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Overview | Speakers | Venue | Sponsors | Poster Submissions


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Day 1 Day 2
Day 1 - Monday, November 16, 2015
7:00 Continental Breakfast & Registration
7:55 Welcome & Opening Remarks
Plenary Session
Moderator: Charles Theuer, TRACON Pharmaceuticals
  The Discovery and Development of EZH2 Inhibitor Tazemetostat (EPZ-6438) and Identification and Optimization of First-In-Class PRMT5 Inhibitor EPZ015666 (GSK3235025)

Richard Chesworth

Vice Present, Molecular Discovery

  1. Targeting Lysine Methyltransferases: Identification and Optimization of First-In-Class
EZH2 Inhibitor Tazemetostat (EPZ6438)
The presentation will discuss the discovery of tazemetostat going from bench to bedside. It will include the role of Y641 EZH2 mutants on H3K27 methylation along with an overview of the multi-parametric optimization of a HTS hit in the discovery of Tazemetostat

2. Targeting Arginine Methyltransferases: Identification and Optimization of First-In-Class PRMT5 Inhibitor EPZ015666 (GSK3235025)
EPZ015666 demonstrated potent killing of cancer cells in vitro and efficacy in animal models of MCL, with correlation to methyl mark inhibition. EPZ01566 is orally bioavailable and demonstrated dose-dependent inhibition of intracellular symmetric arginine di-methylation of SmD3, a PRMT5 substrate that plays a critical role in RNA processing. In pre-clinical studies, Z138 and Maver MCL cell lines were shown to be sensitive to EPZ015666, and in xenografts of these cell lines, the compound showed dose-dependent tumor growth inhibition.
  Transcriptional Modulation of Oncogenic Pathways as a Therapeutic Strategy

James Audia
Chief Scientific Officer
Constellation Pharmaceuticals
  Pharmacologic inhibition of BET family bromodomains (BETi) results in the abrogation of cancer cell growth through the specific disruption of oncogenic gene expression. CPI-0610 is a potent and selective BET bromodomain inhibitor with favorable pharmacokinetic (PK) and pharmacodynamic (PD) properties currently in phase I clinical trials. Interim results from the clinic will be discussed, including aspects of its PK and PD performance, as well as signals of toxicity and efficacy. As patient selection strategies remain a critical outstanding challenge for developing BET bromodomain inhibitors, molecular features of acquired and de novo resistance will be discussed. In addition, we have examined BRD4 loading at gene control elements and our findings stress a role of promoter-associated BRD4 in pause-release and the selective transcriptional response to BETi. Finally, we discuss strategies to target oncogenic gene expression through the utilization of non-BET bromodomain small molecule inhibitors.
9:30 One Carbon Metabolism in Cancer

Mark Manfredi
Chief Scientific Officer
Raze Therapeutics

  Cancer cells co-opt normal metabolic pathways in response to the internal (e.g. oncogene activation) and external (e.g. hypoxia) environment for the benefit of survival and proliferation. Identifying cancer cell specific metabolic vulnerabilities remains the key challenge for therapeutic intervention. The original anti-metabolites included antifolates that inhibit one carbon metabolism (1CM) pathway. Several of these agents are still used today as cancer therapeutics. However, their cytotoxic effects on normal tissues limits their potential. The 1CM pathway derives one carbon units from serine and other sources. One carbon units are carried on a tetrahydrofolate scaffold to diverse utilization pathways including those that regulate epigenetics, redox status and anabolic processes. Several enzymes in the 1CM pathway have recently been implicated in cancer including 3-phosphoglycerate dehydrogenase (PHGDH), a cytosolic enzyme that catalyzes a key step in de-novo serine synthesis and is amplified and overexpressed in several cancers including breast cancer and melanoma. Mitochondrial 1CM enzymes that extract and transform 1C units are also overexpressed in cancer and regulated by oncogenes and hypoxia. For example, both serine hydroxymethyltranserase 2 (SHMT2) and methylene tetrahydrofolate 2 (MTHFD2) have been noted as cancer targets. This presentation will focus on target discovery and progress on the development of next generation inhibitors of the 1CM pathway.
10:15 Morning Networking Break
Epigenetics in Cancer Therapy
Moderator: Thomas Deraedt, Harvard Medical School
10:45 DOT1L as a Therapeutic Target for the Treatment of DNMT3A-Mutant Leukemia

Rachel Rau
Assistant Professor
Texas Children's Cancer and Hematology Center
Baylor College of Medicine

11:10 PRC2 Loss Amplifies Ras Driven Transcription and Confers Sensitivity to BRD4-Based Therapies

Thomas Deraedt
Harvard Medical School

  The polycomb repressive complex 2 (PRC2) exerts oncogenic effects in many tumor types. However, loss-of-function mutations in PRC2 components occur in a subset of hematopoietic malignancies, suggesting that this complex plays a dichotomous and poorly understood role in cancer. Genomic, cellular, and mouse modeling data demonstrate that the polycomb group gene SUZ12 functions as tumor suppressor in PNS tumors and high-grade gliomas by cooperating with mutations in NF1. NF1 encodes a Ras GTPase-activating protein (RasGAP) and its loss drives cancer by activating Ras. We show that SUZ12 loss potentiates the effects of NF1 mutations by amplifying Ras-driven transcription through effects on chromatin. Importantly however, SUZ12 inactivation also triggers an epigenetic switch that sensitizes these cancers to bromodomain inhibitors. Collectively, these studies not only reveal an unexpected connection between the PRC2 complex, NF1, and Ras, but also identify a promising epigenetic-based therapeutic strategy that may be exploited for a variety of cancers.
11:35 Unraveling p53 Transcriptional Networks in Tumor Suppression

Laura Attardi
Professor, Radiation Oncology and Genetics
Stanford University School of Medicine

  The p53 tumor suppressor gene is mutated in over half of all human cancers, of various types. p53 is a cellular stress sensor, inducing cell-cycle arrest, senescence, or apoptosis in response to various stress signals to impede proliferation. However, the molecular details of how p53 acts as a tumor suppressor remain enigmatic, and we have therefore used mouse genetics to define transcriptional programs involved in p53 function in tumor suppression. We have generated a set of p53 transcriptional activation domain (TAD) mutant knock-in mouse strains expressing p53 mutants in the first (p5325,26), second (p5353,54), or both TADs (p5325,26,53,54). We have found that p5325,26 is severely compromised for transactivation of most canonical p53 target genes, but retains the ability to activate a subset of p53 target genes, while p5353,54 is uncompromised in transcriptional activity and p5325,26,53,54 completely lacks transactivation activity. Interestingly, although unable to trigger cell-cycle arrest or apoptosis in response to acute DNA damage signals, p5325,26 retains full activity in suppressing various cancers, indicating that efficient transactivation of most classical p53 targets is dispensable for tumor suppression. As p5325,26 activates only a subset of p53-dependent genes, yet retains tumor suppressor activity, it has helped to pinpoint a small set of novel, direct p53-inducible tumor suppression-associated genes, whose functions we are currently analyzing. This approach will help define the transcriptional networks involved in p53-mediated tumor suppression, which has great significance for developing novel cancer therapeutics.
12:00 Lunch On Your Own
Epigenetic Mechanisms in Cancer
Moderator: Megan Hitchins, Stanford University School of Medicine
1:30 Epigenetic Regulation of Inflammation Modulates KRas-Driven Pancreatic Cancer Progression

Gregory David
Associate Professor, Biochemistry and Molecular Pharmacology
New York University School of Medicine

  Pancreatic ductal adenocarcinoma (PDAC) is virtually invariably a fatal disease, and is characterized by invasive and metastatic progression, as well as a striking resistance to conventional therapeutic approaches. In addition, recent reports have demonstrated that inflammation is a key component of precancerous lesion initiation and progression. However, the downstream events linking oncogenic activation KRas to inflammation are not yet fully understood. Here, we report that the chromatin associated Sin3B co-repressor, previously shown to be required for oncogene-induced senescence, promotes KRas-driven pancreatic lesions formation and progression in the mouse. At the molecular level, Sin3B is necessary for Kras-induced or chemically-induced inflammation of the pancreas. Importantly, we have now extrapolated these results to pancreatic human cells and have correlated Sin3B expression levels and inflammation in preneoplastic and neoplastic human pancreatic samples. Together, these results point to an unexpected tumor promoting function of senescence associated secreted cytokines. In addition, this study indicates that Sin3B and its associated chromatin modifying activities may represent potent therapeutic targets for the prevention of pancreatic cancer and other inflammation-driven cancers.
1:55 Roles for the Sirtuin SIRT5 in Tumor Metabolism

David Lombard
Assistant Professor of Pathology
University of Michigan

  The sirtuins are a family of NAD+-dependent protein deacetylases that regulate metabolism and other diverse aspects of cell biology. Three sirtuins (SIRT3, SIRT4, and SIRT5) are present in the mitochondrial matrix. SIRT5 is an inefficient deacetylase, instead removing succinyl, malonyl, and glutaryl groups from lysines of its target proteins (Du et al., 2011; Park et al., 2013; Peng et al., 2011; Rardin et al., 2013; Tan et al., 2014). To provide insight into SIRT5 functions, together with Dr. Yingming Zhao’s group, we previously carried out proteomics profiling of the cellular succinylome (Park et al., 2013). This revealed potential impacts of succinylation on enzymes involved in mitochondrial metabolism: amino acid degradation, TCA cycle, and fatty acid metabolism. We tested the impact of SIRT5 on two substrates identified in our studies, Pyruvate Dehydrogenase Complex (PDC) and Succinate Dehydrogenase (SDH). SIRT5 inhibited biochemical activities of both complexes, and suppressed overall mitochondrial respiration in the presence of glucose, pyruvate, and succinate (Park et al., 2013).

Metabolism in tumor cells is “reprogrammed” to meet the anabolic demands of uncontrolled cellular proliferation. Reprogramming of mitochondrial metabolism represents a key element of this metabolic rewiring. PDC and SDH are both implicated in neoplasia and cancer cell metabolic reprogramming. In particular, activation of PDC activity in cancer cells reverts tumor cell metabolism, inhibits malignant behavior, and induces apoptosis in diverse cancer types (Bonnet et al., 2007; Kim et al., 2006; McFate et al., 2008; Papandreou et al., 2006; Schulz et al., 2006). Given the role of SIRT5 in regulating PDC and SDH, we hypothesized that SIRT5 might play a role in metabolic reprogramming in cancer. Our progress in testing this idea will be discussed.

Support: NIH (R01GM101171 and R21CA177925 to D.B.L.; T32 AG000114 to J.P.) and the Glenn Foundation for Medical Research.
2:20 A Heterochromatic State Required for the Survival of Drug-Tolerant Cancer Cell Populations

Marie Classon

Scientist, Molecular Oncology
  Establishing and maintaining phenotypic heterogeneity within cell and organismal populations is an evolutionarily conserved strategy that ensures survival of the population following stressful exposures. We previously identified a transient, reversible, drug-tolerant cancer cell subpopulation that survives otherwise lethal drug exposures. Here we show that these drug-tolerant persisters (DTPs) originate in a sub-population of cells that assumes a highly heterochromatic state, following lethal drug exposures. This state is largely restricted to repetitive regions of the genome and requires factors that establish or bind to repressive histone tail modifications. This transcriptionally repressive state, which decreases the expression of retrotransposable elements, is critical for DTP survival and disruption of this heterochromatic state results in the ablation of drug–tolerant cells. These findings implicate epigenetic silencing of transposable elements as a population survival strategy in subpopulations of cancer cells during lethal drug exposures.
2:45 Constitutional Epimutation as an Alternative Mechanism to Genetic Mutation in Cancer Predisposition

Megan Hitchins

Associate Professor, Oncology
Stanford University School of Medicine
  Constitutional epimutation, defined as a heritable change in gene expression due to epigenetic modification and not a genetic mutation, has been identified as an alternative cause for high-risk cancer syndromes in patients without a germline mutation of the disease-associated gene(s). Constitutional epimutation thus represents a novel mechanism for disease causation. The most extensively studied of these is epimutation of the DNA mismatch repair gene, MLH1, which represents a minor cause for Lynch syndrome. Observational studies in the families of carriers of an MLH1 epimutation have revealed different patterns of inheritance of this epigenetic defect. These have included null inheritance, in which the MLH1 epimutation arose de novo in the cancer-affected proband and was not transmitted to the next generation due to germline erasure of the epimutation in the carrier. Non-Mendelian inheritance has been described in two Lynch syndrome families, in which the epimutation was transmitted to a proportion of offspring. Autosomal dominant inheritance has also been demonstrated in families whose epimutation is linked to an underlying genetic alteration in the vicinity of the gene. Although considered rare, the prevalence of MLH1 epimutation, and epimutations in general, is likely to be under-estimated, given this is an under-studied area of cancer etiology. Screening for this type of defect has yet to be implemented on a routine basis. This presentation will provide a brief overview of the role of epimutation in cancer causation, provide case studies of families with distinct inheritance patterns, and provide an outlook for integrating epimutation testing into mainstream diagnostics.
3:10 Afternoon Networking Break
Novel Epigenetic Targets
Moderator: Rachel Rau, Texas Children's Cancer and Hematology Center Baylor College of Medicine
3:40 Inhibitors of Epigenetic Target for Cancer Therapy

Jun Qi
Lead Scientist
Dana-Farber Cancer Institute

  In cancer, epigenetic proteins are intensely studied targets for drug discovery owing to the general view that it is not just the DNA sequence that is altered in epigenetics-based diseases. Our studies havebeen focused on developing small molecule inhibitors of chromatin modifying enzymes, so-called epigenetic “writers”, “readers”, and “erasers”, and have developed small moleculeinhibitors of the BET (for bromodomain and extraterminal domain) epigenetic readers, which recognizes the acetylated lysine side chain on histones. We then further designed and developed a series of small molecules and assays to target other epigenetic proteins, including writers and erasers. By utilizing these chemical and chemical biology tools, we are establishing the mechanistic understanding of these targets in cancer therapy.
4:05 Targeting Ubiquitination Activity of PRC1 Complex with Small Molecule Inhibitors in Cancer

Tomek Cierpicki
Assistant Professor
University of Michigan

  Bmi1 is a stem cell gene, which determines the proliferative capacity and self-renewal of normal and leukemic stem cells. Multiple studies identified Bmi1 to be able to induce oncogenic transformation and promote tumor growth in a variety of in vitro and in vivo animal models. Bmi1 is a central component of the Polycomb Repressive Complex 1 (PRC1) and interacts with Ring1B protein to form an active E3 ligase, which ubiquitinates histone H2A on lysine K119. This epigenetic modification of H2A is required to maintain transcriptionally repressive state of many genes. Small molecule inhibitors of the Ring1B-Bmi1 E3 ligase activity have not been reported to date, but are highly desired as potential therapeutic agents targeting cancer stem cells. We identified small molecule inhibitors of Ring1B-Bmi1 using fragment based drug discovery approach. Using medicinal chemistry, we developed compounds that bind to Ring1B-Bmi1 and inhibit E3 ubiquitin ligase activity with low micromolar affinities.

In this presentation I will describe our newly developed Ring1B-Bmi1 inhibitors. My presentation will cover:
- Characterization and mechanism of action in vitro
- Inhibition of H2A ubiquitination in cells
- Blocking self renewal of leukemia initiating cells
- Potential to develop these compounds into potent anti-cancer agents
4:30 Histone Variant H2A.Z.2: A Novel Driver of Melanoma Progression

Chiara Vardabasso
Senior Postdoc, Oncological Sciences
Bernstein Lab
Icahn School of Medicine at Mount Sinai

  Despite the emergence of effective targeted and immuno-therapies, malignant melanoma remains a largely incurable disease. Much effort has been expended in characterizing and targeting the genetic alterations, however studies have recently begun to shed light on the importance of epigenetic regulation in melanoma pathogenesis.

Histone variants are sequence and structural variants of canonical histones, which replace their conventional counterparts within the nucleosome through the action of dedicated factors known as histone chaperones. Histone variants and their chaperones are rapidly gaining attention in the field of cancer epigenetics. We have established a novel role for the uncharacterized histone variant H2A.Z.2 as a driver of malignant melanoma, the first report to define a biological function for H2A.Z.2 in any tumor type. H2A.Z.2 is highly expressed in metastatic melanoma, correlates with decreased patient survival, and is required for cellular proliferation. Our integrated genomic analyses revealed that H2A.Z.2 controls the transcriptional output of E2F target genes in melanoma cells. These genes are highly expressed and display a distinct signature of H2A.Z occupancy. We further identified the BET (bromodomain and extraterminal domain) protein BRD2 as an H2A.Z-interacting protein, whose levels are also elevated in melanoma. H2A.Z.2-regulated genes are bound by BRD2 and E2F1, and H2A.Z.2 recruits these factors to its targets. Importantly, H2A.Z.2 deficiency sensitizes melanoma cells to chemotherapy and targeted therapies. Collectively, our findings implicate H2A.Z.2 as a mediator of cell proliferation and drug sensitivity in malignant melanoma, holding translational potential for novel therapeutic strategies.
4:55 [Oral Presentation from Exemplary Submitted Abstracts] Inhibition of c-FLIPL Expression by miR-708 Increases the Sensitivity to Anticancer Drug in Renal Cancer Cells

Chang-Hoon Woo
Assistant Professor, Department of Pharmacology and Anatomy
Yeungnam University College of Medicine

  Dysregulation of the antiapoptotic protein, cellular FLICE-like inhibitory protein (c-FLIP), has been proven to be associated with tumorigenesis and chemoresistance in various types of human cancers. Therefore, c-FLIP is an excellent target for therapeutic intervention. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in tumorigenesis, tumor suppression, and resistance or sensitivity to anticancer drugs. It remains unclear whether miRNAs can regulate the expression of c-FLIP. The goal of this study was to identify miRNAs that could inhibit the growth and induce cell death of renal cancer by targeting c-FLIP expression. We show that c-FLIPL and miRNA-708 expressions are inversely correlated, that is, c-FLIPL is upregulated and miRNA-708 is rarely expressed in renal cancer cells. Luciferase report assay demonstrated that miRNA-708 negatively regulated c-FLIPL expression by binding to miRNA-708 binding site in 3' untranslated region of c-FLIPL. We also show that ectopic expression of miRNA-708 increases the accumulation of sub-G1 populations as well as the cleavage of procaspase-3 and PARP, which were prevented by pretreatment with the pan-caspase inhibitor, Z-VAD. Furthermore, ectopic expression of miRNA-708 increases the sensitivities to various apoptotic stimuli such as tumor necrosis factor–related apoptosis-inducing ligand (TRAIL), doxorubicin, thapsigargin in Caki cells. Interestingly, miRNA-708 specifically repressed c-FLIPL without any change on c-FLIPs expression. In contrast, inhibition of endogenous miRNA-708 by use of antago-miRNA results in increase of c-FLIPL protein expression and resistance to TRAIL, doxorubicin, and thapsigargin treatments. We found that c-FLIPL expression was upregulated in renal cell carcinoma tissues compared with normal tissues. Inversely, miRNA-708 expression was reduced in renal cell carcinoma tissues. Moreover, miRNA-708 enhances tumor-suppressive effect of doxorubicin in a xenograft model of human renal cancer. In conclusion, these findings suggest that miRNA-708 should be considered as a tumor suppressor because it negatively regulates the antiapoptotic protein c-FLIPL and regulates sensitivities to various apoptotic stimuli.
5:05 Evening Reception & Poster Session
Day 1 Day 2
Day 2 - Tuesday, November 17, 2015
7:00 Continental Breakfast
Genetic & Epigenetic Changes: Cancer & Stem Cells
Moderator: Marie Classon, Genentech
8:00 Epigenetic Heterogeneity and Treatment-Resistant Cancer Stem Cells

Michael Kladde

Associate Professor, Biochemistry and Molecular Biology
University of Florida Health Cancer Center
  Tumors are comprised of heterogeneous cells with diverse molecular and phenotypic features, such as clinically relevant cancer stem cells. The reversible nature of cancer cell proliferative potential and drug tolerance suggests plasticity; however, the extent to which epigenetic heterogeneity underlies these phenotypes remains largely unexplored. We developed MAPit-patch to map chromatin accessibility and DNA methylation at high resolution on single molecules using targeted next-generation bisulfite sequencing. Using MAPit-patch to profile 71 promoters of cancer-associated genes in human glioblastoma (GBM) and normal neural stem cells (NSC) revealed substantial intra-locus epigenetic heterogeneity, i.e., considering each locus individually across all sequenced reads. This unmasking of epigenetically distinct cellular subpopulations suggests high overall chromatin dynamics in both NSC and GBM. Promoters that were hypermethylated in GBM initially exhibited inaccessibility and variable DNA methylation in NSC, suggesting that promoters with increased nucleosome occupancy in NSC become hypermethylated in GBM. At the MLH1 promoter, a minority subpopulation of GBM cells with inaccessible, but unmethylated, chromatin was detected, which correlated with the fraction of MLH1-negative and temozolomide-tolerant GBM cells and was absent in NSC. Importantly, enrichment of these epigenetically distinct subpopulations in temozolomide-tolerant GBM cells indicated that these cells, in part, contribute to intratumoral phenotypic diversity. Outgrowth of temozolomide-tolerant cells in the presence of a low concentration of the drug selected for cells with hypermethylated MLH1 chromatin. We conclude that temozolomide tolerance of a subpopulation of GBM cells is mechanistically linked to increased nucleosome occupancy and transcriptional repression of the promoter of the mismatch repair gene MLH1.

My presentation will cover:
• MAPit-patch, a cost-effective, scalable, and single-molecule method for targeted detection of DNA methylation and chromatin accessibility at many genes in a single tube.
• The widespread phenomenon of intra-locus epigenetic heterogeneity across cell populations.
• Sensitive detection of subpopulations of epigenetic configurations previously obscured by genome-wide and population-ensemble epigenetic techniques.
• Identification of subpopulation of GBM cells with increased nucleosome occupancy and transcriptional repression of the MLH1 promoter, conferring chemotherapeutic resistance.
• At MLH1 in GBM, increased nucleosome occupancy appears to precede DNA hypermethylation.
8:25 Histone Demethylation Sanctions Enhancer DNA Methylation during Embryonic Stem Cell Differentiation

Humaira Gowher

Assistant Professor
Purdue University
  The timing and location of DNA methylation are critical parameters controlling cell differentiation. Dysregulation of these processes has wide-spread consequences ranging from developmental disorders to cancer. Dnmt3a and 3b methyltransferases (MTases) that primarily catalyze de novo DNA methylation have essential, distinct roles in embryonic development and differentiation. Currently proposed mechanisms of targeting Dnmt3a and 3b to specific genomic sites include the interaction with some DNA and chromatin binding factors. Insights into chromatin-mediated regulation of DNA MTases activity are provided by the genome-wide comparisons of epigenetic marks which show an inverse correlation between DNA methylation and histone H3K4 methylation. In addition, in vitro structural studies show that a specific interaction of the chromatin binding domain (ADD) of Dnmt3a with histones that are unmethylated at H3K4 leads to catalytic activation of the enzyme. However, the physiological role of this histone-mediated modulation of Dnmt3a activity in regulation of gene expression has not been fully defined. When embryonic stem cells differentiate pluripotency genes are repressed which requires their specific enhancers to be decommissioned by lysine demethylase Lsd1 activity. We demonstrate that demethylation of histone H3K4 monomethylation at pluripotency gene enhancers by Lsd1 serves to target Dnmt3a via their ADD domains, thus targeting DNA methylation regionally. Our data here show the functional role of Dnmt3a as a reader and effector in this queue of epigenetic events, where local histone demethylation and deacetylation allows the interaction and specific activation of Dnmt3a to lock the Pp enhancers in a stable repressive state.
8:50 Dynamic Enhancer Landscapes During Pancreatic Differentiation of Human ES Cells

Feng Yue
Assistant Professor
Pennsylvania State University College of Medicine

  Temporal and spatial-specific gene transcription is tightly controlled by cis-regulatory elements such as promoters and enhancers. Here we show that epigenetic priming of enhancers signifies developmental competence using during pancreatic differentiation of human ES cells. We performed RNA-Seq, GRO-Seq and ChIP-Seq for H3K4me3, H4K4me1 and H3K27Ac in each developmental stages, including hESCs, definitive endoderm (DE), primitive gut tube (GT), posterior forgut (FG), and pancreatic endoderm (PE). We observed that poised enhancer state could be used to predict the ability of developmental intermediates to respond to inductive signals. We further find that lineage-specific enhancers are first recognized by transcription factors involved in chromatin priming, while subsequent recruitment of lineage- inductive transcription factors leads to enhancer and target gene activation. Our results identify acquisition of a poised chromatin state at enhancers as a general mechanism by which progenitor cells gain the competence to rapidly activate lineage-specific genes in response to inductive signals.
9:15 [Oral Presentation from Exemplary Submitted Abstracts] Methylation-Driven Subtyping of Head and Neck Squamous Cell Carcinoma

Julie Koenig
M.D. Candidate
Stanford School of Medicine

  Head and neck squamous cell carcinoma (HNSCC), a leading cause of cancer related death, is highly heterogeneous, with multiple distinct etiologies and molecular profiles, suggesting that multiple disease subtypes exist. Established HNSCC risk factors include HPV infection, smoking and age, all of which can alter DNA methylation. Therefore, we hypothesized that profiling of DNA methylation within HNSCC populations could identify HNSCC intrinsic subtypes. We applied our MethylMix algorithm to genome-wide DNA methylation data for 528 HNSCC cases from The Cancer Genome Atlas to identify epigenetic ‘driver genes.’ These are genes that are abnormally methylated in cancer with corresponding alteration of mRNA expression. We then used unsupervised consensus clustering of driver gene methylation to classify patients into robust clusters or putative subtypes. Of 5 subtypes, one had significantly improved survival and was enriched for HPV+ tonsilar and oropharyngeal cancers. This subtype was highly immunogenic and was enriched for overexpression and hypomethylation of meiosis-related genes, suggesting loss of epigenetic silencing of developmental genes as a mechanism of HPV pathogenesis. A putative ‘epigenetically driven’ subtype was discovered, which had a strikingly elevated number of hypermethylated genes and was enriched for CASP8 mutations. Two ‘smoking-related’ subtypes were identified, one of which displayed widespread DNA hypomethylation and inactivating mutations and/or somatic deletions within the histone methyltransferase NSD1. This implies a role of NSD1 in stabilizing DNA methylation levels. This study demonstrates that biologically and clinically distinct epigenetic HNSCC subtypes exist. Further investigation of these subtypes may lead to discovery of subtype-specific biomarkers and therapeutic targets.
9:25 [Oral Presentation from Exemplary Submitted Abstracts] Heterochromatin Protein HP1gamma Promotes Colorectal Cancer Progression and is Regulated by miR-30a

Quan Zhao
Deputy Dean of School of Life Sciences
Nanjing University, China

  Colorectal cancer (CRC) pathogenesis remains incompletely understood. Here we report that the heterochromatin protein HP1? is upregulated commonly in human CRC where it promotes cell proliferation in vitro and in vivo. Gene expression and promoter binding experiments demonstrated that HP1? directly regulated CDKN1A (p21Waf1/Cip1) in a manner associated with methylation of histone H3K9 on its promoter. We identified miR-30 as a tumor suppressive microRNA that targets HP1? in vitro and in vivo to specifically suppress the growth of colorectal cancer in mouse xenograft models. MiR-30a was widely downregulated in primary human CRC tissues where its expression correlated inversely with high levels of HP1? protein. Our results identify a new miR-30a/HP1?/p21 regulatory axis controlling CRC development which may offer prognostic and therapeutic opportunities.
9:35 [Oral Presentation from Exemplary Submitted Abstracts] Epigenetic Regulation of Hypoxia and Heterogeneity, Modifying Metastasis by Regulating Epigenome

M.A. Nezami
Pacific Medical Center of Hope

  The main barrier to effective treatment of many types of solid tumors is heterogeneity and genetic instability of cancer. It has been hypothesized that targeting angiogenesis could manage this problem, as it modifies microenvironmental cross talk with the tumor, reducing the chemoresistance and improving the response to the available therapies. Clinical experience with this approach revealed unexpectedly distinct responses between different tumors and organ sites. It has been postulated that the wide spectrum of pre-clinical and clinical results obtained with anti-angiogenic agents is a result of functional linkage existing between genetic and epigenetic variabilities. The epigenetic regulations controlling tumor and microenvironment cross-talk, resulting in new blood vessel formation, is a dynamic process, causing heterogeneity and progressive characteristics similar with the instability of the cancer cell genome. Research of epigenetic regulations of targets in angiogenesis is relevant and exciting. We present a summary of cases of advanced Stage IV patients with heterogenous cancer who were treated using a novel epigenetic therapy, in a protocol called multi targeted epigenetic therapy (MTET), resulting in independent “antiangiogenic response” identified by disseminated circulatory tumor cells analysis and translated to improved progression free, or overall survival. We follow the serum/plasma VEGF measurements as a biomarker for vasculogenesis, and circulatory tumor cells assay, which can be used as a prognostic marker. We conclude that this small sample presents considerable effect size and can impact the current practice of oncology by providing better prognostic and therapeutic tools targeting angiogenesis in refractory heterogeneous disease, by regulating the epigenome.
Epigenetic Tools & Technologies
Moderator: Feng Yue, Pennsylvania State University College of Medicine
9:45 Visualizing Human Transcription at Nucleotide Resolution by Native Elongating Transcript Sequencing

Stirling Churchman
Assistant Professor, Genetics
Harvard Medical School

  Major features of transcription by human RNA Polymerase II (Pol II) remain poorly defined due to a lack of quantitative approaches for visualizing Pol II progress at nucleotide resolution. We developed a simple and powerful approach for performing native elongating transcript sequencing (NET-seq) in human cells that globally maps strand-specific Pol II density at nucleotide resolution. NET-seq exposes a mode of antisense transcription that originates downstream and converges on transcription from the canonical promoter. Convergent transcription is associated with a distinctive chromatin configuration and is characteristic of lower-expressed genes. Integration of NET-seq with genomic footprinting data reveals stereotypic Pol II pausing coincident with transcription factor occupancy. Finally, exons retained in mature transcripts display Pol II pausing signatures that differ markedly from skipped exons, indicating an intrinsic capacity for Pol II to recognize exons with different processing fates. Together, human NET-seq exposes the topography and regulatory complexity of human gene expression.
10:10 Morning Networking Break
10:45 Profiling Epigenomic Landscapes and Gene Regulatory Networks

Johanna Samuelsson
Senior Research Scientist
Active Motiff

  Chromatin immunoprecipitation followed by next generation sequencing (ChIP-Seq) and other genome wide technologies have been integral in advancing our understanding of how epigenetic phenomena are regulated and how they affect gene expression. However, as we ask more complex questions the limitations of traditional genome-scale approaches have motivated researchers to develop new and improved methodologies for the characterization of the epigenomic landscape.

This presentation will cover some of the progress that we and others have made in advancing the traditional ChIP-based assays and will include a new ChIP-Seq spike-in method for improved normalization and quantitation, a unique engineered DNA-binding molecule-mediated ChIP technology using the CRISPR system for dissecting the chromatin structure of your genomic regions of interest, and the development of a novel transposase based ChIP assay enabling the investigation of multiple targets within the same sample. These advanced technologies can be applied in new fields and diverse models to gain a deeper understanding of the complex regulatory mechanisms governing our genomes.
11:10 Single-Cell Chromatin Accessibility Reveals Principles of Regulatory Variation

William Greenleaf
Assistant Professor, Genetics
Stanford University School of Medicine

  Recent proliferation of powerful methods for interrogating single cells has allowed detailed characterization of phenotypic molecular variation at the single cell level, and provided deep insight into characteristics underlying cancer heterogeneity and drug resistance. In parallel, genome-wide mapping of regulatory elements in large ensembles of cells have unveiled variation in chromatin structure across cell-types, particularly at distal regulatory regions. However, the lack of methods to probe DNA accessibility within individual cells has prevented dissection of single cell regulatory variation – variation the likely drives phenotypic heterogeneity. We have developed single-cell ATAC-seq (scATAC-seq), a robust method for mapping the accessible genome of individual cells via assay for transposase-accessible chromatin using sequencing (ATAC-seq). Maps aggregated from data generated from hundreds of single-cells closely resemble accessibility profiles from tens of millions of cells and provides insights into cell-to-cell variation. We have developed novel single cell analysis methods that can associate variability in chromatin accessibility with sets of genomic loci. Accessibility variance is associated with specific trans-factors and cis-elements, and combinations of trans-factors are associated with either induction or suppression of cell-to-cell variability. We further identify sets of trans-factors associated with cell-type specific accessibility variance across cell types. Surprisingly, genomic regions that are correlated in accessibility variation in cis across the genome are likely to lie in the same topological domain, providing a link between single-cell accessibility variation and three-dimensional genome organization. All together, single-cell analysis of DNA accessibility provides new insight into cellular variation of the “regulome” at ultimate sensitivity, with powerful potential applications to basic biology and clinical investigation.
11:35 Lysine Methylation from Epigenetics to mRNA Splicing

Scott Carlson
Postdoctoral Fellow, Gozani Lab
Stanford University School of Medicine

  We recently reported the first proteomic strategy to enrich and identify proteins modified by lysine methylation (Kme), revealing hundreds of proteins potentially regulated by this modification. Splicing factors featured prominently among proteins modified by Kme, as well as dozens of protein functionally linked to splicing regulation. Pre-mRNA splicing is a key step in transcriptional regulation and the splicing machinery is responsible for complex decisions about alternative exon utilization. Since Kme is a central part of the epigenetic “histone code” we hypothesize that Kme may act similarly as part of a post-translational “splicing code”.

We have developed a range of proteomic and candidate-based approaches to discover which methyltransferase enzymes (KMTs) regulate splicing factor Kme, and to identify the downstream “reader” proteins that recognize specific methylation sites. We will present data showing methylation and recognition of core spliceosome proteins by specific KMTs and reader domains.

• New proteomic techniques to characterize the activity of methyltransferase enzymes
• New approaches to identify interactions regulated by methylation of non-histone proteins
• Lysine methylation regulates splicing factors that are often mutated in diverse cancers
• Non-histone methylation pathways may present new targets for cancer therapy
12:00 Lunch Provided by GTCbio
1:30 Conference Concludes
Day 1 Day 2

cancer-epigenetics Agenda