3rd Cancer Epigenetics
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Day 1 Day 2 Day 3
Day 1 - Wednesday, November 6, 2013


Check In & Registration


Welcome & Opening Remarks

Joint Session with 8th Cell Based Assay & Screening Technologies

3D Cell Cultures & Physiologically Relevant Model Systems
Moderator: Geoffrey Bartholomeusz, Assistant Professor & Director of the siRNA Core Facility
University of Texas MD Anderson Cancer Center


Rational Design and Interrogation of Physiologically Relevant 3D Co-Culture Models

Jan Lichtenberg
CEO & Co-Founder
InSphero AG

3-dimensional (3D) cell culture technology enables engineering of organotypic in vitro models for drug discovery and pre-clinical safety assessment. As a tissue consists not only of a single cell type, key parameters for successful model generation are the right choice of cell types – which can be either cell lines, differentiated primary cells or stem cell-derived cells – and their relative ratios within the model. Moreover, histological structuring of 3D models and microenvironment design, including the culture medium, are important determinants of tissue engineering. The resulting models can vary substantially in complexity and resulting biological information. In the presentation we provide practical guidance for rational 3D cell model design. Application examples for the assessment of targeted immunomodulatory antibodies and the detection of idiosyncratic liver toxicity will demonstrate the importance of multi-cell type organotypic models to improve in vitro biology.

Generating 3D cell-based in-vitro models is only one step towards answers to specific biological questions. The equally important second step is the efficient and biologically relevant interrogation of these models – ideally leveraging existing assay technology and instrumentation to facilitate the implementation of 3D cell-based assays into existing workflows. A wide spectrum of currently used assay technology has been investigated, reaching from biochemical assays to histology, RNA and protein expression profiling and high-content analysis. The presentation will provide guidelines for choosing appropriate assays and for multiplexing them to gain high information content of 3D screens and to reduce overall costs.

Benefits of this presentation include practical guidance on designing 3D microtissues to correspond to the underlying biological questions and on developing robust and efficient read-out strategies.


Validating Performance of Cytotoxicity Assays Applied to 3D Cell Culture Models

Terry Riss
Senior Product Specialist
Cell Health
Promega Corporation

Cells cultured in 3D model systems often acquire relatively large in vivo-like structures compared to the thickness of a 2D monolayer of cells grown on standard plastic plates. Multicellular 3D culture systems containing more than one cell type and exhibiting formation of a complex extracellular matrix represent a more physiologically relevant environment, yet provide a challenge for assay chemistries originally designed for measuring events from monolayers of cells. There is an unmet need for guidelines for design and verification of convenient and effective assays useful for larger 3D microtissues. Critical factors to consider for each model system and cell type include effective penetration of detection reagents and/or complete lysis of microtissue structures using combinations of detergent and physical disruption. We will present the approach used to verify performance of the bioluminescent ATP detection assay for measuring cell viability, a caspase assay for detecting apoptosis, and cell stress assays to detect mechanisms leading to cytotoxicity. Recommendations for factors to consider when verifying performance of cell health assays on 3D culture models will be presented.

Attendees will learn about:
-Critical aspects to consider when using commercial assays designed for cell monolayers and attempting to apply them to 3D culture models
-The importance of knowing the stability of the marker you are trying to measure
-Methods to achieve effective lysis of microtissues
-New assay chemistries being developed to measure viability of cells in 3D cultures


A Human Breathing Lung-on-a-Chip for Drug Screening and Nanotoxicology Applications

Dan Dongeun Huh
Wilf Family Term Chair & Assistant Professor
University of Pennsylvania

A major problem slowing the development and regulatory approval of new and safer medical products is the lack of experimental in vitro model systems that can replace costly and time-consuming animal studies by predicting drug efficacy and toxicity in humans. Here we describe a biomimetic microsystem that reconstitutes the critical functional alveolar-capillary interface of the human lung. This microdevice reproduces complex integrated organ-level responses to bacteria and inflammatory cytokines introduced into the alveolar space by inducing expression of intercellular adhesion molecule-1 (ICAM-1) on the microvascular endothelium surface, adhesion of circulating blood-borne neutrophils, their transmigration across the capillary-alveolar interface, and phagocytosis of the infectious pathogens. Using this approach, we developed novel nanotoxicology models and revealed that physiological cyclic mechanical strain greatly accentuates toxic and inflammatory responses of the lung to silica nanoparticles. Mechanical strain also enhances nanoparticle uptake by the epithelial cells and stimulates their transport into the underlying microvasculature. Importantly, similar effects of physiological breathing on nanoparticle absorption were observed in whole lung using a mouse lung ventilation-perfusion model. We also explored the potential use of this microsystem for the development of microengineered models of human lung disease for applications in drug screening. This mechanically active biomimetic microsystem represents valuable new model systems for in vitro analysis of various physiological functions and disease processes, in addition to providing low-cost alternatives to animal and clinical studies for drug screening and toxicology applications.


Human Organo-Typical Co-Culture System Platform for Functional Substance Activity Profiling in Immunopharmacology and Immunotoxicology

Manfred Schmolz
Managing Director & CSO
EDI GmbH (a subsidiary of Myriad RBM)

Our proprietary test platform, developed in the past decade and used for contract research under certified conditions for more than 10 years, consists of co-cultures of human immune cells (whole-blood) plus cells of different tissues, such as fully differentiated intestinal epithelia, bronchial epithelia, 3D-epidermis, synovial cells, or endothelial cells, allows a far more relevant, while physiological insight into the highly complex communication network between the immune system and the target organs of acute and chronic inflammatory processes than any other currently available in-vitro test model. These models are widely tunable to meet the testing conditions of all sorts of test compounds and modes of action, including the creation of pathophysiologically relevant (inflammatory) conditions.

The audience will learn that organo-typic complexity, even when using primary cells, is achievable without impairing reproducibility and throughput. All systems can be custom-tailored in a wide range in order to allow a more relevant, comprehensive in vitro profiling of drug substances.


Afternoon Networking & Coffee Break


Hemodynamic Blood Flow, Transport and Heterotypic Cell-to-Cell Communication are Necessary for Restoring In Vivo Cell Responsiveness In Vitro

Brian Wamhoff
Vice President, Research & Development

Static primary cell culture systems, widely used as tools for drug efficacy, metabolism and toxicity, are known to dedifferentiate over time and lose their metabolic phenotype, which may partly explain the suboptimal predictive ability of many of these systems. This, coupled with the non-physiological dosing profile limitation of static cultures may contribute to dramatic differences noted between efficacious and toxic drug concentrations in vitro and corresponding in vivo or clinical plasma concentrations. By interfacing high fidelity physiological parameters and primary human cell types, we have developed 3D multi-cellular human vascular and liver systems. For both systems, in their simplest form, tissue-specific endothelial cells are co-cultured with vascular smooth muscle cells or liver hepatocytes allowing for heterotypic cell-to-cell communication. The system exposes the endothelium to organ-specific blood flow hemodynamics (derived by high-resolution MRI or ultrasound) while also replicating human organ intercellular transport and perfusion in the 3D multicellular-culture.

After a prescribed time period, in vivo morphology, phenotype, biology and metabolism are restored in both cell types in vitro compared to static co-culture comparators. Importantly, the cell types now respond to drugs and hormones at concentrations that approximate human in vivo levels, which are often more than one to two orders of magnitude different from standard 2D co-culture systems. In this presentation we will focus on the importance of heterotypic cell-cell communication, hemodynamic flow and transport for restoring in vivo primary cell response in vitro, interfacing biomedical engineering with physiology. We have screened over 150 compounds in both systems and will show data that predicts human responsiveness at clinical Cmax drug exposure levels for both safety and efficacy and how these systems are being using to actively move drugs through the drug discovery pipeline. Additionally, we will discuss how these fundamental principles can be applied across other organ systems, such as the blood-brain-barrier or the tumor microenvironment, and non-human species, such as rat, dog and monkey, which are key for interspecies assessment as it pertains to the FDA Animal Rule.


A Novel Approach to Overcome Oncogenic Addiction via 3D Model Systems

Hakim Djaballah
High-Throughput Screening Core Facility
Memorial Sloan-Kettering Cancer Center

Classical drug discovery pathways for oncology have relied heavily on killing cancer cells; the approach has worked extremely well in some cases but not as good as predicted in others with many failures reported the clinic. Therefore, there is a sense of urgency in discovering novel small molecule therapeutics for combat cancer. We have taken an opportunistic approach looking for small molecules which would selectively revert the oncogenic addictive state of the cancer cell yielding a vulnerable phenotype; thought to be easily targetable with common chemotherapeutics agents. I will describe the approach and discuss our findings thus far with the ultimate goal of progression to the clinical.

Learning benefits include and not limited to:
1. High content assay approaches monitoring 3D cell formation in 384-well microtiter plates.
2. Screening for compounds able to reverse the cluster phenotype of 3D cells.
3. Importance of biomarkers to compare and contrast 3D cells versus those growing in 2D; critical parameter to confirm that the cell pile-up or cluster is indeed a 3D outcome.



The Third Dimension for High Throughput RNAi-Driven Target Identification

Geoffrey Bartholomeusz
Assistant Professor & Director
siRNA Screening Service
University of Texas MD Anderson Cancer Center

The tumor microenvironment is a complex 3D microenvironment. Although two-dimensional (2D) model systems have contributed to our understanding of tumor biology these models fall short of reproducing the complex and dynamic environments of the tumor. This has prompted the development of three-dimensional (3D) models. The most commonly used 3D model is the spheroid model. This model is of intermediate complexity between in-vivo tumors and monolayer cultures and takes advantage of the natural tendency of cells to aggregate. The cellular organization within spheroids emulates the heterogeneity of solid tumors with necrosis and radiation-resistant hypoxic regions. We have developed a 3D spheroid cell culture model to address our hypothesis - silencing targets that regulate tumor architecture will alter the integrity of the tumor, reduce the hypoxic state and sensitize the tumor to radiation and/or chemotherapy. We performed a high throughput RNAi screen utilizing our spheroid model in which the activation of HIF-1a was used as the readout for the selection of hits from the primary screen and alterations of hypoxic status of the inner core of the spheroid was used in the final validation and selection of the top ranked hits. Utilizing our selection criteria for this study we identified and validated 5 unique targets whose silencing alters the integrity of the spheroid architecture.

In conclusion, the features of the third dimension, hypoxia, morphology and the heterogeneous growth characteristics of spheroids not present in 2D monolayer cell cultures makes this model a necessary model for studies in tumor biology.

Benefits of study
1. Good model to be used in high throughput screening
2. Good model for target identification with clinical relevance
3. Novel therapeutic approach
4. Minimizes the use of animals in the determining the therapeutic efficacy of small molecules


Day 1 of Summit Concludes

Day 1 Day 2 Day 3
Day 2 - Thursday, November 7, 2013


Registration & Continental Breakfast


Welcome & Opening Remarks

Epigenetic Mechanisms in Cancer
Session Chair: David Lombard, University of Michigan


The Chromatin Associated Sin3B Protein Promotes Oncogene-driven Inflammation and Pancreatic Cancer Progression

Gregory David

Associate Professor of Pharmacology
New York University School of Medicine

Inflammation is critical for the progression of KRas-induced pancreatic preneoplastic lesions towards invasive pancreatic ductal adenocarcinoma (PDAC). KRas itself directs the inflammatory response in PDAC, although the underlying mechanisms remain largely unknown. Here we report that genetic inactivation of histone deacetylase (HDAC)-associated Sin3B suppresses KRas-induced senescence in a mouse model of PDAC. Surprisingly, impaired senescence is accompanied with a significant delay in PDAC progression, correlating with the suppression of inflammation. At the molecular level, a Sin3B-HDAC pathway mediates cell autonomous IL-1? production, acting to orchestrate an inflammatory program downstream of activated Kras. These results indicate that the senescence-associated inflammatory program is critical for PDAC progression and point to Sin3B-associated HDAC activity as a therapeutic target to inhibit inflammation-driven tumorigenesis. Therefore, this study highlights the potential for targeting epigenetic regulators in order to prevent pancreatic cancer progression, and by inference, other diseases triggered by aberrant inflammation.


How Do Changes of the Epigenome Contribute to Cancer?

Gerd P. Pfeifer

Professor, Cancer Biology
City of Hope, Beckman Research Institute

Although changes of the epigenome, such as perturbation of DNA methylation patterns, are common events in human cancer, their contribution to the initiation and progression of malignant tumors has remained unclear. We analyzed histone and DNA cytosine modifications (5-methylcytosine and 5-hydroxymethylcytosine) in paired samples of human colorectal cancer and normal tissue. 5-hydroxymethylcytosine, an oxidation product of 5-methylcytosine and a putative intermediate in DNA demethylation pathways, was quantitated globally and was mapped at high resolution, but we could not confirm a role of this oxidized base or its tumor-associated depletion in locus-specific DNA hypermethylation. To explore alternative scenarios, we mapped the Polycomb mark H3K27me3, a repressive histone modification, and the active histone mark H3K4me3 in the same sample pairs. DNA methylation was strongly targeted to bivalent, H3K4me3 and H3K27me3-associated promoters, which loose both histone marks and acquire DNA methylation. We related the tumor-associated changes in DNA methylation and histone modifications to alterations in gene expression seen in the colorectal tumors. We propose a model in which specific epigenetic changes are shown to be important for the selection of tumor-driving events that play a major role in cancer progression.

Benefits of this talk:
Gives an overview of DNA methylation changes in cancer.
Characterizes the role of 5-hydroxymethylcytosine in cancer.
Introduces the importance of histone methylation changes in cancer.
Describes a new model of cancer progression based on chromatin instability.


An Epigenetic Switch in Sonic Hedgehog Signaling Regulates Gene Activation and Medulloblastoma Growth

Jiang Wu

Assistant Professor, Physiology and Developmental Biology
UT Southwestern Medical Center

The Sonic hedgehog (Shh) signaling pathway plays important roles during normal development and in cancer progression. Mutations in Shh pathway proteins lead to developmental diseases and cancers. Recent studies suggest an increasing link between active Shh pathways with the growth of various kinds of cancers. The mitogenic function of Shh signaling requires Gli-mediated transcriptional activation. We have found a Shh-induced epigenetic switch that functions together with Gli proteins to control transcription outcomes. Prior to induction, poised Shh target genes are marked by a bivalent chromatin domain containing a repressive histone H3 lysine 27 trimethylation (H3K27me3) mark and an active histone H3 lysine 4 trimethylation (H3K4me3) mark. Upon signal activation, Shh induces a local switch of epigenetic co-factors from the H3K27me3 methyltransferase polycomb repressive complex 2 (PRC2) to the demethylase Jmjd3/Kdm6b, which is crucial for removing H3K27me3 and activating gene expression. Interestingly, activation of gene expression by Jmjd3 also requires its non-enzymatic activities. We provided evidence showing that Jmjd3 recruits and functions coordinately with the MLL H3K4 methyltransferase complexes to resolve the bivalent domain in a Shh-dependent manner. In vivo, we demonstrated that Jmjd3 is required for Gli activator-dependent normal neural tube and cerebellum development as well as for the growth of Shh-type medulloblastoma cells. Thus we discovered a novel regulatory mechanism underlying the signaling-induced transition of Shh target genes from poised to the activated states, which provides potential therapeutic targets for various cancers with elevated Shh signaling.

9:15 Epigenetic Study of Mouse Brain Aging


Xiangru Xu, Assistant Professor of Anesthesiology, Yale

9:40 Novel Functions for Sirtuin Proteins

David Lombard

Assistant Professor of Pathology
University of Michigan


Networking and Refreshment Break

New Clinical Development of Epigenetic Drugs
Session Chair: David Stokoe, Genentech


Enhancer Malfunction in Cancer

Ali Shilatifard
Stowers Institute for Medical Research

Gene expression by RNA polymerase II (Pol II) is regulated at multiple levels in order to allow for the faithful transmission of genomic information throughout development. Factors interacting with Pol II both at the level of the promoters and enhancers are central for the regulation of gene expression. Enhancers constitute genomic elements that are promoter-distally located, and in many instances, are necessary for the induction and maintenance of gene expression. Enhancers are often bound by developmental transcription factors, which through looping bring these distal regulatory elements into close proximity to the promoter-proximal regions regulating their transcriptional activities. Therefore, enhancers provide an important regulatory cog for optimal transcriptional coherence to allow for tissue- and context-specific transcription of important developmental genes in a time sensitive and optimized manner. I will discuss our recent studies in the identification of factors that function in the regulation of the chromatin state and the activities of enhancers during development. These studies have resulted in the identification of the Drosophila Trr/COMPASS and its mammalian homologue, the MLL3/4 COMPASS-like complexes, as enhancer monomethyltransferases that cooperate with the histone demethylase UTX to regulate the transition from inactive/poised to active enhancers. I will also discuss our findings on role the Trr/MLL3/MLL4-specific “Trrific” enhancers play in the pathogenesis of cancers as a result of MLL3/MLL4 mutations.


SGI-110, A Novel Second Generation Hypomethylating Agent in Phase 2 Clinical Development for Hematological and Solid Tumor Malignancies

Tom Heightman
Senior Director, Medicinal Chemistry
Astex Pharmaceuticals

SGI-110 is a novel second generation DNA methylation inhibitor currently in Phase I/II clinical study for treatment of myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). SGI-110 is a dinucleotide prodrug of decitabine designed to be less prone to deamination by cytidine deaminase. Potential advantages over decitabine include improved stability, prolonged exposure to decitabine with potential for improved efficacy, and a more convenient subcutaneous administration.

The SGI-110 clinical trial design incorporates dose escalation and dose expansion studies in MDS and AML patients. The dose escalation study was designed to evaluate the biological activity, preliminary safety, and efficacy of SGI-110 with two dosing schedules in relapsed or refractory MDS or AML patients. The PK profiles show efficient conversion of SGI-110 to decitabine with achievable therapeutic exposures, longer apparent half life, and lower Cmax than predicted equivalent decitabine doses given IV. Global hypomethylating effects have been observed at all dose levels evaluated to date, and preliminary efficacy has been observed in heavily pretreated MDS and AML patients. The dose expansion study is ongoing and includes patients randomized to receive either the biologically effective dose or maximum tolerated dose to better evaluate both efficacy and safety in MDS and AML patients.

In addition, SGI-110 is being tested in solid tumors as a chemotherapy sensitizer in ovarian cancer in combination with carboplatin, and as a single agent in hepatic cancer patients, supported by pre-clinical data demonstrating demethylation and re-expression of aberrantly silenced tumour suppressor genes.


4SC-202: A Novel Epigenetic Modulator Translates into Clinic

Hella Kohlhoff
Manager Translational Pharmacology

4SC-202 is an oral epigenetic modulator of the Wnt and Hedgehog signaling pathway currently tested in a dose escalation phase I clinical trial in hemato-oncologic indications (TOPAS). Primary endpoints comprise safety, tolerability and pharmacokinetics. As secondary objective several PD/biomarker readouts are evaluated including ex vivo assays to determine the extent of enzyme inhibition in whole blood cells, protein acetylation in PBMCs and regulation of gene expression. First hints of anti-cancer efficacy could be determined as well by partial response of one patient and long term stabilization of heavily pretreated patients. 4SC-202 inhibits the Wnt signaling pathway by coordinated transcriptional regulation of positive and negative regulators of the signaling cascade, mediated by specific inhibition of HDAC1, 2 and 3 enzymes. Comparison with other class I specific HDAC inhibitors shows huge differences in gene regulation. By inhibition of the Wnt and Hedgehog signaling pathway, 4SC-202 provokes the inhibition of stemness-related properties like anchorage independent growth, spheroid formation, invasion and metastasis.

• Enzyme inhibition leads to complex mode of action
• Epigenetic modulator tested in phase I
• Biomarker development for epigenetic drugs


Lunch On Your Own

New Clinical Development of Epigenetic Drugs (Continued)
Chair: Adam S. Bristol, Aquilo Capital Management


Development of Histone Demethylase Inhibitors for Oncological and Neurodegenerative Disease

Tamara Maes

LSD1 is a histone demethylase and regulator of gene expression. LSD1 over-expression has been described in cancer and associated with bad prognosis. Oryzon LSD1 inhibitors were shown to selectively abrogate the clonogenic potential of acute myeloid leukemia cells with MLL translocations, sparing the repopulating potential of normal hematopoietic stem cells (Harris et al., 2012). ORY-1001 is a potent selective LSD1i with excellent pharmacological characteristics. ORY-1001 reduces leukemic stem cell potential, potently inhibits colony formation, overcomes the differentiation block in AML cell lines, and induces apoptosis / inhibits proliferation at sub-nanomolar concentrations in selected AML cell lines. ORY-1001 has received a positive opinion for orphan drug status for AML from the EMA and will start Phase I studies in 4Q 2013.

The potential of LSD1 inhibitors is not limited to cancer; LSD1 partners with Co-REST and REST/NRSF, a gene that represses neuronal genes in non-neuronal cells. Aberrant levels of REST/Co-REST are implicated in diseases like Huntington’s disease, Rett syndrome, and increased REST expression was found in the brain of Alzheimer disease patients. We have developed ORY-2001, an orally available dual LSD1/MAOB inhibitor with mid-nanomolar potency. ORY-2001 is well tolerated and can be administered chronically. ORY-2001 increases survival and improves motor function in models of HD and rescues memory and learning defects in SAMP8 mice, a model for Alzheimer disease.
These results indicate the potential for use of LSD1 inhibitors in oncological and neurodegenerative disease.


Markus Templin, Head Protein Profiling & Assay Development, NMI - Natural and Medical Sciences, Institute at the University of Tuebingen

Epigenetic Biomarkers and Diagnostics


An Integrated Quantitative Proteomics Strategy for Systems-biology of Protein Post-translational Modification (PTM)

Jack Cheng

Chief Executive Officer
PTM Biolabs

The specific inhibitors of Protein Post-translational Modification (PTM) regulative enzymes including HDAC, HMT, kinase and E3 ligase represent promising anti-cancer drugs, while great challenges of how to effectively screen the biomarker and targets of those inhibitors still exist. Using combinative advantages of high quality PTM pan antibodies based affinity enrichment and mass spectrometer- based quantitative proteomics, the PTM substrate profile in response to the inhibitors treatment can be intensively delineated, which provides the direct impacts on biomarker and target discovery in biomedical and biopharmaceutical pre-clinic studies.

1) high quality antibody development against PTM and “histone code”;
2) Integrated quantitative proteomics approach to identify novel PTM and “histone code”
3) Integrated quantitative proteomics approach to comprehensively profile PTM substrates and “histone code” in various pathophysiological conditions (both in cell and tissue level);
4) A systematic strategy for screening novel PTM-based biomarker and drug target


Networking and Refreshment Break

Chair: Gerd P. Pfeifer, City of Hope, Beckman Research Institute
3:45 Targeting Chromatin Modifiers to Overcome Resistance to Anti-Cancer Drugs

David Stokoe
Senior Scientist

The identification of specific molecular abnormalities in human tumors has recently given rise to the development of novel targeted therapeutics. Some of these have shown dramatic responses in defined patient population expressing these abnormalities, including Gleevec, Herceptin, Tarceva and Zelboraf. Unfortunately, in almost all cases, resistance to these agents develops, thereby hampering full potential clinical benefit. There is emerging evidence that one common mechanism underlying resistance to many of these agents involves changes in the chromatin landscape, including altered methylation and acetylation of key residues in Histone tails. Understanding the exact types of alterations, as well as the relevant modifying enzymes, represents a potential source of novel targets for therapeutic intervention. We have identified a requirement for specific histone modifying enzymes in establishing a state of reversible drug tolerance within a subpopulation of tumor cells. By combining targeted therapies with inhibitors of these enzymes, it is possible to prevent or delay the emergence of drug resistance. Through RNAi screening with a library that targets chromatin regulators, we have identified additional proteins required to engage the drug-tolerant state, which may represent additional targets for therapeutic intervention.

1. Understanding mechanisms of drug resistance
2. Profiling epigenetic alterations in drug tolerant cells
3. Identifying chromatin modulators critical for the drug tolerant state
4. Use of therapeutic agents to reverse drug resistance

4:10 A Paradox in Cancer Cells Enables Safe and Highly Effective Non-cytotoxic Epigenetic-differentiation Therapy

Yogen Saunthararajah
Staff member
Cleveland Clinic
Professor, Medicine
Lerner College of Medicine of Case Western Reserve University

A very basic problem with most oncotherapy is dependence on key apoptosis genes (e.g., TP53, p16/CDKN2A) to mediate cell cycle exit of cancer cells. This is a problem since such genes are amongst the most frequently deleted genes in cancer. Meanwhile, these same genes are intact in normal stem cells. Accordingly, most cancer treatment is substantially toxic, yet durable responses are only seen in the few cancers that have generally intact apoptosis genes (e.g., testicular cancer, pediatric acute lymphoblastic leukemia). Clearly therefore, there is a need for treatments that are novel not just with regards to proximal molecular targets, but also in terms of the downstream pathway used for cell cycle exit. Another important, but less well-appreciated characteristic of cancer cells, including cancer ‘stem’ cells, is very high expression of master lineage-specifying transcription factors. This is presumably because lineage-commitment is favorable for high-grade MYC activity and persistent cell growth and division. Metazoa (multicellular organisms), however, contain multiple late-differentiation genes that powerfully antagonize MYC to terminate proliferation (this is essential for coordinated multi-cellularity). Thus, cancer cells display a paradox: high expression of key lineage-specifying transcription factors yet aberrant epigenetic repression of the proliferation-terminating target-genes of these same transcription factors. In this presentation, using myeloid cancer as an example, the specific molecular mechanics underlying this paradox are detailed. Most importantly, we show how these molecular mechanics can be exploited for very safe and highly effective non-cytotoxic (p53-independent, normal stem cell sparing) epigenetic-differentiation therapy, illustrated by the results from the first interventional clinical trial that set out to translate these principles.

- understand cancer biology fundamentals
- appreciate core reasons for treatment resistance
- explore rational molecular bases to guide application of epigenetic drugs
- discover the clinical potential of such treatments

Epigenetic Tools and Technologies
4:35 ChIP-Seq on 10,000 Cells Using Diagenode’s MicroChIP and MicroPlex Library Preparation Protocols

Sharon Squazzo
Applications and Business Development Scientist

Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) has become the gold standard for whole-genome mapping of protein-DNA interactions. However, conventional ChIP and library preparation protocols associated with current high-throughput sequencing platforms require large amounts of starting material (at least hundreds of thousands of cells per immunoprecipitation) making it difficult for researchers to apply this technology when working with various limited cancer cell populations.
To overcome this barrier, Diagenode has developed a small-scale ChIP-Seq protocol compatible with 10,000 cells of starting material. Several experimental parameters have been optimized in the True MicroChIP protocol to enable successful low cell number ChIP-Seq such as; low concentration chromatin shearing, antibody titration, use of different carriers and wash stringencies. The MicroPlex Library Preparation protocol requires only picogram amounts of immunoprecipitated DNA inputs for sequencing library preparation and it is compatible with Illumina® platforms and barcodes.
During this presentation, we will present new ChIP-Seq tools for genome-wide analysis using the True MicroChIP protocol followed by MicroPlex Library Preparation protocols. The performance of this optimized method was evaluated for read mapping, sensitivity and specificity at a range of starting cell numbers covering three orders of magnitude of 1 million cells per IP down to 10,000 cells per IP. The combination of the True MicroChIP and MicroPlex Library protocols will now enable cancer researchers to perform epigenomic experiments who were previously unable to do so due to limited amounts of starting material.

1. Shearing chromatin from low cell number samples.
2. Optimization strategies for low cell ChIP protocols.
3. Preparing sequencing libraries from picogram amounts of starting material.
4. Comparing low input ChIP-Seq data to standard ChIP-Seq reference datasets.
5:00 An Epigenetic Mark Goes Global – New Tools for Proteome-wide Analysis of Lysine Methylation

Scott Carlson
American Cancer Society Postdoctoral Fellow
Gozani Lab, Department of Biology
Stanford University

Methylation of lysine on non-histone proteins has recently been found to have many important functions in mammalian systems. In addition, many lysine methyltransferases (KMTs) and demethylases are implicated in diseases including and cancer and neurodegenerative disorders. Despite the growing importance of lysine methylation, the physiological substrates of most KMTs have not been defined and, until recently, no proteome-wide strategies existed to identify proteins carrying lysine methylation. To address these questions we have developed a novel affinity reagent based on the triple Malignant Brain Tumor (MBT) domain of L3MBTL1 that recognizes mono- and di-methylated lysine with exquisite specificity. In the first proteome-wide analysis of methyl-lysine we have enriched methylated proteins from cell nuclei for identification by liquid chromatography and tandem mass spectrometry (LC-MS/MS). Quantitative analysis using stable isotope labels has shown that hundreds of proteins are enriched relative to a control reagent lacking affinity for methyl-lysine, and in many cases we have directly identified novel sites of methylation. These results prove that lysine methylation occurs far more widely across the proteome than previously realized. We have also shown that this approach can be used to identify non-histone substrates of KMTs. To understand how lysine methylation acts in disease progression we are monitoring methylation following over-expression or knock-down of oncogenic KMTs in cancer cell lines. The same approach is applicable to any organism or biological system, and will be a powerful tool to understand the role of lysine methylation in cell signaling.

• How do classical epigenetic post-translational modifications act through non-histone proteins to regulate cancer?
• What strategies are emerging to understand the biology of clinically-relevant enzymes that regulate lysine methylation?
• How diverse are the processes regulated by non-histone lysine methylation?
• How can novel strategies be used to understand the global effects of lysine methyltransferase


Networking Reception & Poster Session

Day 1 Day 2 Day 3
Day 3 - Friday, November 8, 2013
7:30 Continental Breakfast
Novel Epigenetic Targets in Early Development
Chair: Roy Pollock, Epizyme
8:00 Targeting Histone Methyltransferases in Cancer Therapy

Roy Pollock
Senior Director, Biological Sciences

Histone methyltransferases (HMTs) are a promising new class of therapeutic targets, especially for oncology indications where specific genetic alterations affecting HMT activity drive tumorigenesis. Epizyme has synthesized potent and selective small molecule inhibitors of several HMTs, including DOT1L and EZH2, as targeted therapeutics for human cancers bearing defined genetic lesions. The properties of such inhibitors, including their ability to selectively kill tumor cells bearing specific genetic alterations in cell culture and animal models, will be discussed.
8:45 Epigenetic Characterization of Genome Undergoing DNA Damage

Christopher Hale
Postdoctoral Fellow, Jacobsen Lab
University of California, Los Angeles

The eukaryotic genome has multiple pathways to maintain genome stability throughout the cell cycle. Epigenetically, many organisms employ a combination of DNA- and histone-level modifications as part of the transcriptional aspect of this maintenance. Eukaryotes also utilizes multiple layers of regulation to ensure that the DNA content of the cell remains constant. This regulation includes pathways relating to DNA replication liscensing and progression, as well as DNA repair pathways. Loss of either the epigenetic or genetic stability of the genome has been previously implicated in various aspects of cancer biology. In Arabidopsis we have identified mutations in histone methyltransferases that impact both the epigenetic and genetic stability of the nucleus. These mutations lead to aberrant DNA replication and engender a constituative DNA damage response. These phenotypes have allowed us to use whole-genome sequencing approaches to characterize the behavior of a genome undergoing DNA damage as well as carry out a forward genetic screen for factors that mediate the relationship between DNA replication and epigentic silencing. Furthermore, as part of this work, we have identified epigenetic responses to exposure to DNA damage and identified a role for the Arabidopsis BRCA1 ortholog in maintaining the epigenome.

Benefits of the talk:
How can non-human systems be used to gain insight into cancer biology?
What is the relationship between DNA damage and epigenetic marks such as DNA methylation?
How does BRCA1 influence the epigenome?
How does loss of epigentic stability affect DNA stability?

Oral Presentations from Exemplary Submitted Abstracts

9:10 EpiGnome Methyl-Seq: A Novel Post Bisulfite Conversion Library prep Method for Methylation Analysis

Fraz Syed, Illumina

9:20 HDAC1/2-Selective Inhibitor K-560 Exhibited a Cytostatic Anticancer Activity

Shinichi Uesato, Kansai University

9:30 Networking and Refreshment Break
Epigenetic Tools and Technologies (Cont.)
10:15 DNA Methylation in Development and Disease: What Is the Next Wave?

Guoping Fan
Professor, Human Genetics
University of California Los Angeles

Aberrant DNA methylation has been linked to human diseases such as cancer. Indeed, promoter hypermethylation in cancer cells are associated with the silencing of tumor-suppressor genes and global hypomethylation is related to cancer genome instability. Our understanding of DNA methylation in normal development and tumorigenesis is greatly enhanced by the technology of genomic bisulfite sequencing via next-generation sequencing. Genome bisulfite sequencing has provided unprecedented detail into the DNA methylation profile in normal somatic/stem cells as well as cancerous cells. These studies have yielded insights into: 1) wide-spread methylation changes during tumorigenesis and cell differentiation; 2) pervasive allele-specific methylation; and 3) high levels of non-CpG methylation in normal somatic cells such as neurons. More recently, the re-discovery of DNA hydroxymethylation catalyzed by Tet enzymes also leads to the role of DNA oxidation in the pathway of DNA demethylation. In this talk, I will present our recent studies of DNA methylation in cell reprogramming and in genetic disorders such as ICF Syndrome. I will also discuss the technology development in DNA methylation analysis including genome-scale base-resolution DNA methylation patterns in single-cells. The development of high-throughput tools in DNA methylation assays will further promote many exciting research fields including stem cell biology and cancer epigenetics.
10:40 A First in Class Chemical Probe for SETD7
John Tatlock, Senior Principal Scientist, Worldwide Medicinal Chemistry, Pfizer
Protein methyltransferases play a diverse role in epigenetic regulation of gene transcription, silencing, chromatin structure, DNA repair and replication. SETD7 was among the first histone lysine methyltransferases discovered and was originally characterized as a lysine 4 histone 3 (H3K4) monomethyltransferase. More recently, the role of SETD7 as a protein function regulator via the methylation of non-histone proteins has emerged.

While SETD7-mediated catalysis of protein methylation has been broadly characterized in vitro, the in vivo relevance has yet to be established. To directly evaluate the biological significance of SETD7 methyltransferase activity, a small molecule inhibitor of its catalytic function was developed. Structure guided optimization of a high throughput screening hit yielded (R)-PFI-2 as a potent (IC50 <10 nM) and selective inhibitor of human SETD7.

The Case for Pre-Competitive Chemical Probes
HTS to chemical probe story for a first in class HMT inhibitor
World’s First Small Molecule SETD7 Co-Crystal
11:05 Bioorthogonal Chemical Probes of Protein Acetylation

Y. George Zheng
Bioorthogonal Chemical Probes of Protein Acetylation
College of Pharmacy, University of Georgia

Dynamic lysine acetylation of proteins is involved in a variety of fundamental biological processes including epigenetic programing, cell cycle, apoptosis, metabolism, and signal transduction. Acetylation is introduced by protein lysine acetyltransferases (KATs) which transfer the acetyl group from the co-substrate acetyl-coenzyme A (acetyl-CoA, Ac-CoA) to the epsilon-amino group of specific lysine residues in proteins. Elucidating biological and pathological functions of protein lysine acetyltransferases (KATs) greatly depends on the knowledge of the dynamic and spatial localization of their enzymatic targets in the cellular proteome. We report the design and application of chemical probes for facile labeling and detection of substrates of the three major human KAT enzymes. In this approach, In this approach, a series of acetyl-CoA analogs with functional groups were synthesized for selective labeling and detection of KAT substrates. Meanwhile, we replaced the bulky residues in the cofactor-binding site of several KATs with smaller residues by site-directed mutagenesis in order to expand the cofactor binding pocket for AcCoA derivatives. By using sensitive fluorescent assays, we screened and identified several active enzyme-cofactor pairs. Rationally designed KAT-cofactor pairs are applied in conjunction with “clickable” reporters for KAT substrate labeling. The functionalized and transferable acyl moiety of the Ac-CoA analogs further allowed the labeled substrates to be probed with alkynyl or azido-tagged fluorescent reporters by the copper-catalyzed azide-alkyne cycloaddition. The AcCoA analogs, in combination with the rational protein-engineering approach, provide powerful molecular tools for labeling and mapping KAT targets in the context of complex biological mixtures at the proteomic level.
11:30 Targeting Leukemia Oncogene Expression with Inhibitors of Chromatin Reader Proteins

Christopher Ott
Research Fellow, Medical Oncology
Dana-Farber Cancer Institute, Harvard Medical School

The development of chemical inhibitors of proteins involved in epigenetic regulation of gene expression has led to the emergence of a limited number of promising therapeutic agents designed to target aberrant gene regulatory factors. These factors generally modify either chromatin or DNA by adding ('writing') or removing ('erasing') post-translational modifications of histones. Recently, small molecule inhibitors of 'reader' chromatin effector proteins have been developed. We have investigated the therapeutic potential of inhibitors of the BET class of human bromodomain proteins in several models of cancer, including B- and T-cell acute lymphoblastic leukemia (ALL). Bromodomains bind to post-translationally acetylated lysines of histones and mediate assembly of macromolecular protein complexes required for transcriptional activation and polymerase elongation. A first-in-class BET inhibitor developed in our laboratory, JQ1, is a thieno-triazolo-1,4-diazapine that binds with high affinity into the acetyllysine binding pocket of BET bromodomains. JQ1 potently reduces the viability of ALL cell lines with high-risk cytogenetics. JQ1 induces the transcriptional downregulation of leukemia oncogenes MYC and IL7R. Genome-wide expression profiling demonstrated a restricted effect of JQ1 on transcription with MYC and IL7R being among the most downregulated genes. In mouse models of ALL, JQ1 suppresses c-Myc expression and STAT5 phosphorylation, and significantly prolongs survival. These results demonstrate that BET bromodomain inhibition is a promising therapeutic strategy for cancer.

1) Discussion of small molecule inhibition of chromatin signaling.
2) Discussion of the chemical inhibition of chromatin ‘reader’ domains.
3) Application of chromatin inhibitors to cancer models.


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