• Epigenetics: Cancer and Beyond

    Epigenetics: Cancer and Beyond

    Organizers: Donald McCaffery (Resverlogix Corp.), Norman Wong (Resverlogix Corp.), Sarah Zapotichny (Resverlogix Corp.), George Zavoico (Jones Trading Institutional Services), Sonya Dougal (The New York Academy of Sciences), and Caitlin McOmish (The New York Academy of Sciences)
    Keynote Speaker: Craig B. Thompson (Memorial Sloan Kettering Cancer Center)
    Presented by the Biochemical Pharmacology Discussion Group and the Cancer & Signaling Discussion Group
    Reported by Robert Frawley | Posted July 7, 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)

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    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)

    How to cite this eBriefing

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

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