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Epigenetics: Cancer and Beyond

Epigenetics: Cancer and Beyond
Reported by
Robert Frawley

Posted July 07, 2016


DNA transcription is regulated by epigenetic processes which alter chromatin structure, including posttranslational modifications like acetylation. These modifications affect the interactions between histones and DNA, remodeling chromatin and recruiting proteins for transcription. Epigenetic abnormalities contribute to tumorigenesis and are known to be involved in other diseases. The Epigenetics: Cancer and Beyond symposium, presented by the Academy's Cancer and Signaling Discussion Group on April 28, 2016, gathered researchers to discuss therapeutic targeting of epigenetic modifiers via small molecule inhibitors.

Craig Thompson of Memorial Sloan Kettering Cancer Center began the first session, on epigenetics in health and disease, with a summary of his work on the cellular energetics and metabolism of epigenetic marks. Thompson noted that modifications such as acetylation and methylation, which attach an acetyl or methyl group, respectively, use high-energy molecules as inert markers; acetyl and methyl groups can both produce considerable amounts of ATP, and storing such ATP sources would be dangerous for cells in a low-energy state. Thus, Thompson asserts that acetylation specifically is a rheostat for cellular metabolism, with an abundance of acetate increasing acetylation and transcriptional activity. He demonstrated that acetylation is driven by glucose abundance in cells. He also identified specific mutations in the enzyme isocitrate dehydrogenase (IDH) that lead to neomorphic enzyme activity—in which the mutated enzyme not only loses its original function but also gains a new, often pathologic function—creating the pathologic metabolite 2-hydroxyglutarate (2-HG). Acetate and IDH are required in the tricarboxylic (TCA) cycle for mitochondrial metabolism, and the mutation creates stress on ATP synthesis that may drive cancer progression. Thompson described drugs created to target IDH mutations and thus to decrease 2-HG production; in human trials, about 40% of individuals in an acute myeloid leukemia (AML) study went into remission because the drug induced bone marrow cells to differentiate rather than maintain cancer stem cell potential and then proliferate. Methylation changes are closely tied to cell fate and cancer phenotypes in several cancers, particularly AML.

The epigenetic code refers to secondary modifications to chromatin components that regulate chromatin activity: transcription is regulated by addition, removal, or recognition of these modifications (writers, erasers, readers); acetylation is associated with active transcriptional regions of chromatin; BET bromodomains (readers) bind to acetylated lysines on histones and recruit additional transcription factors. (Image courtesy of Ewelina Kulikowski)

Roberto Pili from the Indiana University School of Medicine described the benefits of combinatorial therapies pairing epigenetic modifiers with tyrosine kinase inhibitors (TKIs), interleukin-2 immunotherapy, or checkpoint blockade. Pili hypothesized that targeting transcription factors with histone deacetylase (HDAC) inhibitors would improve the antitumor response to standard therapies. He examined antiangiogenic resistance, in which tumors proliferate despite having their blood supply cut off via medical intervention, and demonstrated that this resistance is reversible and could be driven by epigenetics. Using kinomic analysis, an assessment of kinases in action, he found that the likelihood of developing resistance to TKIs used to treat angiogenesis is different for each drug, possibly because of epigenetic drug interactions. Pairing HDAC inhibitors such as entinostat, which regulates immunosuppressive regulatory T (Treg) cells, with high-dose IL-2 immunotherapy increased antitumor activity. There is evidence that the drug also increases antitumor activity in human primary tumors treated with checkpoint blockade inhibitors.

Epigenetic targeting may therapeutically enhance current immunotherapies. (Image courtesy of Roberto Pili)

Keiko Ozato of the National Institute of Child Health and Human Development spoke about bromodomain-containing protein 4 (BRD4) and the important role it plays in blood- and stem-cell differentiation, in cell proliferation, and in cell-cycle gene regulation. The protein is epigenetically regulated by acetylated histones. Discovery of the bromodomain protein family, which includes the bromodomain and extra terminal domain (BET) family, and of BRD inhibitors led to a spike in interest several years ago, but global BET knockout models are embryonic lethal. To describe the functions of Brd4, Ozato's group generated Brd4 knockout in erythroid lineages, macrophages, and hematopoietic stem cells (HSCs) in mouse models. The group found that BRD4 is required for myeloid and lymphoid cell differentiation from HSCs. Interestingly, Brd4 is required for the development of some thymocytes (Th2 and Th17) but not others (Th1 and Tregs). RNA sequencing in erythroblasts showed that Brd4 promotes cell division and cell-cycle progression (knockout cells stalled before the G2 phase), and thousands of genes were differentially regulated between wildtype and knockout lineages. ChIP sequencing showed direct interaction between BRD4 and a large number of the genes assayed. Ozato found that BRD4 is essential for specific-lineage cell proliferation and for early development of immune cells and red blood cells, and that it drives transcription broadly.

Christopher Vakoc from Cold Spring Harbor Laboratory closed the morning session with a discussion of specific activities of BRD4. His group used the gene editing technology CRISPR to knock out functional protein domains to elucidate bromodomain activity. The screens identified a lineage-specific response linked to the activity of Myc protein, which though ubiquitously expressed is regulated according to lineage. Leukemia is particularly susceptible to BRD4 inhibition, and Vakoc found that the target genes for acetylation by the p300 acetyltransferase are BRD4-dependent. This specificity may be controlled by recognition of super-enhancer regions of DNA. While the presence of super-enhancer regions is necessary for blood-cell specificity, it is not sufficient; other events specific to BRD4 inhibition correlated to expression of a transcriptional regulator and other factors implicated in leukemia. Discovering why BRD4 inhibition is so specific to leukemic cells could lead to therapeutic opportunities.

In the second session, on epigenetic therapies, Michael Elowitz from the California Institute of Technology discussed epigenetic memory, the durability of epigenetic change with proliferation and inheritance. Researchers know many of the interacting partners in epigenetics but do not have the functional understanding that would be needed to therapeutically rewire the pathways. Elowitz's group studied epigenetic regulation in single cells using time-lapse imaging with a yellow florescent protein reporter, testing four chromatin regulators with a range of activities (histone methylation, DNA methylation, and HDAC4 deacetylation). The group found that gene silencing occurs in an all-or-nothing response that includes rapid deactivation. Only some epigenetic transitions were reversible. Using flow cytometry data, the group developed a 3-state model for gene transcription (active, inactive-reversible, inactive non-reversible) and determined reaction constants for each type of modification (methylation, deacetylation, and so on). Elowitz's research, by creating a unified model of epigenetic modification, is a step toward the development of usable synthetic biology for cell memory and epigenetic recording.

Daniel Vitt of the biotech company 4SC AG presented his group's work on a small molecule designed to treat small cell lung cancer and hematological tumors. The molecule, 4SC-202, is a well-tolerated immunomodulator that sensitizes checkpoint inhibitors; it broadly regulates deacetylases, enhancing tumor accessibility and reducing immunosuppression. Combination treatment with 4SC-202 and anti-PD1 therapy decreased tumor volume notably in several tumor models in vitro and in mouse models. The combination drug boosted immune cell numbers, increasing immune response and reducing immune suppression in the mice. It also limited cancer cells' clonogenic potential via hedgehog pathway inhibition, which blocks tumor colony formation in vitro. The drug's combination of immune-priming and clonogenic-cell targeting has produced a robust response in clinical trials.

Ewelina Kulikowski of Resverlogix described the company's drug apabetalone (RVX-208), which produces phenotypic changes in cardiovascular disease. Another BET-inhibitor, apabetalone binds preferentially to the second bromodomain of BRD4, thereby inhibiting the bromodomain–lysine interaction. Apabetalone is known to modulate several pathways, with effects on the complement and fibrin clotting pathways, in vascular inflammation and calcification, and in diabetes. Apabetalone downregulated numerous proteins in the complement and fibrin clotting pathways and reduced the expression of signature genes important in acute cardiac infarction and vascular inflammation. Kulikowski described several clinical studies by her group in which modulation of these pathways contributed to lower rates of major adverse cardiac events (MACE). She also described an ongoing phase III trial in cardiovascular disease. She emphasized that epigenetic markers play a key role in physiology, with applications beyond oncology and immunotherapy.

Eric Campeau of Zenith Epigenetics continued the discussion of BRD4, presenting data on the company's pan-BET-inhibitor ZEN-3694, which was developed with the help of a computational screen for small molecule inhibitors. This molecule, which targets androgen receptor (AR) signaling, has activity in prostate cancer, but unlike existing AR antagonists or drugs that target the androgen ligand, ZEN-3964 acts through epigenetic mechanisms. It blocks the BRD4 interaction that is stimulated by AR binding, to prevent the transcription of oncogenes. The drug is efficacious in various preclinical models of prostate cancers that are resistant to current therapies, and acts through different mechanisms across prostate cancer subtypes. ZEN-3694 is currently in phase I clinical trials in metastatic castration-resistant prostate cancer. This novel mechanism of inhibition is predicted to offer good combinatorial potential with current and future cancer therapies, including chemotherapy, targeted agents, checkpoint blockade, and immunotherapies.

While other prostate cancer drugs limit androgen receptor activity, either by reducing androgen synthesis or by antagonizing the receptor, the pan BET-inhibitor blocks the ligand-binding AR signal and the activity of the constitutively active AR V7 isoform. (Image courtesy of Zenith Epigenetics)

Patrick Trojer of Constellation Pharmaceuticals concluded the symposium with a talk on a new drug, CPI-1205, that targets the methyltransferase enhancer of zeste homolog 2 (EZH2). The EZH2 protein is part of the polycomb repressive complex 2 (PRC2), which promotes tumor progression and suppresses chemokine production through transcriptional regulation. EZH2 inhibitors, such as CPI-1205, block the catalytic site without breaking down the PRC2 complex. An EZH2-inhibitor and PRC2 co-crystal structure confirmed that the EZH2 inhibitor binds in the EZH2 SET domain and partially overlaps with the SAM binding site, thereby confirming the SAM-competitive mechanism of inhibition. CPI-1205 is a potent, selective EZH2 inhibitor that reduces global histone H3K27 trimethylation in a reversible way. It selectively kills many, though not all, B-cell lymphoma cell lines. In vivo data from B-cell lymphoma xenograft models show tumor regression with CPI-1205 treatment as a single agent and synergistic antitumor effects with chemotherapy. In a 10-patient phase I trial, the drug had anti-lymphoma activity at various doses and no adverse effects. Researchers are hopeful that the therapy will continue to be effective in subsequent trial phases.

CPI-1205 targets the methyltransferase enhancer of zeste homolog 2 (EZH2), part of the polycomb repressive complex 2 (PRC2), which promotes tumor progression and suppresses chemokine production through transcriptional regulation. (Image courtesy of Patrick Trojer)

Use the tabs above to find multimedia from this event.

Presentations available from:
Eric Campeau, PhD (Zenith Epigenetics)
Ewelina Kulikowski, PhD (Resverlogix Corp.)
Roberto Pili, MD (Indiana University School of Medicine)
Christopher Vakoc, MD, PhD (Cold Spring Harbor Laboratory)
Daniel Vitt, PhD (4SC AG)

The Biochemical Pharmacology Discussion Group is proudly supported by:

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How to cite this eBriefing

The New York Academy of Sciences. Epigenetics: Cancer and Beyond. Academy eBriefings. 2016. Available at:

Exploiting Epigenetic Therapies for Drug Resistance and Immunomodulation

Roberto Pili (Indiana University School of Medicine)

Chromatin Regulators as Cancer Dependencies

Christopher Vakoc (Cold Spring Harbor Laboratory)

Epigenetic Regulators of Tumor Microenvironment: New Ways for Enhancing Responses to Immunotherapies

Daniel Vitt (4SC AG)

New Horizons for BET Inhibition: Apabetalone for the Treatment of High-risk Cardiovascular Disease

Ewelina Kulikowski (Resverlogix Corp.)

Preclinical Characterization of ZEN-3694, a Novel BET Bromodomain Inhibitor in Phase I Studies for mCRPC

Eric Campeau (Zenith Epigenetics)

Journal Articles

Alsarraj J, Walker RC, Webster JD, et al. Deletion of the proline-rich region of the murine metastasis susceptibility gene Brd4 promotes epithelial-to-mesenchymal transition- and stem cell-like conversion. Cancer Res. 2011;71(8):3121-31.

Bhadury J, Nilsson LM, Muralidharan SV, et al. BET and HDAC inhibitors induce similar genes and biological effects and synergize to kill in Myc-induced murine lymphoma. Proc Natl Acad Sci U S A. 2014;111(26):E2721-30.

Bintu L, Yong J, Antebi YE, et al. Dynamics of epigenetic regulation at the single-cell level. Science. 2016;351(6274):720-4.

Devaiah BN, Case-Borden C, Gegonne A, et al. BRD4 is a histone acetyltransferase that evicts nucleosomes from chromatin. Nat Struct Mol Biol. 2016;23(6):540-8.

Duffy BC, Liu S, Martin GS, et al. Discovery of a new chemical series of BRD4(1) inhibitors using protein-ligand docking and structure-guided design. Bioorg Med Chem Lett. 2015;25(14):2818-23.

Figueroa ME, Abdel-Wahab O, Lu C, et al. Leukemic IDH1 and IDH2 mutations result in a hypermethylation phenotype, disrupt TET2 function, and impair hematopoietic differentiation. Cancer Cell. 2010;18(6):553-67.

Gilham D, Wasiak S, Tsujikawa LM, et al. RVX-208, a BET-inhibitor for treating atherosclerotic cardiovascular disease, raises ApoA-I/HDL and represses pathways that contribute to cardiovascular disease. Atherosclerosis. 2016;247:48-57.

Kanno T, Kanno Y, LeRoy G, et al. BRD4 assists elongation of both coding and enhancer RNAs by interacting with acetylated histones. Nat Struct Mol Biol. 2014;21(12):1047-57.

Kharenko OA, Gesner EM, Patel RG, et al. RVX-297 — a novel BD2 selective inhibitor of BET bromodomains. Biochem Biophys Res Commun. 2016. [Epub ahead of print]

Kim DH, Marinov GK, Pepke S, et al. Single-cell transcriptome analysis reveals dynamic changes in lncRNA expression during reprogramming. Cell Stem Cell. 2015;16(1):88-101.

Lu C, Thompson CB. Metabolic regulation of epigenetics. Cell Metab. 2012;16(1):9-17.

McGrath J, Trojer P. Targeting histone lysine methylation in cancer. Pharmacol Ther. 2015;150:1-22. Review.

McGrath JP, Williamson KE, Balasubramanian S, et al. Pharmacological inhibition of the histone lysine demethylase KDM1A suppresses the growth of multiple acute myeloid leukemia subtypes. Cancer Res. 2016;76(7):1975-88.

Muralidharan SV, Bhadury J, Nilsson LM, et al. BET bromodomain inhibitors synergize with ATR inhibitors to induce DNA damage, apoptosis, senescence-associated secretory pathway and ER stress in Myc-induced lymphoma cells. Oncogene. 2016. [Epub ahead of print]

Rathert P, Roth M, Neumann T, et al. Transcriptional plasticity promotes primary and acquired resistance to BET inhibition. Nature. 2015;525(7570):543-7.

Rosenfeld N, Young JW, Alon U, et al. Gene regulation at the single-cell level. Science. 2005;307(5717):1962-5.

Shen L, Orillion A, Pili R. Histone deacetylase inhibitors as immunomodulators in cancer therapeutics. Epigenomics. 2016;8(3):415-28.

Shi J, Vakoc CR. The mechanisms behind the therapeutic activity of BET bromodomain inhibition. Mol Cell. 2014 5;54(5):728-36. Review.

Tasdemir N, Banito A, Roe JS, et al. BRD4 connects enhancer remodeling to senescence immune surveillance. Cancer Discov. 2016;6(6):612-29.

Trojer P. Epigenetics: Disrupting histone lysine methylation. Nat Chem Biol. 2015;11(8):552-4.

Turcan S, Rohle D, Goenka A, et al. IDH1 mutation is sufficient to establish the glioma hypermethylator phenotype. Nature. 2012;483(7390):479-83.

Venneti S, Thompson CB. Metabolic modulation of epigenetics in gliomas. Brain Pathol. 2013;23(2):217-21. Review.

Vinogradova M, Gehling VS, Gustafson A, et al. An inhibitor of KDM5 demethylases reduces survival of drug-tolerant cancer cells. Nat Chem Biol. 2016;12(7):531-8.

Weiss A, Ding X, van Beijnum JR, et al. Rapid optimization of drug combinations for the optimal angiostatic treatment of cancer. Angiogenesis. 2015;18(3):233-44.


Donald McCaffrey

Resverlogix Corp.

Norman Wong, MD

Resverlogix Corp.

Sarah Zapotichny

Resverlogix Corp.

George Zavoico, PhD

Jones Trading Institutional Services

Sonya Dougal, PhD

The New York Academy of Sciences

Caitlin McOmish, PhD

The New York Academy of Sciences

Session Chairs

Dominique Verhelle, PhD, MBA

Third Rock Ventures

Norman Wong, MD

Resverlogix Corp.

Keynote Speaker

Craig B. Thompson, MD

Memorial Sloan Kettering Cancer Center
website | publications

Craig B. Thompson is the president and chief executive officer of Memorial Sloan Kettering Cancer Center. Thompson is a board-certified internist and medical oncologist with extensive research experience in cancer, immunology, and translational medicine. His current research focuses on the regulation of cellular metabolism during cell growth/differentiation and on the role that metabolic changes play in the origin and progression of cancer. Thompson is a member of the Institute of Medicine, the National Academy of Sciences, the American Academy of Arts and Sciences, and the Medical Advisory Board of the Howard Hughes Medical Institute. He holds an MD from the University of Pennsylvania.


Eric Campeau, PhD

Zenith Epigenetics

Eric Campeau is director of biology at Zenith Epigenetics and a member of the advisory board of Addgene, a nonprofit global plasmid repository. He obtained his PhD from McGill University, Canada, followed by a postdoctoral fellowship at Lawrence Berkeley National Laboratory, where he was later hired as a scientist. He was also previously an instructor at the University of Massachusetts Medical School. His research interests include developing new tools and technologies to elucidate biological mechanisms and determining the mechanisms of action of small molecule inhibitors of epigenetic targets. He is also interested in identifying and developing molecular markers of response to epigenetic inhibitors, as well as translating these findings into patient stratification strategies for single-agent and combination therapies for various cancers.

Michael Elowitz, PhD

California Institute of Technology
website | publications

Michael Elowitz is a Howard Hughes Medical Institute Investigator and professor of biology and biological engineering and applied physics at California Institute of Technology. Elowitz's laboratory uses synthetic biology approaches, together with dynamic, quantitative single-cell imaging, to identify fundamental design principles that enable gene circuits to function in living cells and tissues. Elowitz developed the Repressilator, an artificial genetic clock that generates gene expression oscillations in individual E. coli cells, and since then has continued to design and build other synthetic genetic circuits for programming or rewiring functions in living cells. His lab showed that gene expression is intrinsically stochastic, or noisy, and revealed how this noise functions to enable a variety of cellular functions that would be difficult or impossible without it, from probabilistic differentiation to time-based regulation. Elowitz received his PhD in physics from Princeton University and did postdoctoral research at the Rockefeller University.

Ewelina Kulikowski, PhD

Resverlogix Corp.
website | publications

Ewelina Kulikowski is the senior vice president of research and development at Resverlogix Corp., an epigenetics-focused company that created first-in-class small molecule therapeutics for BET inhibition. Kulikowski contributes to various aspects of the clinical and business development of novel drugs for the treatment of cardiovascular, inflammatory, orphan, and neurodegenerative diseases. She has been involved in the discovery, development, investigational new drug (IND) application, and clinical path of apabetalone (RVX-208). Kulikowski previously served as the director of research development, leading the vascular inflammation and ophthalmology programs at Resverlogix, and as director of business development and scientific affairs. She received her PhD in oncology from the University of Calgary, Canada, in 2004 and has been at Resverlogix since 2005.

Keiko Ozato, PhD

National Institute of Child Health and Human Development, NIH
website | publications

Keiko Ozato received her PhD from Kyoto University, Japan, and trained in developmental biology and immunology at Carnegie Institution of Washington and at the National Cancer Institute before starting an independent laboratory in the National Institute of Child Health and Human Development, NIH. She has been a tenured senior investigator in NICHD since 1987. The focus of her laboratory has been chromatin and epigenetic regulation of innate immunity. Her current studies center on three nuclear proteins: the chromatin-binding factor BRD4, a DNA binding transcription factor IRF8, and the variant histone H3.3. Her laboratory isolated a murine Brd4 in 2000 and did pioneering research on BET bromodomain proteins.

Roberto Pili, MD

Indiana University School of Medicine
website | publications

Roberto Pili is the medical director of genitourinary oncology and coleader of the developing research program in genitourinary malignancies at the Indiana University Melvin and Bren Simon Cancer Center, which supports research for prostate, bladder, and kidney cancers. He is also the Robert Wallace Miller Professor of Oncology at the Indiana University School of Medicine and a translational researcher at the IU Simon Cancer Center. His research focuses on the development of novel therapeutic agents, including epigenetic agents such as histone deacetylase inhibitors, and on understanding their immunomodulatory effects. He also conducts phase I/II clinical trials of novel agents for the treatment of genitourinary malignancies. Pili serves as a reviewer for study sections of the NCI and the Department of Defense.

Patrick Trojer, PhD

Constellation Pharmaceuticals
website | publications

Patrick Trojer is vice president at Constellation Pharmaceuticals, where he was a founding scientist. He oversees oncology target identification and validation, drug discovery, and clinical biomarker discovery. He has expertise in epigenetics, chromatin biology, and cancer biology. Trojer was the project team lead on the EZH2 discovery program that successfully progressed to the clinic. He did his postdoctoral studies in Danny Reinberg's laboratory at NYU School of Medicine, working on understanding the functional consequences of dynamic changes in histone lysine methylation states. He obtained his PhD in biology with a focus on protein biochemistry and molecular biology at the Leopold Franzens University in Innsbruck, Austria.

Christopher Vakoc, MD, PhD

Cold Spring Harbor Laboratory
website | publications

Christopher Vakoc earned PhD and MD degrees from the University of Pennsylvania. His dissertation research was performed in the laboratory of Gerd Blobel, where he studied basic mechanisms of long-range enhancer function, hematopoietic transcription factors, and histone lysine methylation. In 2008, Vakoc accepted a position as a Cold Spring Harbor Laboratory Fellow, in a program that allows young scientists to pursue independent research before taking a faculty position. During this time, he initiated research into how chromatin modifications support the pathogenesis of leukemia. A key focus of this work has been to leverage functional genomics approaches to reveal unique chromatin regulator dependencies in cancer cells. This work has led to the identification of several chromatin regulator pathways that are essential to maintain the leukemia cell state, which includes the discovery of BRD4 as a drug target in acute myeloid leukemia. This work has also revealed novel mechanisms of transcriptional regulation, such as identifying a role for MLL as a mitotic bookmark and a role for TRIM33 in enhancer decommissioning. Vakoc is currently an associate professor at CSHL.

Daniel Vitt, PhD

website | publications

Daniel Vitt is the chief scientific officer of 4SC AG in Germany, a company he cofounded in 1997. As a member of the executive board, he is responsible for preclinical and clinical development activities at 4SC group, particularly the therapeutic pipeline, bringing several projects in oncology and immunology toward the clinical development stage, among them 4SC-202, an epigenetic cancer drug, and vidofludimus, which has completed phase II studies in irritable bowel disease and rheumatoid arthritis. Vitt completed his PhD in organic chemistry at the Institute of Organic Chemistry at the University of Würzburg, Germany. He is member of the supervisory board of Quattro Research GmbH in Munich and member of the scientific advisory board of CI3 Cluster for Individualized Immune Intervention in Mainz, Germany.

Robert Frawley

Robert Frawley holds a BS in biomedical engineering from Columbia University and is completing a PhD in physiology, biophysics and systems biology at Weill Cornell Graduate School. He enjoys writing for a broad audience and teaching high school science after school.


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