bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract SY08-01: AMPK and mTOR in nutrient signaling, metabolism, and autophagy regulation.
Authors: Kim Joungmok , Russell Ryan , Yuan Haxin , Guan Kun-Liang
Published: August 2014

Autophagy is a stress response protecting cells from unfavorable conditions, such as nutrient starvation. The cellular energy sensor AMPK and key growth controller mTOR, both are regulated by nutrient status, play major roles in autophagy regulation. The class III phosphatidylinositol-3 kinase, Vps34, forms multiple complexes and regulates both intracellular vesicle trafficking and autophagy induction. We show that AMPK plays a key role in regulating different Vps34 complexes. AMPK inhibits the non-autophagy Vps34 complex by phosphorylating Vps34 and suppresses overall PI(3)P production and protects cells from starvation. In parallel, AMPK activates the pro-autophagy Vps34 complex by phosphorylating Beclin1 to induce autophagy. Atg14L, an autophagy essential gene present only in the pro-autophagy Vps34 complex, inhibits Vps34 phosphorylation but increases Beclin1 phosphorylation by AMPK. As such, Atg14L dictates the differential regulation (either inhibition or activation) of different Vps34 complexes in response to glucose starvation. In addition, the Vps34 complex is regulated by mTOR, which is inhibited by nutrient starvation. mTOR phosphorylates Beclin to inhibit the autophagy specific Vps34 complex. Our study reveals an intricate molecular regulation of Vps34 complexes by AMPK and mTOR in nutrient stress response and autophagy.

Citation Format: Joungmok Kim, Ryan Russell, Haxin Yuan, Kun-Liang Guan. AMPK and mTOR in nutrient signaling, metabolism, and autophagy regulation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY08-01. doi:10.1158/1538-7445.AM2013-SY08-01
Abstract SY09-01: Inflammatory networks and cancer immune surveillance.
Authors: Vonderheide Robert H.
Published: August 2014

Cancer-associated inflammation is felt to restrain tumor immunity, particularly in pancreatic ductal adenocarcinoma (PDA) for which a massive infiltration of immunosuppressive leukocytes into tumor stroma accompanies cancer growth. Gr-1+ CD11b+ cells, often called myeloid-derived suppressor cells (MDSCs), are a key feature of cancer inflammation in this disease, but the crosstalk between Gr-1+ CD11b+ cells and PDA tumor cells remains incompletely understood. In a genetically engineered mouse model of invasive PDA, Gr-1+ CD11b+ myeloid cells accumulate in primary and metastatic lesions and potently inhibit proliferation of antigen-specific T cells. Among the many cytokines and chemokines produced by PDA tumors, GM-CSF was both necessary and sufficient to drive proliferation and differentiation of c-kit+Gr-1negCD11bneg splenocytes from tumor-bearing mice into functional MDSCs. In vivo, abrogation of GM-CSF accomplished either with neutralizing antibodies or shRNA inhibited the recruitment of Gr-1+ CD11b+ cells to the tumor microenvironment and blocked tumor development. This inhibition of tumor growth was reversed by in vivo CD8 T cell depletion. These findings demonstrate that tumor-derived GM-CSF regulates a major component of myeloid inflammation and T cell immunity in PDA and suggest new targets with therapeutic potential for disrupting cancer inflammation and enhancing immunity. To test if these findings extend to other gastrointestinal cancers, such as esophageal squamous cell carcinoma, a second genetically engineered mouse model of cancer that recapitulates esophageal squamous cell carcinoma was evaluated. In this model, desmoplasia and a prominent myeloid inflammatory response around early and advanced lesions are observed, associated with tumor prand strikingly, tumor progression was significantly halted when this inflammation was eliminated. Thus, there is a common theme among epithelial-derived upper gastrointestinal tumors by which tumor-derived factors drive an inflammatory program that fosters tumorigenesis and tumor invasion, at least in part through the inhibition of anti-tumor immunity.

Citation Format: Robert H. Vonderheide. Inflammatory networks and cancer immune surveillance. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY09-01. doi:10.1158/1538-7445.AM2013-SY09-01
Abstract SY11-03: Circulating tumor DNA as a non-invasive diagnostic and research tool.
Authors: Rosenfeld Nitzan
Published: August 2014

In patients with solid malignancies, cell-free DNA carrying tumor-specific sequences can be found in body fluids such as blood plasma. This circulating tumor DNA (ctDNA) contains representation of the entire cancer genome, broken down into short mutation-carrying fragments, whose relative levels vary in response to treatment and progression. Different approaches to the study of ctDNA can work together to provide a window for noninvasive analysis of the cancer genome and its dynamics. Quantification of ctDNA levels may be useful as a prognostic biomarker and to assess response to treatment. When cancer overcomes treatment and progresses, the fraction of mutant alleles in plasma DNA can reach levels of 10%~20% or more, which makes it possible to study cancer evolution noninvasively by whole-genome or exome analysis of plasma DNA. Sensitivity and resistance to targeted therapies are often associated with known mutations in hotspot loci or genes. In advanced cancer patients, the status of pre-defined mutations can be analyzed noninvasively using circulating plasma DNA as a liquid biopsy. Targeted sequencing of limited regions in circulating DNA can allow identification of de novo mutations, even when these are present as rare fragments. This also facilitates parallel quantification of multiple mutations in plasma, which can indicate the relative systemic burden of different mutations or clones. Routine monitoring for sensitizing and resistance-conferring mutations may provide a framework for rational adaptive cancer therapy, providing real-time information on mutation levels to guide treatment against the most aggressive or treatable clones.

Citation Format: Nitzan Rosenfeld. Circulating tumor DNA as a non-invasive diagnostic and research tool. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY11-03. doi:10.1158/1538-7445.AM2013-SY11-03
Abstract SY12-03: Multi-tumor analysis of TCGA data identifies expression commonalities across tumor types.
Authors: Hoadley Katherine A. , Fan Cheng , Wilkerson Matthew D. , Mose Lisle E. , Jefferys Stuart R. , Auman J. Todd , Parker Joel S. , Hayes D. Neil , Perou Charles M.
Published: August 2014

High dimensional assays, such as DNA microarrays for gene expression, have helped delineate the complexity and diversity of most major cancer types. The Cancer Genome Atlas (TCGA), with its goal to comprehensively genomically characterize over twenty different types of tumors, gives us a wealth of data to start looking at similarities across tumor types. Much of the current data is now generated using Massively Parallel Sequencing (MPS) approaches, which yields tens of millions of “reads” per sample. Illumina Hiseq-based mRNA-sequencing data for twelve tumor types representing over 3,500 samples were analyzed for patterns unique to each tumors, and patterns present across multiple tumor types. Tumor types tested included rectal adenocarcinoma, colon adenocarcinoma, lung squamous cell carcinoma and adenocarcinoma, uterine corpus endometrial carcinoma, ovarian serous cystadenocarcinoma, kidney clear cell, breast ductal and lobular carcinoma, glioblastoma multiforme, bladder carcinomas, acute myeloid leukemia, and head and neck squamous cell carcinoma. Tissue-specific expression was the dominant signature; however, there were similarities across sets of tumors including a common “squamous” pattern that was populated by tumors from three distinct anatomic sites.

Pathway or Module-based analysis also provided methods to comparisons across tissues. Cross tissue analysis of mRNA-seq data provides interesting observations of common tumor biology with potential insights into treatment.

Citation Format: Katherine A. Hoadley, Cheng Fan, Matthew D. Wilkerson, Lisle E. Mose, Stuart R. Jefferys, J. Todd Auman, Joel S. Parker, D. Neil Hayes, Charles M. Perou. Multi-tumor analysis of TCGA data identifies expression commonalities across tumor types. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY12-03. doi:10.1158/1538-7445.AM2013-SY12-03
Abstract SY17-01: BRAF and RAS signaling in melanoma: Basic biology to clinical exploitation.
Authors: Marais Richard M.
Published: August 2014

BRAF is mutated in about 45% of melanomas and its upstream activator NRAS is mutated in a further 30% of cases. We have developed mouse models of melanoma driven by these oncogenes. Expression of oncogenic BRAF in mature mouse melanocytes induces melanoma, whereas its expression in embryonic mouse melanocytes is embryonically lethal. In contrast, expression of oncogenic NRAS in mature melanocytes does not induce melanoma, but its expression in embryonic mouse melanocytes induces melanomas of the brain that bear the cardinal features of leptomeningeal melanocytosis in children. We are using these models and human xenograft of patient melanomas grown in immuno-compromised mice to study mechanisms of resistance to BRAF pathway inhibitors. We find that up-regulation of EGF receptor pathway signaling can mediate resistance to BRAF inhibitors by signaling through the PI3 kinase AKT pathway. A combination of a BRAF and EGF receptor inhibitor, or a broad specificity SRC inhibitor can overcome resistance, potentially offering second-line treatments to relapsed patients.

Citation Format: Richard M. Marais. BRAF and RAS signaling in melanoma: Basic biology to clinical exploitation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY17-01. doi:10.1158/1538-7445.AM2013-SY17-01
Abstract SY19-01: Chemical genetic investigations of protein and lipid kinase signaling.
Authors: Shokat Kevan M.
Published: August 2014

Kinases are highly regulated enzymes with diverse mechanisms controlling their catalytic output. Inhibitor discovery efforts for kinases have produced ATP-competitive compounds, allosteric regulators, irreversible binders, and highly specific inhibitors. These distinct classes of small molecules have revealed many novel aspects about kinase-mediated signaling and some have progressed from simple tool compounds into clinically validated therapeutics. My presentation will explore several small-molecule inhibitors for kinases highlighting elaborate mechanisms by which kinase function is modulated. A complete surprise of targeted kinase drug discovery has been discovery of ATP-competitive inhibitors that behave as agonists, rather than antagonists, of their direct kinase target. These studies hint at a connection between ATP binding site occupancy and networks of communication that are independent of kinase catalysis. Indeed, kinase inhibitors have been found that induce changes in protein localization, protein-protein interactions, and even enhancement of catalytic activity of the target kinase. The relevance of these findings to the therapeutic efficacy of kinase inhibitors and to the future identification of new classes of drug targets will be discussed.

Citation Format: Kevan M. Shokat. Chemical genetic investigations of protein and lipid kinase signaling. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY19-01. doi:10.1158/1538-7445.AM2013-SY19-01
Abstract SY25-03: Haploinsufficiency in cancer: When half simply isn't good enough.
Authors: Solimini Nicole , Pavlova Natalya N. , Mermel Craig , Xu Qikai , Burrows Anna E. , Luo Ji , li Mamie , Beroukhim Rameen , Meyerson Matthew , Elledge Stephen J.
Published: August 2014

Breast cancer is a collection of diseases with distinct clinical behaviors and underlying genetic causes. We have searched genetically for genes that have cancer relevant phenotypes including genes that alter cellular proliferation, cellular transformation, cell survival, cellular senescence, and genes that are essential for the proliferation of cancer cells. We have approached this by the generation of libraries of shRNAs for loss of function experiments and libraries of ORFs for gain of function experiments. We will discuss these technologies and how they can be applied to the functional dissection of genes important for breast cancer. Using these technologies, we found evidence for wide-spread haploinsufficiency in cancer due to recurring hemizygous deletions. We propose a tumor suppressor hypothesis called the Cancer Gene Island model whereby, hemizygous deletions selledge regions containing a high density of negative growth regulators (STOP genes) and a low density of essential genes (GO genes). The deletions provide a proliferative advantage to cells through cumulative haploinsufficiency.

Citation Format: Nicole Solimini, Natalya N. Pavlova, Craig Mermel, Qikai Xu, Anna E. Burrows, Ji Luo, Mamie li, Rameen Beroukhim, Matthew Meyerson, Stephen J. Elledge. Haploinsufficiency in cancer: When half simply isn't good enough. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY25-03. doi:10.1158/1538-7445.AM2013-SY25-03
Abstract SY26-03: Innate immune sensing of tumors via the host STING pathway as a bridge to a spontaneous adaptive T cell response.
Authors: Gajewski Thomas Frank
Published: August 2014

Recent evidence has suggested that clinical responses to immunotherapeutic interventions occur predominantly in patients having a pre-existing anti-tumor immune response and a T cell-inflamed tumor microenvironment. It has therefore become critical to understand the mechanism by which a subset of patients generates this phenotype, which should instruct on how to deliberately facilitate generation of appropriate tumor-based inflammation and thereby expand the fraction of patients deriving clinical benefit from immunotherapy. However, the mechanisms by which innate immune responses become activated in response to cancer, and how they can bridge to spontaneous T cell priming against tumor antigens, have remained poorly defined. We recently reported that host type I IFN signals are required for a spontaneous T cell response against tumors. In the current work, we have mapped the sensing mechanism to one involving STING/TBK1/IRF3 which drives IFN-β transcription by host APCs. Spontaneous T cell responses against tumors were eliminated in mice deficient in STING or IRF3, and rejection of immunogenic tumors was abrogated. The tumor-derived ligand that triggers this pathway appears to be DNA. Using the DNA-specific dye DRAQ5 or EDU labeling and single cell ImageStream analysis, we demonstrated that in vivo transfer of tumor DNA to host APCs occurs in vivo; this was confirmed using PCR specific for tumor DNA. APC uptake of tumor DNA was associated with IFN-β mRNA induction and nuclear translocation of IRF-3, which were dependent upon host STING. We hypothesized that autophagosome provide a mechanism for delivery of protected tumor DNA into APCs. Inhibition of tumor cell autophagy using the chemical 3-MA, or shRNA silencing of Becn or ATG5, led to a marked reduction in the induction of host IFN-β production and spontaneous T cell responses in vivo. Our data suggest that tumor cell autophagy is required for optimal T cell priming against tumor antigens in vivo, that occurs through DNA-mediated activation of host IFN-β production through a STING-dependent pathway. Analysis of these biologic steps in human melanoma metastases is ongoing. Strategies to mimic activation of the STING pathway should be considered as a mechanism to create an inflamed tumor microenvironment with the hope of expanding the clinical efficacy of T cell-based immunotherapies.

Citation Format: Thomas Frank Gajewski. Innate immune sensing of tumors via the host STING pathway as a bridge to a spontaneous adaptive T cell response. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr SY26-03. doi:10.1158/1538-7445.AM2013-SY26-03
Abstract A01: Evaluation of H3K27me3 and EZH2 in pediatric glial and glioneuronal tumors shows decreased H3K27me3 in H3F3A K27M mutant GBM
Authors: Venneti Sriram , Garimella Mihir , Sullivan Lisa , Martinez Daniel , Huse Jason , Heguy Adriana , Santi Mariarita , Thompson Craig , Thompson Craig , Alexander Judkins
Published: August 2014

Histone H3F3A mutations are seen in ~30% of pediatric glioblastoma (GBMs) and involve either the lysine residue at position 27 (K27M) or glycine at position 34 (G34R/V). Sixteen genes encode histone H3, each variant differing in only a few amino acids. Therefore how mutations in a single H3 gene contribute to carcinogenesis is unknown. H3F3A K27M mutations are predicted to alter methylation of H3K27. H3K27me3 is a repressive mark critical to stem cell maintenance and is mediated by EZH2, a member of the polycomb-group (PcG) family. We evaluated H3K27me3 and EZH2 expression using immunohistochemistry in 76 pediatric brain tumors. H3K27me3 was lowered/absent in tumor cells but preserved in endothelial cells and infiltrating lymphocytes in 6/20 GBMs. H3K27me3 showed strong immunoreactivity in all other tumor subtypes. Sequencing of GBMs showed H3F3A K27M mutations in all 6 cases with lowered/absent H3K27me3. Further, these 6 GBMs with lowered/absent H3K27me3 in tumor cells showed a worse prognosis than GBMs with preserved H3K27me3. EZH2 expression was high in GBMs, but absent/focal in other tumors. However, no significant differences in EZH2 expression were observed between H3F3A K27M mutant and wild type GBMs, suggesting that EZH2 mediated trimethylation of H3K27 is inhibited in GBM harboring K27M mutations. Our results indicate that H3F3A K27M mutant GBMs show decreased H3K27me3 that may be of diagnostic, prognostic and biological relevance.

Citation Format: Sriram Venneti, Mihir Garimella, Lisa Sullivan, Daniel Martinez, Jason Huse, Adriana Heguy, Mariarita Santi, Craig Thompson, Craig Thompson, Judkins Alexander. Evaluation of H3K27me3 and EZH2 in pediatric glial and glioneuronal tumors shows decreased H3K27me3 in H3F3A K27M mutant GBM. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A01.
Abstract A23: Repression of Id4 (inhibitor of DNA binding 4) by the polycomb group protein EZH2 in prostate cancer
Authors: Chinaranagari Swathi , Sharma Pankaj , Chaudhary Jaideep
Published: August 2014

Introduction: Inhibitor of DNA binding/differentiation protein 4 (Id4) is dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. Id4 shares the homology of HLH domain with other Ids and lack the basic DNA binding region. Unlike Id1, Id2 and Id3, Id4 acts as a tumor suppressor in prostate cancer by attenuating cell proliferation and promote apoptosis. We have previously shown that decreased Id4 expression in prostate cancer is due to its promoter hypermethylation. In this study we investigated the molecular mechanism by which Id4 promoter is hypermethylated. Here, we demonstrate that Id4 promoter methylation is initiated by EZH2 dependent tri-methylation of histone 3 at lysine 27 (H3K27me3).

Experimental procedures: Id4 protein expression was analyzed on human prostate adenocarcinoma samples by Immunohistochemistry and prostate cancer cell lines (LNCaP, C81 and DU145) by Western blot. EZH2 was silenced in DU145 cells and confirmed through western blot and Immunocytochemistry. Chromatin Immunoprecipitation (ChIP) was performed with IgG, RNA polymerase, EZH2 and H3K27Me3 antibodies in both prostate cancer cell lines and prostate cancer tissue samples.

Results: IHC demonstrated decreased Id4 protein expression in human prostate tissue samples whereas higher nuclear Id4 expression was found in normal prostate tissues. Re-expression of Id4 in EZH2 silenced Du145 demonstrated that Id4 is regulated in an EZH2 dependent manner. ChIP data on prostate cancer samples and cell lines suggested EZH2 occupancy and H3K27Me3 marks in the Id4 promoter region in cell lines and prostate cancer tissue specimens.

Conclusions: Id4 appears as a potential tumor suppressor gene that is epigenetically silenced during prostate cancer development. H3K27Me3 marks and EZH2 occupancy suggest a PRC2 dependent mechanism in Id4 promoter silencing in prostate cancer.

Acknowledgement: The research was supported by NIH/NCI-RO1CA128914 and in part by NIH/NCRR/RCMI G12RR03062

Citation Format: Swathi Chinaranagari, Pankaj Sharma, Jaideep Chaudhary. Repression of Id4 (inhibitor of DNA binding 4) by the polycomb group protein EZH2 in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A23.
Abstract A25: Yin Yang 1 promotes AKT activation through direct protein binding
Authors: Zhang Qiang , Wan Meimei , Horita David A. , Miller Lance D. , Kridel Steven J. , Kute Timothy E. , Kulik George , Sui Guangchao
Published: August 2014

As a polycomb group protein, Yin Yang 1 (YY1) plays an important role in cancer epigenetics. YY1 regulates both gene expression and protein modifications, including histone acetylation and methylation. We recently reported a proliferative role of YY1 and its cytoplasmic presence in breast cancer. In the current study, we demonstrated that YY1 promotes AKT phosphorylation at S473, a marker of AKT activation. The immunostaining signal of YY1 and AKT(S473) phosphorylation positively correlated and colocalized in breast cancer tissues and cells. The oncogene protein binding (OPB) domain (G201-S226) of YY1 bound to AKT and was necessary for YY1 in promoting AKT phosphorylation. This novel function of YY1 is independent of its transcriptional activity because YY1 promoted AKT phosphorylation in an in vitro setting using purified proteins and YY1 mutants deficient in binding to DNA retained this activity. Our further studies revealed that YY1 interacted with the PH domain of AKT where PIP3 binds and its role in promoting AKT phosphorylation was abolished when we depleted mSIN1, a specific component of mTORC2 that mediates AKT(S473) phosphorylation. Thus, we discovered a PI3K-independent mechanism of mTORC2-mediated AKT activation stimulated by YY1 binding to its PH domain, which supports the proliferative role of YY1 in mammary oncogenesis.

Citation Format: Qiang Zhang, Meimei Wan, David A. Horita, Lance D. Miller, Steven J. Kridel, Timothy E. Kute, George Kulik, Guangchao Sui. Yin Yang 1 promotes AKT activation through direct protein binding. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A25.
Abstract A27: Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells
Authors: Marcinkiewicz Katarzyna M. , Gudas Lorraine J.
Published: August 2014

It is estimated that 41,380 new patients will be diagnosed with cancer of the oral cavity and the pharynx and 7,890 will die of those diseases in 2013 in the United States alone. More than 90% of all oral cavity cancers are of the squamous cell carcinoma (SCC) type. We identified numerous homeobox genes as differentially expressed between non tumorigenic oral keratinocytes OKF6-TERT1R and tumorigenic SCC9 cells. Data from Oncomine, a cancer microarray database, suggest that homeobox (HOX) genes are also dysregulated in oral SCC patients. The activities of Polycomb repressive complexes (PRC), which cause epigenetic modifications, and retinoic acid (RA) signaling can control HOX gene transcription in stem cells. HOXB7, HOXC10, HOXC13, and HOXD8 transcripts are higher in SCC9 than in OKF6-TERT1R cells; using ChIP (chromatin immunoprecipitation) we detected the PRC2 protein SUZ12 and the epigenetic H3K27me3 mark on histone H3 at these genes in OKF6-TERT1R, but not in SCC9 cells. In contrast, IRX1, IRX4, SIX2, and TSHZ3 transcripts are lower in SCC9 than in OKF6-TERT1R cells. We detected SUZ12 and the H3K27me3 mark at these genes in SCC9, but not in OKF6-TERT1R cells. SUZ12 and H3K27me3 levels were not altered by RA at these HOX genes in SCC9 and OKF6-TERT1R cells. We conclude that altered activity of PRC2 is associated with dysregulation of homeobox gene expression in human SCC cells, and that this dysregulation potentially plays a role in the neoplastic transformation of oral keratinocytes. An exciting future direction would be the elucidation of the molecular mechanisms driving aberrant homeobox gene expression in transformed oral keratinocytes by assessing additional epigenetic histone and DNA modifications present at these genes. Such experiments are currently underway.

Citation Format: Katarzyna M. Marcinkiewicz, Lorraine J. Gudas. Altered epigenetic regulation of homeobox genes in human oral squamous cell carcinoma cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A27.
Abstract A47: Investigating Zbtb protein recognition of epigenetically modified DNA
Authors: Terooatea Tommy W. , Pandya Maya J. , Chugg Alan C. , Buck-Koehntop Bethany A.
Published: August 2014

Methylation of CpG dinucleotides is essential for genomic stability, control of gene expression and the regulation of chromatin structure in normal eukaryotic cells. Aberrant alterations in these genomic methylation patterns have been linked with many diseases, including cancer. This correlation has prompted the need to better understand the regulatory mechanisms of DNA methylation in gene transcription. The Zbtb family, consisting of Zbtb33, Zbtb4 and Zbtb38, is a set of methyl-CpG binding proteins (MBPs), which recognize both methylated as well as sequence-specific non-methylated DNA sites through conserved Cys2His2 zinc fingers. DNA recognition subsequently recruits chromatin remodeling machinery to the target site resulting in chromatin compaction and gene silencing. While sufficient evidence suggests that the Zbtb proteins play a role in cancer, an extensive analysis of the gene targets and signaling pathways regulated by each protein has yet to be investigated. Here we utilize an interdisciplinary approach combining cellular biology, molecular biology and structural biology to determine the epigenetic targets regulated by the Zbtb MBPs and to identify the protein region(s) necessary for high-affinity DNA recognition. Utilizing western blot analysis, we have determined that the three Zbtb proteins exhibit differential endogenous expression levels in a variety of cancer cell lines. Further, ChIP qRT-PCR analysis demonstrated that the occupancy of Zbtb33 at various methylated and sequence-specific genes was highly variable between the different cancer cell lines. Combined, these results suggest that the Zbtb proteins likely mediate differential signaling pathways in different cancers, possibly by targeting specific regulatory gene elements. Mapping of the global gene occupation for Zbtb33 in various cancer cell lines is in progress. Additionally, we have designed multiple protein constructs around the conserved Cys2His2 zinc finger DNA binding region as well as additional zinc fingers located in the C-terminal regions of Zbtb4 and Zbtb38, as the functions of the latter have not yet been identified. The constructs have been analyzed by solution NMR and EMSA to determine whether they are amenable for further structural analysis and to characterize their DNA binding interactions. This multidisciplinary approach provides the means for advancing our understanding of the molecular basis by which these proteins interpret and translate epigenetic signals in the cancerous state.

Citation Format: Tommy W. Terooatea, Maya J. Pandya, Alan C. Chugg, Bethany A. Buck-Koehntop. Investigating Zbtb protein recognition of epigenetically modified DNA. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A47.
Abstract B03: Loss of the Polycomb mark at bivalent promoters leads to activation of intestinal stem cell genes in colorectal cancer
Authors: Hahn Maria A. , Li Arthur X. , Wu Xiwei , Rosenberg Daniel W. , Pfeifer Gerd P.
Published: August 2014

During tumor formation, transcription of many genes becomes deregulated. In order to elucidate epigenetic mechanisms responsible for this phenomenon, we analyzed genome-wide patterns of several histone marks, 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) in human stage II colorectal cancer and matching normal tissue samples. By using liquid chromatography—mass spectrometry (LC-MS/MS), we determined that colon tumors have strongly reduced global levels of 5hmC. However, Tet-assisted bisulfite sequencing showed that site-specific loss of 5hmC in tumors is not associated with localized aberrant CpG island hypermethylation. We observed that DNA hypermethylation is generally targeted to bivalent promoters, which loose H3K27me3 and the associated genes are repressed effectively. Importantly, loss of H3K27me3 at bivalent promoters is not always coupled with gain of 5mC. Instead, H3K27me3 loss is also frequently associated with activation of intestinal stem cell markers and stem cell regulators including LGR5, SOX9, CDX1, CDX2, KLF5, and SALL4 as well as activation of many genes linked to colorectal cancer progression such as MET, CLDN1, PTGS2 (COX-2) and CCND1 (cyclin D1). Activation of these genes correlates with accumulation of the active chromatin mark H3K4me3 at their promoters. Our study demonstrates two distinct features of H3K27me3 loss in cancer. Loss of H3K27me3 at bivalent genes can be accompanied by either gain of 5mC and strong repression or by persistence or gain of H3K4me3 and gene activation. We propose that instability of H3K27me3 coupled with selection for tumor-promoting events plays a major role in colorectal cancer progression most notably by causing activation of cancer driving genes including factors involved in stem cell maintenance.

Citation Format: Maria A. Hahn, Arthur X. Li, Xiwei Wu, Daniel W. Rosenberg, Gerd P. Pfeifer. Loss of the Polycomb mark at bivalent promoters leads to activation of intestinal stem cell genes in colorectal cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B03.
Abstract B18: Western diet-fed mice with inherited cancer predisposition reveal early epigenetic changes and protein markers for colon oncogenesis
Authors: Bebek Denis Dermadi , Valo Satu , Pussila Marjaana , Reyhani Nima , Sarantaus Laura , Nystrom Minna
Published: August 2014

Colorectal cancer (CRC) is the second most common cause of cancer-related deaths in the Western world. Interactions between genetic and environmental factors, such as diet, are suggested to play a critical role in its etiology. However, the mechanisms that mediate the effects of diet on oncogenesis are largely unknown. We conducted a long-term feeding experiment in the mouse to address epigenetic changes arising in normal colonic mucosa and their functional consequences as putative cancer-predisposing events available for early detection.

The quantitative analysis of proteomes and the expression of 94 growth-regulatory genes previously linked to human CRC and aberrant hypermethylation were studied at two time points (5 weeks and 12 months of age) in the heterozygote Mlh1+/- mice analogous to human Lynch syndrome, and wild type Mlh1+/+ littermates, fed with Western-style (WD) or AIN-93G control diet. In the proteome study the most significant expression changes were found in the Mlh1+/- WD mice compared to mice fed with AIN-93G control diet and the 19 identified proteins mainly point to abnormities in energy metabolism and protein unfolding. We further found that in mice carrying Mlh1 mutation and/or fed with WD, histologically normal proximal colonic mucosa exhibited a significant expression decrease in tumor suppressor genes, Dkk1, Slc5a8, Hoxd1, and Socs1. Especially in Dkk1, a secreted antagonist of the Wnt/β-catenin pathway the reduced expression was associated with its promoter hypermethylation. The fact that changes in Dkk1 seem to predispose to neoplasia in the proximal colon and that 5 out of 6 mice with neoplastic colonic lesions were fed with WD suggests that the found expression changes are early markers for oncogenesis.

Citation Format: Denis Dermadi Bebek, Satu Valo, Marjaana Pussila, Nima Reyhani, Laura Sarantaus, Minna Nystrom. Western diet-fed mice with inherited cancer predisposition reveal early epigenetic changes and protein markers for colon oncogenesis. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B18.
Abstract B41: Methylome profiling identifies unique patterns of DNA methylation in endometrial cancers
Authors: Trimarchi Michael P. , Yan Pearlly S. , Huang Kun , Groden Joanna , Bundschuh Ralf , Goodfellow Paul J.
Published: August 2014

Introduction: DNA methylation is a stable epigenetic mark often perturbed during carcinogenesis. Growing evidence demonstrates a role for DNA methylation both as a regulator of gene expression and as a potential biomarker. Widespread accumulation of methylation in regulatory elements in certain cancers (termed the CpG island methylator phenotype) may play a role in carcinogenesis. Few studies, however, have investigated genome-wide methylation in a clinical cohort, as the methodology for analyzing these large datasets remains poorly developed. Endometrial cancer is the most common female gynecological cancer in the US, with sporadic endometrioid as the predominant subtype.

Methods: This study profiles the methylomes of 76 sporadic endometrioid endometrial primary tumors and 12 normal endometrium using sequenced methylated fragments captured by the MBD protein (MethylCap-seq).

Results: Analysis of bulk methylation in promoter CpG islands (CGI) identified a subset of tumors with a putative methylator phenotype. We are currently developing a gene signature to more easily detect endometrial tumors with a methylator phenotype, perform cross-platform analyses, and further characterize clinicopathological variables associated with the phenotype. We have also developed a pipeline for quality analysis and show that exclusion of poor quality samples increases statistical power to call differential methylation and reduces analysis artifacts.

Conclusion: The results of our study highlight MethylCap-seq as a useful tool for profiling methylation patterns in large clinical datasets.

Citation Format: Michael P. Trimarchi, Pearlly S. Yan, Kun Huang, Joanna Groden, Ralf Bundschuh, Paul J. Goodfellow. Methylome profiling identifies unique patterns of DNA methylation in endometrial cancers. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B41.
Abstract B48: Targeting SETD2 knockout cells: A synthetic lethal approach to treating ccRCC
Authors: Fahey Catherine C. , Hacker Kathryn E. , Jin Jian , Davis Ian J. , Rathmell W. Kimryn
Published: August 2014

Background: Kidney cancer is estimated to affect 65,150 newly diagnosed individuals in 2013. Approximately 70% of these diagnoses will be clear cell renal cell carcinoma (ccRCC). Many targeted therapies for ccRCC are currently being pursued, as ccRCC is resistant to radiation and often does not respond to chemotherapy. The most commonly mutated tumor suppressor gene in ccRCC is VHL, located on chromosome 3. The loss of chromosome 3p is a high frequency event in ccRCC, which often also encompasses the genes PBRM1, BAP1, and SETD2. Mutations in one or more of these genes represent the next most common common genetic alterations in ccRCC. In our preliminary data, we have observed that mutations in SETD2, in particular, area associated with global deficits in chromatin packaging and organization.

Methods: We are pursuing the opportunity to exploit SETD2 loss as a source of cellular fragility by performing a small molecule screen on SETD2 wild type and SETD2 knockout cell lines. We will use a screening set of epigenetically targeted compounds to perform a high throughput screen with cell viability as the endpoint, as well as use a selected few compounds to compare effect on wild type and knockout cells in a more in depth manner.

Results: Preliminarily, we have tested SETD2 knock-down lines to confirm that we can effectively preform all aspects of this screen. In our current studies, we are repeating testing with the selected few compounds and beginning the high throughput screen with a SETD2 knockout cell line.

Conclusion: Mutations in chromatin modifying genes in ccRCC present an opportunity for synthetic therapeutic approaches to induce reduced cell viability, as well as to identify unique meaningful interactions between chromatin modifier genes.

Citation Format: Catherine C. Fahey, Kathryn E. Hacker, Jian Jin, Ian J. Davis, W. Kimryn Rathmell. Targeting SETD2 knockout cells: A synthetic lethal approach to treating ccRCC. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B48.
Abstract B50: Selectively targeting prostate cancer with antiandrogen-equipped histone deacetylase inhibitors
Authors: Gryder Berkley E. , Akbashev Michelle J. , Rood Michael K. , Dillard Paulette , Khan Shafiq , Oyelere Adegboyega K.
Published: August 2014

Diverse cellular processes relevant to cancer progression are regulated by the acetylation status of proteins, such as chromatin remodeling via histone proteins, controlled by opposing histone deacetylase (HDAC) and histone acetyltransferase (HAT) enzymes. Histone deacetylase inhibitors (HDACi) show great promise in preclinical cancer models, but clinical trials for solid tumors have failed to improve patient survival. This is due in part to an inability of HDACi to effectively accumulate in cancerous cells. To address this problem we designed HDACi with secondary pharmacophores to facilitate selective enrichment in malignant cells. We present the first example of HDACi compounds targeted to prostate tumors by equipping them (via “click” chemistry) with the secondary ability to bind the androgen receptor (AR) with non-steroidal antiandrogen moieties. Leads among these new duel-acting molecules bind to the AR and halt AR transcriptional activity at lower concentrations than clinical antiandrogens. They inhibit key isoforms of HDAC with improved potency over clinically approved HDACi, SAHA. Fluorescent microscopy reveals varying degrees of AR nuclear localization in response to these compounds. These biological properties translate into anticancer activity against hormone independent (AR-) DU145 prostate cancer, and even more so against hormone dependent (AR+) LNCaP, while having greatly reduced toxicity in non-cancerous cells. These compounds illustrate a “smart bomb” approach towards achieving selective, safe, and potent cancer therapies.

Citation Format: Berkley E. Gryder, Michelle J. Akbashev, Michael K. Rood, Paulette Dillard, Shafiq Khan, Adegboyega K. Oyelere. Selectively targeting prostate cancer with antiandrogen-equipped histone deacetylase inhibitors. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B50.
Abstract B53: Selective BET bromodomains epigenetic signaling inhibition as a therapeutic strategy in primary bone tumors
Authors: Lamoureux Francois , Baud'huin Marc , Rodriguez Lidia , Jacques Camille , Berreur Martine , Rédini Francoise , Bradner James E. , Heymann Dominique , Ory Benjamin
Published: August 2014

Osteosarcoma is the most frequent primary bone tumor that develops mainly in young adults. Despite recent improvements of the therapeutic advances, the survival rate at 5 years is below 30% for patients with poor response to treatment or with metastasis.

The histones modifications are of critical importance in maintaining of the transcription program of both normal and tumor cells. The bromodomain and extra-terminal domain (BET) protein family is an important class of “histone reading protein” capable to recognize the N-acetylation of lysine residues on histone tails. BET bromain proteins have recently been described as regulators of MYC expression in various tumors. In this study, we present the therapeutic opportunity to pharmacologically target the BET bromodomain family in primary bone tumors.

The consequence of this pharmacologic inhibition of bromodomains is a wide gene expression alteration, but it is believed to selectively target malignant cells by disrupting transcription at intense activity, most notably MYC and Runx2 in our model. Considering MYC and Runx2 are of particular importance for the oncogenic potential of primary bone tumors, a therapeutic strategy targeting those networks might be extremely relevant and potent.

In osteosarcoma tumor cell lines, BET inhibitor reduced cell growth in a dose-dependent manner and induced apoptosis with an increase of sub-G1 fraction and PARP cleavage. These biological events were accompanied by decreased expression and activity of both MYC and Runx-2, and by an expression modulation of their target genes. Moreover, BET inhibitor affects bone remodeling process by disrupting both osteoclast and osteoblast differenciation. In vivo, BET inhibitor (IP; 50mg/kg) significantly inhibits tumor growth by 70% and prolongs survival in both POS-1 syngenic and HOS xenograft models compared to control. Additionnally, these results were accompanied by a decrease of associated bone lesions.

These findings demonstrate that dual pharmacologic inhibition of MYC and Runx-2 is achievable through targeting BET bromodomains to treat osteosarcoma.

Note: This abstract was not presented at the conference.

Citation Format: Francois Lamoureux, Marc Baud'huin, Lidia Rodriguez, Camille Jacques, Martine Berreur, Francoise Rédini, James E. Bradner, Dominique Heymann, Benjamin Ory. Selective BET bromodomains epigenetic signaling inhibition as a therapeutic strategy in primary bone tumors. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B53.
Abstract B58: Implementation of a synthetic reagent to mimic chromatin provides a chromatin immunoprecipitation quantitative control
Authors: Rosenfeld John M. , Li Zirong , Taganov Konstantin , Cooke Tracy , Thyagarajan Bhaskar , Solache Alejandra
Published: August 2014

Chromatin immunoprecipitation (ChIP) is an analytical method used to investigate interaction of proteins with specific genomic DNA regions in vivo to provide a better understanding of mechanisms of gene regulation, DNA replication and DNA repair. However, variations in the efficiency of immunoprecipitation, background signals from carryover in immunoprecipitation, variability between chromatin preps, and loss of material during immunoprecipitation and purification of the ChIP DNA are sources of variability that restrict the use of ChIP as a quantitative tool. To address these limitations, we have developed a simple spike-in control that enables labs to generate results that are more consistent and more quantitative. The approach we have developed uses a synthetic peptide-DNA reagent that is spiked into chromatin immunoprecipitation reactions. Because this reagent mimics the behavior of cross-linked chromatin and can be quantitatively recovered by immunoprecipitation, it can be readily incorporated in to virtually any ChIP protocol. To utilize this internal control, a fixed amount of the synthetic peptide-DNA complex is spiked into the chromatin and co-ChIPed simultaneously with the antibody of interest. The recovered synthetic DNA is then used to normalize the results obtained with the antibody of interest. This spike-in control can be designed using a peptide specific to the ChIP antibody of interest and recovered using a single antibody. Alternatively, a universal spike-in control can be designed using a peptide that is recognized by an epitope tag antibody. This universal spike-in control and epitope tagged antibody are used along with the antibody of interest in a standard ChIP reaction. Our data show that the inclusion of our internal control significantly reduces variability between ChIP assays, thus increasing the accuracy of the ChIP data. Because of the universal feature, this method can also be used to compare ChIP data between different antibodies or to normalize data variations that often result between users and multiple experiments. Furthermore, experiments are underway to confirm that such a spike in control can be incorporated upstream of chromatin preparation, and is capable of being adapted for NGS library construction to aid in normalization between chromatin NGS experiments.

Citation Format: John M. Rosenfeld, Zirong Li, Konstantin Taganov, Tracy Cooke, Bhaskar Thyagarajan, Alejandra Solache. Implementation of a synthetic reagent to mimic chromatin provides a chromatin immunoprecipitation quantitative control. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B58.