bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract SY21-04: Wnt signaling, stem cells, and cancer.
Authors: Nusse Roel
Published: August 2014

We are interested in the regulation of growth, development and integrity of animal tissues. We study multiple different organs, trying to identify common principles, and extend these investigations to cancer and injury repair. In most organs, different cell types are generated by stem cells; cells that also make copies of themselves and thereby maintain the tissue. A right balance between the number of stem and differentiated cells is essential for the proper function of the organs. Locally acting signals are important to maintain this balance in a spatially organized manner and these signals are key to understanding the regulation of growth. A common theme linking the work together is the Wnt family of signals. Work from many laboratories has shown that Wnt proteins are essential for the control over stem cells. How this is achieved is far from clear and is the subject of studies in the lab, both in vivo and in cell culture. In vivo, a particular question we address is how physiological changes, such as those occurring during hormonal stimuli, injury or programmed tissue degeneration have an impact on the self-renewal signals and on stem cell biology.

Current research includes: 1) Identifying and tracing Wnt-responsive stem cells in tissues; 2) mapping cis-acting transcriptional control elements (enhancers) that control Wnt gene expression in normal and injured tissues; 3) the use of active Wnt proteins to maintain stem cells (including embryonic stem cells) in cell culture; 4) presenting Wnt protein in a vectorial manner to stem cells to direct asymmetric cell division.

Citation Format: Roel Nusse. Wnt signaling, stem cells, and cancer. [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 SY21-04. doi:10.1158/1538-7445.AM2013-SY21-04
Abstract SY27-04: Engineering improved cancer vaccines.
Authors: Dranoff Glenn
Published: August 2014

Efficacious cancer immunotherapies will likely require combinations of strategies that enhance tumor antigen presentation and antagonize negative immune regulatory circuits. We demonstrated that vaccination with irradiated, autologous melanoma cells engineered to secrete GM-CSF followed by antibody blockade of CTLA-4 accomplishes clinically significant tumor destruction with minimal toxicity in a majority of stage IV metastatic melanoma and some advanced ovarian carcinoma patients. The extent of tumor necrosis in post-treatment biopsies was linearly related to the natural logarithm of the ratio of infiltrating CD8+ effector T cells to FoxP3+ Tregs, suggesting that further Treg inhibition might increase the frequency of clinical responses. Through an analysis of cytokine deficient mice, we delineated a critical role for GM-CSF in Treg homeostasis. GM-CSF is required for the expression of the phosphatidylserine binding protein MFG-E8 in antigen presenting cells, whereas the uptake of apoptotic cells by phagocyte-derived MFG-E8 maintains peripheral Treg activity. The pharmacologic inhibition of MFG-E8 function through genetic or engineering-based approaches blocks Treg induction, which intensifies vaccine-induced responses, leading to the regression of established tumors in mice. The clinical translation of these therapeutic strategies to Phase I testing in humans is underway.

The detailed analysis of patients achieving sustained clinical benefits from irradiated, autologous GM-CSF secreting tumor cell vaccines and CTLA-4 antibody blockade also affords a rich opportunity to identify antigens associated with immune-mediated tumor destruction and to delineate mechanisms of therapeutic immunity. We elucidated several of the molecular pathways that underlie these anti-tumor effects, including the NKG2D system and multiple secreted/or cell surface proteins that contribute to tumor promoting inflammation. The therapy-induced antibodies manifest functional activity in vitro, antagonizing tumor cell survival, invasive potential, and angiogenesis. These findings support a key role for humoral immunity in tumor destruction, and highlight interest in more detailed characterization of the anti-tumor B cell repertoire in vaccinated patients.

Citation Format: Glenn Dranoff. Engineering improved cancer vaccines. [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 SY27-04. doi:10.1158/1538-7445.AM2013-SY27-04
Abstract SY29-03: Following single mRNAs from birth to death in living cells and tissues.
Authors: Singer Robert
Published: August 2014

Imaging has been an essential tool to analyze the dynamic properties of RNA. New technologies in optical microscopy and tagging methods have allowed us to detect and track individual mRNA molecules in single living cells, yielding insights that could not have been obtained through any ensemble measurement: for instance in measuring the time for transcription elongation or nuclear pore transport. We have been dedicated to developing and implementing these technologies to further the understanding of dynamics of mRNA regulation in cells. We have utilized various microscopic approaches to analyze the components of real-time transcription of endogenous genes in living tissues of a transgenic mouse where the mRNAs are labeled by a fluorescent RNA binding protein. These approaches employ a plethora of imaging techniques, ranging from confocal and multiphoton microscopy, long-term cell imaging, high-speed real-time widefield microscopy, single molecule tracking, super-registration microscopy and fluorescence fluctuation analysis. The kinetics of the key elements of RNA metabolism: initiation, elongation, termination, as well as nuclear pore export, cytoplasmic trafficking, localization, translation and decay are now tractable at the single molecule level in vivo. By using mathematical modeling to extract the quantitative parameters of these kinetics, we can describe these processes precisely in living cells, leading to an understanding of the regulatory events governing gene expression-fundamental to the mechanism of disease.

Supported by NIH GMS Grants to RH Singer.

Citation Format: Robert Singer. Following single mRNAs from birth to death in living cells and tissues. [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 SY29-03. doi:10.1158/1538-7445.AM2013-SY29-03
Abstract SY33-02: Understanding the dynamics of cancer: From developmental pathways to in vivo imaging.
Authors: Reya Tannishtha
Published: August 2014

Our research focuses on the signals that control hematopoietic stem cell self-renewal and differentiation, how these signals are utilized during regeneration and how their dysregulation may lead to cancer. Using a multitude of genetic models, we have shown that classic developmental signaling pathways such as Wnt, Hedgehog and Notch play key roles in stem cell growth and regeneration and are dysregulated during leukemia development. In addition, we have used real-time imaging strategies to show that hematopoietic stem cells have the capacity to undergo both symmetric and asymmetric division, and that shifts in the balance between these modes of division are controlled by the microenvironment and subverted by oncogenes. Importantly, we have shown that regulators of this process, including the cell fate determinant Musashi, can promote aggressive cancers and could serve as critical targets for diagnostics and therapy. Most recently, we have developed a high resolution in vivo imaging system that has allowed us to begin to map the behavior and interactions of hematopoietic stem cells with the microenvironment within living animals, and to define how these change during regeneration and cancer formation.

Citation Format: Tannishtha Reya. Understanding the dynamics of cancer: From developmental pathways to in vivo imaging. [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 SY33-02. doi:10.1158/1538-7445.AM2013-SY33-02
Abstract SY36-01: Exploiting intra-tumor heterogeneity through agent-based models of tumor growth to infer properties of human malignancies.
Authors: Sottoriva Andrea , Ma Zhicheng , Kang Haeyoun , Curtis Christina
Published: August 2014

In recent years, intra-tumor heterogeneity (ITH) has emerged as a fundamental property of many solid tumors. This feature is the product of cancer evolution and may be implicated in malignant progression and clinical outcome. ITH not only reflects the sequence of malignant events that have occurred in the neoplasm, but it also embeds a signature of the past proliferative history of tumor cells. This signature can be exploited to infer tumor dynamics and to measure properties of the cellular organization that render each cancer distinct. In particular, by integrating multiple types of genomic data derived from different regions of an individual tumor, with a 3-dimensional agent-based model of tumor growth, we can infer clinically relevant cancer parameters directly in human samples. This approach is highly flexible, enabling several malignant processes to be considered, and provides a natural framework with which to interpret the apparent complexity of cancer genomic data, with implications for understanding mechanisms of tumor progression. We demonstrate the utility of this approach on a cohort of breast and colorectal cancers.

Citation Format: Andrea Sottoriva, Zhicheng Ma, Haeyoun Kang, Christina Curtis. Exploiting intra-tumor heterogeneity through agent-based models of tumor growth to infer properties of human malignancies. [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 SY36-01. doi:10.1158/1538-7445.AM2013-SY36-01
Abstract A06: A novel cell line model of the alternative lengthening of telomeres (ALT) telomere maintenance mechanism
Authors: Heaphy Christopher M. , Haffner Michael C. , Meeker Alan K.
Published: August 2014

Unlimited replicative capacity is a key hallmark of cancer that requires the abrogation of telomere attrition due to the end-replication problem. The vast majority of tumors maintain telomere lengths by expressing the enzyme telomerase; however, a subset utilizes a cancer-specific, telomerase-independent telomere maintenance mechanism, termed Alternative Lengthening of Telomeres (ALT). We recently uncovered a strong correlation between ALT and recurrent cancer-associated somatic inactivating mutations in the ATRX and DAXX genes. The ATRX and DAXX proteins form a chromatin remodeling complex with histone chaperone activity, targeting incorporation of the histone variant H3.3 to G-rich repetitive regions of the genome, including the telomeres, which are known to normally be maintained in a repressed heterochromatic state. Here, we have developed an isogenic cell line pair derived from first generation telomerase knock-out (KO) mouse embryonic fibroblasts. Compared to early-passage telomerase KO cells and late passage WT control cells, late-passage telomerase KO cells spontaneously developed the ALT phenotype. These ALT(+) cells display increased DNA damage, as observed by dramatically increased accumulation of γH2AX foci. We plan to use this unique cell line to identify drugs exhibiting selective toxicity for ALT(+) cells. Preliminary results indicate that ALT(+) late-passage telomerase KO cells exhibit an increased sensitivity to the DNA-PK inhibitor NU7441 compared to the isogenic early-passage ALT(-) telomerase KO cells. The identification of bona fide ALT-specific drugs will pave the way for the development of new targeted treatments for the significant number of cancers that harbor this unique molecular phenotype.

Citation Format: Christopher M. Heaphy, Michael C. Haffner, Alan K. Meeker. A novel cell line model of the alternative lengthening of telomeres (ALT) telomere maintenance mechanism. [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 A06.
Abstract A07: Characterizing the specific oncogenic and tumor suppressor roles of H3K27me3 epigenetic modulators in T cell leukemia
Authors: Ntziachristos Panagiotis , Tsirigos Aris , Trimarchi Thomas , Loizou Evangelia , Luyao Xu , Vlierberghe Pieter Van , King Bryan , Welstead Grant , Mullighan Charles , Jaenisch Rudolf , Ferrando Adolfo , Aifantis Iannis
Published: August 2014

T cell acute lymphoblastic leukemia (T-ALL) is an immature hematopoietic malignancy driven mainly by oncogenic activation of NOTCH1 signaling. In a recently published work, we identified and characterized a tumor suppressor role for the Polycomb Repressive Complex 2 (PRC2), that was underlined through the identification of loss-of-function mutations and deletions of members of the PRC2 complex in 25% of the patients with T cell leukemia. Further inhibition of PRC2 action in T cell leukemia comes through competition with master oncogenes, such as Notch1, for chromatin binding. Extending our work on the H3K27me3 mark, we now show that the activity of the corresponding H3K27me3 demethylases is also very important for the initiation, progression and maintenance of the disease. More specifically, transcriptional data analysis and exome sequencing of primary T-ALL samples demonstrates a significant correlation between levels and mutational status of these demethylases and major oncogenic pathways in leukemia. Functionally, genomic ablation of these epigenetic modulators as well as targeting with a specific-recently produced and characterized-inhibitor-GSKJ4, leads to apoptosis and cell cycle arrest of T-ALL lines in vitro and in vivo, accompanied by major epigenetic and transcriptional changes at specific anti-apoptotic, oncogenic and metabolic genes. Furthermore, genetic ablation of these epigenetic modulators leads to slower initiation of the disease in a classic mouse model of leukemia with highly improved survival rates. In light of recent developments of novel epigenetic inhibitors against these molecules, these findings pave the way to specific pharmacological targeting of T cell leukemia.

Citation Format: Panagiotis Ntziachristos, Aris Tsirigos, Thomas Trimarchi, Evangelia Loizou, Xu Luyao, Pieter Van Vlierberghe, Bryan King, Grant Welstead, Charles Mullighan, Rudolf Jaenisch, Adolfo Ferrando, Iannis Aifantis. Characterizing the specific oncogenic and tumor suppressor roles of H3K27me3 epigenetic modulators in T cell leukemia. [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 A07.
Abstract A17: Glycosaminoglycans regulates histone acetylation status in multiple myeloma
Authors: Purushothaman Anurag , Sanderson Ralph D.
Published: August 2014

Histone acetylation modulates gene expression, cellular differentiation, and cell survival and is regulated by the opposing activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDAC inhibition results in accumulation of acetylated nucleosomal histones and induces differentiation and/or apoptosis in transformed cells and therefore HADC inhibitors are being actively studied as novel therapies in multiple myeloma. However, the mechanisms that regulate the acetylation levels of histones in myeloma remain unclear. We recently discovered that glycosaminoglycan (GAGs) chains of myeloma cells play an important role in regulating histone acetylation status. GAGs, such as chondroitin sulfate and heparan sulfate, which are expressed in the form serglycin and syndecan-1 proteoglycans, respectively, are highly expressed by myeloma cells and are present in the nucleus of these cells. Because exogenous GAGs are shown to both inhibit HAT activity and bind to the acetylation sites of histones, we hypothesized that reduction in GAGs would increase HAT activity and histone acetylation. We found that removal of GAGs from the nucleus of myeloma cells either by treatment with GAG degrading bacterial enzymes or by expressing the heparan sulfate degrading enzyme heparanase, significantly increased HAT activity and histone acetylation in myeloma cells. Further, the levels of acetylated histones are highly upregulated in tumors formed from cells expressing high levels of heparanase or cells that are silenced for syndecan-1 expression. BIACore assay showed that both chondroitin and heparan sulfates strongly bind to HAT enzyme p300. By dot blot assay we also demonstrate that both syndecan-1 and serglycin bind strongly to histones. Previous studies have shown that the negatively charged GAGs can bind to positively charged lysine residues (acetylation sites) on histone proteins. Overall our findings clearly demonstrate that GAGs decreases histone acetylation by both inhibiting HAT activity and by binding to acetylation sites of histones. This represents a novel mechanism for histone hypoacetylation, which is traditionally considered due to HDAC activity.

Citation Format: Anurag Purushothaman, Ralph D. Sanderson. Glycosaminoglycans regulates histone acetylation status in multiple myeloma. [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 A17.
Abstract A19: The histone acetyltransferase PCAF is required for Hedgehog-Gli-dependent transcription and proliferation
Authors: Malatesta Martina , Steinhauer Cornelia , Squatrito Massimo , Helin Kristian
Published: August 2014

The Hedgehog (Hh) signaling pathway plays an important role in embryonic patterning and development of many tissues and organs as well as in maintaining and repairing mature tissues in adults. Uncontrolled activation of the Hh-Gli pathway has been implicated in developmental abnormalities as well as in several cancers, including brain tumors like medulloblastoma and glioblastoma. Inhibition of an aberrant Hh-Gli signaling has thus emerged as an attractive target for anticancer therapy, however the mechanisms that mediate Hh-Gli signaling in vertebrates remain still poorly understood. Here, we show that the histone acetyltransferase (HAT) PCAF/KAT2B is an important regulator of the Hh pathway. Specifically, we demonstrate that PCAF depletion leads to an impairment of Hh activity and reduced expression of Hh target genes. Consequently, downregulation of PCAF in medulloblastoma and glioblastoma cell lines leads to decreased cell proliferation and increased apoptosis. Furthermore, we show that PCAF interacts with GLI1, the downstream effector in the Hh-Gli pathway, and that PCAF or GLI1 loss reduces the levels of H3K9 acetylation on Hh target gene promoters. Our study identifies the acetyltransferase PCAF as a positive cofactor of the Hh-Gli signaling pathway, and we propose PCAF as a potential interesting novel therapeutic target for the treatment of patients with medulloblastoma and glioblastoma.

Citation Format: Martina Malatesta, Cornelia Steinhauer, Massimo Squatrito, Kristian Helin. The histone acetyltransferase PCAF is required for Hedgehog-Gli-dependent transcription and proliferation. [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 A19.
Abstract A21: Orphan nuclear receptor TLX recruits lysine-specific demethylase 1 to repress androgen receptor gene transcription and functions to promote hormone-resistant growth of prostate cancer cells
Authors: Jia Lin , Wu Dinglan , Yu Shan , Chan Franky Leung
Published: August 2014

Prostate cancer (PCa) is the most frequently diagnosed type of cancer and the second leading cause of death from cancer in males in the United States. The initial growth of most localized PCa is slow and androgen-dependent. Thus, surgical treatment (prostatectomy) and hormone therapy (androgen deprivation) are effective initially. However, many patients relapse and progress to a fatal androgen-independent and bone-metastatic stage shortly when both primary and secondary hormone therapies fail. Unfortunately, the current options for the metastatic hormone-refractory PCa are still limited and mostly ineffective, resulting in high mortality of this disease. Although dysregulated androgen receptor (AR) signaling pathway has been proposed to contribute to the development of hormone-resistance, the involved mechanisms are still not fully defined. Our present findings revealed that the orphan nuclear receptor TLX (NR2E1) exhibited increased expression patterns in androgen-independent and antiandrogen-resistant PCa cells, and also the high-grade and hormone-refractory clinical PCa tissues. Stable overexpression of TLX in AR-positive LNCaP cells could promote both androgen-independent and antiandrogen-resistant cell growth in vitro, and enhance tumor growth capacity in castrated male SCID mice in vivo. Decreased expression of AR and AR target genes in LNCaP-TLX cells were identified, while results of AR-promoter driven luciferase reporter assay also revealed that TLX overexpression could suppress the AR transcription in non-prostatic HEK293 cells. Furthermore, we also identified the direct binding site of TLX on the AR promoter by chromatin immunoprecipitation analyses in TLX-transfected HEK293 cells. The molecular mechanism may involve the TLX-mediated recruitment of lysine-specific demethylase 1 (LSD1) as treatment with an inhibitor of LSD1 pargyline could reverse the transrepressive regulation of TLX on AR-promoter driven luciferase activity and restore AR gene expression levels in LNCaP cells. In summary, our results showed for the first time that overexpression of TLX might contribute to the hormone-resistant PCa cell growth and advanced progression of PCa. LSD1 might serve as a corepressor in TLX-mediated direct transrepression on AR gene expression.

This work is partly supported by an RGC-General Research Fund CUHK461012.

Citation Format: Lin Jia, Dinglan Wu, Shan Yu, Franky Leung Chan. Orphan nuclear receptor TLX recruits lysine-specific demethylase 1 to repress androgen receptor gene transcription and functions to promote hormone-resistant growth of prostate cancer 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 A21.
Abstract A45: Chromatin dynamics of the zebrafish retina during development and aging
Authors: Wei Xiangyun
Published: August 2014

During organogenesis, the retina develops from a single sheet of undifferentiated neuroepithelium into a layered mature tissue composed of Müller, photoreceptor, horizontal, bipolar, amacrine, interplexiform and ganglion cells. This differentiation of various retinal cell types requires the transcription of distinct sets of genes in precise temporal orders. It is unclear how chromatin organization and modifications are specifically regulated at global and regional levels to facilitate the generation of distinct transcriptional profiles required for different retinal cell types. Nor do we know to what extent chromatin structure and organization are altered during retinal aging, or how such alterations lead to unbalanced gene expression profiling and subsequent cell degeneration. In this study, we analyzed the dynamic characteristics of nuclear structures and chromatin organizations of zebrafish retinal cells during development and aging. We show that the degrees of chromatin condensation vary significantly among different retinal cell types, as suggested by varying nuclear volumes and heterochromatin/euchromatin organization. These structural features are altered during the aging process as well. For example, the nuclear volumes of cone photoreceptors increase significantly during aging. The distribution patterns of H3K9me3 and H4K20me3 heterochromatin modifications also vary dramatically among different retinal cell types and at different ages. The gross alterations in nuclear structures and chromatin condensation may suggest extensive changes in gene expression activities. These findings provide insight into how chromatin organization and modifications underlie balanced global transcriptional profiles and consequently, the overall fitness of retinal cells, as well as how such regulations might be altered during the aging process. Our study may have significant social impact because with increasing life expectancy, more and more older people will suffer from age-related macular degeneration and the identification of proper interventions to slow down the retinal aging process would dramatically enhance quality of the life for the elderly.

Citation Format: Xiangyun Wei. Chromatin dynamics of the zebrafish retina during development and aging. [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 A45.
Abstract A57: Dual role of the histone methyltransferase PR-SET7/SETD8 in the local regulation of Pol II promoter-proximal pausing
Authors: Kapoor Priya , Vertino Paula M.
Published: August 2014

Tumor suppressor gene silencing in cancers is often achieved through epigenetic mechanisms which involve the hypermethylation of promoter-associated CpG islands and a local shift from permissive to repressive histone modifications. Recently, we showed that epigenetic silencing of the tumor suppressor gene TMS1/ASC in human breast cancer cells is accompanied by a local shift from histone H4 lysine 16 acetylation (H4K16Ac) to H4 lysine 20 trimethylation (H4K20me3). H4K16Ac and H4K20me3 were found to play antagonistic roles in the regulation of RNA polymerase II promoter-proximal pausing, a critical post-initiation regulatory step that controls the rate of release of initiated Pol II into productive elongation and ultimately transcriptional output. We found that H4K16Ac, mediated by the histone acetyltransferase hMOF and acting in the MSL complex, facilitates the release of paused Pol II by promoting the recruitment of the BRD4/pTEFb complex whereas the local deposition of H4K20me3, mediated by the histone methyltransferase SUV420H2, enforces the pause and promotes gene repression by inhibiting the recruitment of MSL and subsequent H4K16Ac. However, how the local balance between hMOF-mediated H4K16Ac and SUV420H2-mediated H4K20me3 is achieved and converge to regulate Pol II pausing remains unclear. Here, we demonstrate that the histone methyltransferase PR-SET7/SETD8, which monomethylates histone H4 lysine 20 (H4K20me1), controls both H4K16Ac and H4K20me3, and as a result, regulates Pol II pausing and release. Specifically, we show that PR-SET7 and/or H4K20me1 is necessary for the local recruitment of the MSL complex, subsequent H4K16Ac and release from pausing. Although dispensable for SUV420H2 recruitment, PR-SET7 is required for SUV420H2-mediated H4K20me3, playing a role in enforcing Pol II pausing and subsequent epigenetic gene silencing. Whereas depletion of SUV420H2 is sufficient to deplete H4K20me3 and relieve H4K20me3-mediated Pol II pausing, pausing is maintained in the absence of PR-SET7 and local H4K20me1 due to a failure to recruit the MSL complex. These findings highlight the requirement for PR-SET7 and H4K20me1 in establishing both the H4K16Ac and H4K20me3 marks and points to its dual role in the regulation of Pol II pausing dynamics. Furthermore, our data suggests that the reversible repression imposed by the local conversion of H4K20me1 to H4K20me3 is a critical intermediate step in the epigenetic silencing of tumor suppressor genes in cancers.

Citation Format: Priya Kapoor, Paula M. Vertino. Dual role of the histone methyltransferase PR-SET7/SETD8 in the local regulation of Pol II promoter-proximal pausing. [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 A57.
Abstract A60: Analysis of gene copy number changes in chronic lymphocytic leukemia
Authors: Dalay Nejat , Isin Mustafa , Cetin Guven , Aktan Melih
Published: August 2014

Chronic lymphocytic leukemia (CLL) is the most frequent type of leukemia in the elderly and is accompanied by the accumulation of malignant monoclonal B-cells in the bone marrow, peripheral blood or lymph nodes which harbor various genetic aberrations. Genomic instability is an important characteristic of leukemia cells and plays a role both in its development as well as in the response to therapy. Somatic copy number changes in distinct genes are associated with the development of specific leukemias. Copy number variations are important diagnostic tools and help in classifying the disease and guiding the therapy. Current strategies for the detection of chromosomal aberrations in CLL are laborious and time consuming. Using multiplex ligation dependent probe amplification (MLPA) copy number changes at multiple loci can be detected simultaneously in a single simple PCR reaction. In this study, we investigated the copy number alterations in several key genes in CLL by MLPA.

DNA was isolated from the venous blood samples from 92 patients with CLL and 10 healthy controls. Copy number changes of 20 genes were analyzed at 29 different sites by MLPA. DNA samples were hybridized with the sequence specific probes for each gene and the hybridized probe pairs were ligated. PCR reaction was performed to amplify the ligated probes. PCR products were separated by capillary electrophoresis using the ABI Prism 310 genetic analyzer. The peaks were evaluated for the genomic gains and losses after normalization of each peak. Reference genes were used for normalization of each gene signal. The data from the patients were compared with control samples from healthy individuals.

Various genetic aberrations were detected in the patients with CLL. The most frequently observed genetic aberrations were losses in the chromosomal regions 13q14.3 and 13q14.2 and involved the DLEU7 (49 %), DLEU2 (41 %), KCNRG (35.9 %) and RB1 (14 %) genes. Copy number losses in the MYCN, ESR1, EIF3S3, CDKN2A, CDKN2B, ATM, CHFR and TP53 genes were lower with a frequency ranging between 1-10 %.

The most frequently amplified gene was the MYC gene (30.4 %), followed by the LRMP (17.4 %), ESR1 (15 %) and CHFR (14 %) genes. Gains in the MYCN, IGF2R, PARK2, CCND2, CDK4, IFNG and LDLR genes were observed with a lower frequency.

Our data indicate that copy number changes in several important genes are frequent in the pathogenesis of CLL. Simultaneous analysis and characterization of these genetic alterations can be performed using MLPA and the test can provide valuable clinical information when employed as a first-line screening approach for CLL patients.

Citation Format: Nejat Dalay, Mustafa Isin, Guven Cetin, Melih Aktan. Analysis of gene copy number changes in chronic lymphocytic leukemia. [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 A60.
Abstract B02: Telomere-driven chromosome instability impacts on microRNA expression through epigenetic mechanisms
Authors: Jouravleva Karina , Castro-Vega Luis , Londono-Vallejo Arturo
Published: August 2014

Telomere shortening is a major source of chromosome instability (CIN) at early stages during carcinogenesis. However, the mechanisms through which telomere-driven CIN (T-CIN) contributes to the acquisition of tumor phenotypes remain uncharacterized.

We have used human epithelial kidney cells (HEK cells), a well-established in vitro model of progressive telomere instability, to study the impact of T-CIN in the expression of miRs and the biological consequences that might be relevant for the acquisition of tumor phenotypes.

We discovered that human epithelial kidney cells undergoing T-CIN display massive microRNA (miR) expression changes that are not related to local losses or gains. This widespread miR deregulation encompasses a miR-200-dependent epithelial-to-mesenchymal transition (EMT) that confers to immortalized pretumoral cells phenotypic traits of metastatic potential. Since deregulation of miRs in HEK CIN+ cells was widespread, orchestrated, and not related with copy number changes we hypothesized that this phenomenon utilizes epigenetic cues. Indeed, investigation of the mechanisms controlling miR-200 expression revealed that both DNA methylation and histone modifications changed significantly in cells undergoing T-CIN. Unbiased approaches are now being used to describe the epigenome changes genome-wide that are associated with CIN during cell transformation.

Citation Format: Karina Jouravleva, Luis Castro-Vega, Arturo Londono-Vallejo. Telomere-driven chromosome instability impacts on microRNA expression through epigenetic mechanisms. [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 B02.
Abstract B08: Tipping the balance: Factors that influence the ER signaling network in breast cancer
Authors: Jasper Jeff S.
Published: August 2014

Our lab has generated a tamoxifen resistant cell line (TamR) from MCF7 cells by serial passage in a nude xenograft model in the presence of tamoxifen. The TamR tumor model is stimulated by tamoxifen and does not require estradiol for growth. Transcriptional control of ER in this model is shifted to a ligand-independent state as ER target genes are basally increased and do not induce much higher with estradiol or tamoxifen. We hypothesized that the transcriptional program of ER was altered and that candidate factors could be identified with DNase Hypersensitivity analysis by DNase-Seq. Following Identification of DNase Hypersensitivity Regions, motif enrichment analysis, footprinting, and comparison to to known ChIP-Seq data were performed. These analyses indicate a an increased role for the FOXA family members and RNA-Seq indicates a dramatic upregulation of the FOXA2 transcript. Studies are ongoing to determine the relevance of FOXA2 in tamoxifen resistance and impact on the ER transcriptional program.

Citation Format: Jeff S. Jasper. Tipping the balance: Factors that influence the ER signaling network in breast 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 B08.
Abstract B11: Coupling between the DNA methylation and the alternative splicing
Authors: Mauger Oriane , Muchardt Christian , Batsché Eric
Published: August 2014

Pre-messenger RNAs (pre-mRNAs) maturation is initiated cotranscriptionally and chromatin-borne information participates in alternative splicing. We have found recently that elevated levels of trimethylation of histone H3 on Lys9 (H3K9me3) and the chromodomain protein HP1γ are a characteristic of the alternative exons of several genes including CD44 (Saint-André et al. 2011). On this gene, we have shown that the endogenous RNA interference pathway is involved in the H3K9me3 targeting on the variant exons through the recruitment of the nuclear argonaute proteins (AGO1 or AGO2) (Ameyar-Zazoua et al. 2012).

Because the DNA methylation is an epigenetic mark accompanying the transcriptional silencing, we now explore whether the DNA modification into the active genes could play a role in the regulation of the alternative splicing.

Studying the transcriptome and the genome of murine ES cells, allowed us to show that the enzymes that methylate DNA (DNMT1, 3a and 3b) regulate the splicing of many genes. In Hela cells the DNMT1 depletion causes a decrease in the inclusion of alternative exons of CD44. Similar results were obtained after depletion of MBD1 and MBD2, two proteins that bind to methylated DNA. Together these results suggest that DNA methylation regulates splicing of CD44 by a direct mechanism involving MBD1 and MBD2. The disregulation of the DNA methylation on CpGs is a hallmark of the tumorigenesis. However, the effect of DNA methylation changes within coding regions of the genes is poorly understood. We used the well-known model of breast tumor progression based on the MCF10A cells, to investigate the changes into the DNA methylation of the CD44 coding region. We found that the intragenic DNA methylation is modified between the MCF10A parental cells and the derived DCIS and CA1A transformed cells and this is correlated with the inclusion of the CD44 alternative exons. Altogether these results suggest that modifications of DNA methylation could contribute to the disregulations of alternative splicing decisions contributing to the tumor progression.

Ameyar-Zazoua M, Rachez C, Souidi M, Robin P, Fritsch L, Young R, Morozova N, Fenouil R, Descostes N, Andrau JC, Mathieu J, Hamiche A, Ait-Si-Ali S, Muchardt C, Harel-Bellan A, Batsché E (2012) Argonaute proteins couple chromatin silencing to alternative splicing. Nat. Struc. Mol. Biol 19:998-1004.

Saint-André V., Batsché E., Rachez C. and Muchardt C. (2011) Histone H3 lysine 9 tri-methylation and HP1γ favor inclusion of alternative exons. Nat. Struc. Mol. Biol 18:337-44.

Citation Format: Oriane Mauger, Christian Muchardt, Eric Batsché. Coupling between the DNA methylation and the alternative splicing. [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 B11.
Abstract B16: Phenotypic plasticity in the EML cell culture system as a result of HSP90 inhibition
Authors: Napper Jennifer , Sollars Vincent E.
Published: August 2014

The process of canalization whereby phenotypic traits are buffered against stochastic fluctuations to preserve the evolutionarily advantageous “normal” level can be potentially exploited during cancer progression. Release of canalization through pharmacologic or genetic down-regulation of Hsp90 removes this buffer and increases variation, allowing for rapid changes in traits as evidenced in Drosophila, Arabidopsis, and maize. If cancer is viewed as a Darwinian struggle of the premalignant cell to acquire the hallmarks of a fully malignant cell or be extinguished, then increasing variation or heterogeneity has a beneficial effect on the fitness of the premalignant cell. It is the goal of this work to test the hypothesis that Hsp90-derived canalization, though its ability to buffer phenotypic variance and thus reduce cell plasticity, is involved in hematopoietic cell differentiation. Furthermore, we wish to determine if the canalization mechanism has an epigenetic component. We are currently using a murine hematopoietic stem cell culture system, EML cells, to investigate this hypothesis. EML cells have been induced to differentiate into macrophage and granulocyte lineages with and without pharmacologic inhibition of HSP90 through 17-AAG. We found that 17-AAG treatment increases the ability of these differentiating cells to survive in a selective medium, during culturing conditions that suggest an epigenetic mechanism. If our hypothesis is true, then loss of canalization could be a potentially powerful mechanism driving cancer progression. Fully understanding the mechanism involved would allow for new therapeutic insights and provide vital information regarding the use of HSP90 inhibitors.

Citation Format: Jennifer Napper, Vincent E. Sollars. Phenotypic plasticity in the EML cell culture system as a result of HSP90 inhibition. [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 B16.
Abstract B23: Epigenetic regulation of OPA1 sensitizes hepatocellular carcinoma to sorafenib-induced apoptosis
Authors: Puszyk William Matthew , Dong Hui-Jia , Zhao Frank , Ogunwobi Olorunseun , Liu Chen
Published: August 2014

Sorafenib is a multi-kinase inhibitor used in the treatment of un-resectable hepatocellular carcinoma (HCC). Patient response to this therapy is variable. In this study we show that treatment with Sorafenib induces rapid mitochondrial fragmentation, which is associated with the deregulation of mitochondria-fusion-related protein OPA1, thus inducing apoptosis. Exposure of HCC cells or isolated mitochondria to Sorafenib induces cytochrome c release. Our data indicate that siRNA-mediated OPA1 knockdown significantly sensitizes HCC cells to Sorafenib-induced apoptosis. Furthermore, Sorafenib inhibits HCC xenograft tumor growth in vivo, and does not induce apoptosis in healthy primary hepatocytes. Murine xenograft tumor tissue samples revealed mitochondria fusion protein OPA1 expression levels are strongly associated with the sensitivity of HCC to sorafenib treatment. We have shown that Sorafenib suppresses the tumorigenesis of HCC through the induction of mitochondrial injury via OPA1. Epigenetic analysis of the OPA1 gene promoter indicates a role for DNA methylation in the suppression of OPA1 expression, and a potential biomarker which can be utilized for the identification of patients whom may benefit from treatment with Sorafenib. Our results provide new insights into the pathogenesis of HCC and suggest that OPA1 is a novel therapeutic target in patients with HCC.

Citation Format: William Matthew Puszyk, Hui-Jia Dong, Frank Zhao, Olorunseun Ogunwobi, Chen Liu. Epigenetic regulation of OPA1 sensitizes hepatocellular carcinoma to sorafenib-induced apoptosis. [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 B23.
Abstract B32: Dynamic whole-genome methylomes reveal hidden diversity of CpG islands at multiple biological levels
Authors: Zeng Jia , Nagrajan Hema , Yi Soojin V.
Published: August 2014

CpG islands mark epigenetic regulatory hotspots of mammalian genomes. Numerous studies have demonstrated essential roles of CpG islands in regulation of gene expression, developmental processes, genomic imprinting, aging, and cancer. These regulatory effects rest critically on correct (or the lack of) DNA methylation of CpG islands, and aberrant DNA methylation of CpG islands are hallmarks of cancer. However, how DNA methylation of CpG islands occurs across different cell types, and how variation of CpG islands methylation relates to their functions, remain poorly understood. Here we present a global analysis of DNA methylation of CpG islands in the human genome. We examined variation of CpG island methylation across five complete methylomes of distinctive origins (whole blood, prefrontal cortex, sperm, embryonic stem cells and fibroblast). Our goal was to identify distinctive characteristics of CpG islands with respect to their epigenetic variation in normal tissues. Our analysis reveals that, contrary to the prevailing notion, CpG islands mark the most highly variably methylated regions in the human genome. Many CpG islands exhibit highly methylome-specific patterns of DNA methylation. Remarkably, DNA methylation patterns of CpG islands reflect their distinctive nature at many biological levels, including genomic characteristics such as lengths and nucleotide composition, functional differences as evidenced by distinctive gene ontology enrichments, and evolutionary features. Moreover, CpG islands implicated in cancers and those associated with aging show intriguing differences in their genomic, epigenomic and functional features. These findings indicate distinctive nature of CpG islands at genomic, epigenomic and functional levels. These new knowledge provides novel insights into deciphering the regulatory mechanisms of CpG islands in human health and diseases and may be used to improve empirical studies of DNA methylation variation across different biological conditions, and furthermore ultimately to aid development of better diagnostic markers.

Note: This abstract was not presented at the conference.

Citation Format: Jia Zeng, Hema Nagrajan, Soojin V. Yi. Dynamic whole-genome methylomes reveal hidden diversity of CpG islands at multiple biological levels. [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 B32.
Abstract B39: Methylation of BRCA2 gene promoter CpG units through ZAR2-dependent DNMT1 recruitment
Authors: Misra Smita , Mittal Mukul K. , Khedkar Sheetal V. , Chaudhuri Gautam
Published: August 2014

BRCA2 gene expression is stringently regulated during the cell cycle. BRCA2 expression is proportional to the rate of cell proliferation. Looking at the human BRCA2 gene minimal promoter sequence we found that it has over 45 CpG units which could be potentially methylated regulating the recruitment of different transcription factors affecting BRCA2 gene expression. We discovered that BRCA2 gene promoter has bi-directional activity and the product of the reverse activity (a ZAR1-like protein, we named ZAR2) silences the forward promoter (for BRCA2). ZAR2 is a C4-type zinc finger-containing transcription factor that regulates BRCA2 in a cell cycle dependent manner. We found that ZAR2 enters the nucleus of the G0/G1 cells binds to the promoter of BRCA2 and silences the BRCA2 gene expression. ZAR2 has a dsRNA binding domain. We report here that BRCA2 gene promoter, through its bidirectional activity, produces two partially overlapping transcripts (BRCA2 and ZAR2) in the breast cells forming a 111 bp RNA duplex. We postulate that endogenous transcriptional gene silencing RNAs (tgsRNAs) against BRCA2 gene promoter are thus formed from the overlapping RNA duplex in the cell nucleus. These tgsRNAs in turn contribute towards promoter methylation. Our data suggest that ZAR2 upon binding to the tgsRNAs binds to specific locations of BRCA2 gene promoter and recruits DNMT1 which in turn perform CpG methylation. This study will contribute towards the understanding of the epigenetic regulation of BRCA2 gene expression in different cell cycle stages as well as in sporadic cases of breast and other cancer. Supported in part by the DOD-CDMRP IDEA Grants BC990678 and BC050641 to GC and MeTRC pilot project grant 1U54RR026140-01 to SM

Citation Format: Smita Misra, Mukul K. Mittal, Sheetal V. Khedkar, Gautam Chaudhuri. Methylation of BRCA2 gene promoter CpG units through ZAR2-dependent DNMT1 recruitment. [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 B39.