4784
Abstract 4787: Cyclin G2, a novel target of the SNF5/BAF47 tumor suppressor gene
Authors: Wei Darmood , Charboneau Aubri , Chung Ho-yoon , Sakellariou-Thompson Donastas , Davies Brian , Simpson Dennis A. , Kuwahara Yasumichi , Kaufmann William K. , Roberts Charles W. M. , Weissman Bernard E.
Published: August 2015

Malignant Rhabdoid Tumor (MRT) is a rare and aggressive pediatric cancer that most frequently appears in the kidney (RTK) and brain (AT/RT). They are generally characterized by the lack of SNF5 (SMARCB1/INI1) expression, a subunit of the SWI/SNF chromatin remodeling complex. Previous studies have shown that reintroducing SNF5 into MRT cell lines reconstitutes the SWI/SNF complex and results in an inhibition of growth through induction of p21WAF1/CIP1, p16INK4A and p57KIP2 expression. However, the mechanisms behind this growth arrest remain incompletely characterized. In this current report, we used a RT-PCR Cell Cycle SuperArray to identify six candidate genes that showed increased expression after SNF5 reexpression but not after expression of a constitutively active RB gene. One of these genes, Cyclin G2 (CCNG2), a member of the non-canonical Cyclin G family, is thought to act as a negative regulator of the cell cycle. Using a panel of RTK and AT/RT cell lines, we confirmed that SNF5 reexpression leads to induction of CCGN2 in additional MRT cell lines. To determine if CCNG2 is a direct target of SNF5, we used chromatin immunoprecipitation analyses (ChIP) to verify that SNF5 binds to the CCNG2 promoter with peak binding at the transcription start site (TSS) after its reexpression in a MRT cell line. Importantly, primary MRT samples display reduced CCNG2 expression when compared to normal brain tissue or other types of pediatric brain cancers. Therefore, CCNG2 represents a new SNF5 target gene whose downregulation may play a role during MRT development.

Citation Format: Darmood Wei, Aubri Charboneau, Ho-yoon Chung, Donastas Sakellariou-Thompson, Brian Davies, Dennis A. Simpson, Yasumichi Kuwahara, William K. Kaufmann, Charles W. M. Roberts, Bernard E. Weissman. Cyclin G2, a novel target of the SNF5/BAF47 tumor suppressor gene. [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 4787. doi:10.1158/1538-7445.AM2015-4787
Abstract 4799: Variation in mutational landscape among small colonic polyps with differential growth rates
Authors: Sievers Chelsie K. , Pickhardt Perry J. , Matkowskyj Kristina , Albrecht Dawn , Zou Luli , Kim David , Lubner Meghan , Reichelderfer Mark , Halberg Richard
Published: August 2015

Background: Benign adenomatous colonic polyps are transformed into cancers through the stepwise accumulation of mutations. However, not all polyps will progress. A significant number remain static in size, regress, or resolve completely. The mechanisms underlying these differential fates are unknown, and currently there are no biological characteristics that can reliably predict which polyps will grow or progress into invasive cancer. A multiancestral origin contributes to tumor heterogeneity and might provide additional growth promoting signals via increased genetic diversity.

Methods: Utilizing a unique cohort of patients with small (<9mm) colorectal polyps in which growth was followed over a mean of 2.3 years by CT colonography, we performed next generation sequencing followed by micro-dissection and quantitative PCR to determine tumor origin.

Results: The mutational landscape of small polyps is highly heterogeneous. Polyps carried 0 - 5 pathogenic mutations, with the most commonly altered gene being APC, which is considered the gatekeeper of the intestine. Interestingly, the percentage of cells harboring mutations in APC within a single polyp ranged from 2.0% to 70.6% with an average of 22.1%. In contrast, the frequency of a common APC single nucleotide polymorphism was 0% for non-carriers (n = 3), 55.9% for heterozygotes (n = 6), and 97.9% for homozygotes (n = 10). In the presence of an APC mutation, the average frequency of a second pathogenic mutation within a sample was 6.2% (n = 10).

Conclusions: These data indicate that early intratumoral heterogeneity arising as a consequence of multiancestral origin affects tumor formation and growth. By determining the relative fitness advantage of heterogeneous polyps, we can begin to better predict which polyps may be more likely to progress into adenocarcinomas and which patients are predisposed to developing advanced disease.

Citation Format: Chelsie K. Sievers, Perry J. Pickhardt, Kristina Matkowskyj, Dawn Albrecht, Luli Zou, David Kim, Meghan Lubner, Mark Reichelderfer, Richard Halberg. Variation in mutational landscape among small colonic polyps with differential growth rates. [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 4799. doi:10.1158/1538-7445.AM2015-4799