Targeting Histone Methyltransferases and Demethylases header

About This Conference:

Epigenetic drug development has quickly gained widespread interest due to the apparent therapeutic efficacy now being demonstrated in preclinical and clinical studies. Drugging the histone methylome remains at the center of discovery with new findings of somatic mutations and misexpression of genes coding for Histone Methyltransferase and Demethylase enzymes. This, in combination with the chemically tractable nature of these enzymes, is providing a strong impetus for therapeutic intervention via pharmacological modulation. Notably, over the past year a rapid expansion of novel demethylase targets, particularly in the Jumonji C domain-containing demethylase family, has substantially deepened the possibilities of regulating chromatin environments via histone methylation. Histone demethylases are now joining the more-established histone methyltransferase targets for clinical development.

Cambridge Healthtech Institute will once again convene leaders in epigenetic drug development to provide updates on preclinical and clinical programs, introduce novel targets and chemical matter, and discuss strategies for targeting and validating histone methyltransferases and demethylases.

Thursday, October 9

11:30 am Registration

1:00 pm Plenary Keynote Program 

Chas BountraChas Bountra, Ph.D., Professor of Translational Medicine & Head, Structural Genomics Consortium, University of Oxford

Martin TolarMartin Tolar, M.D., Ph.D., Founder, President & CEO, Alzheon, Inc.

Andrew L. Hopkins, Andrew L. Hopkins, D.Phil, FRSC, FSB, Chair of Medicinal Informatics and SULSA Research Professor of Translational Biology, Division of Biological Chemistry and Drug Discovery, College of Life Sciences, University of Dundee

2:45 Refreshment Break in the Exhibit Hall with Poster Viewing

Mechanisms Regulating Gene Expression and Tumorigenesis

3:45 Chairperson’s Opening Remarks

Jonathan D. Licht, M.D., Johanna Dobe Professor & Chief, Division of Hematology/Oncology, Feinberg School of Medicine, Northwestern University

3:55 Disorders of Histone Methylation in Hematological Malignancy

Jonathan D. Licht, M.D., Johanna Dobe Professor & Chief, Division of Hematology/Oncology, Feinberg School of Medicine, Northwestern University

I will discuss the recurrent mutations affecting the pivotal change in hit one methylation between histone 3 lysine 4 (H3K4me- activation) and H3K27me (repression). Among the proteins that will be discussed are EZH2, which is mutated in a substantial fraction of germinal center derived lymphomas, UTX, a histone demethylase for H3K27 that is deleted in multiple myeloma and other tumors and WHSC1/MMSET a gene over expressed in multiple myeloma as well as many solid tumors and mutated in a subset of acute lymphocytic leukemia. MMSET over expression in myeloma causes global changes in chromatin modification and major shifts in gene expression. Similarly EZH2 mutation leads to a change of activity of the enzyme and globe increases in histone methylation. Small molecular inhibitors of EZH2 can reverse the oncogenic effects of EZH2 and I will discuss the path to the development of MMSET inhibitors.

4:25 Epigenetic Reprogramming by Tumor Derived EZH2 Gain-of-Function Mutations Promotes Aggressive 3D Cell Morphologies and Enhances Melanoma Tumor Growth

Robert Rollins, Ph.D., Principal Scientist, Oncology, Pfizer

The discovery of gain-of-function (GOF) mutations of EZH2 in diffuse large B-cell lymphoma, follicular lymphoma, and melanoma strongly suggests an important function for this histone methyltransferase in cancer. To elucidate the role of EZH2 GOF mutations in the solid tumor setting, we ectopically expressed EZH2 GOF mutations in multiple cell models, including melanoma cells. Without having significant effects on cell proliferation, these mutations dramatically altered 3D cell morphology/motility and regulated related intracellular pathways. In addition, melanoma cells expressing ectopic EZH2 GOF mutations form larger tumors than control mice in mouse xenograft studies. These results suggest that EZH2 GOF mutations may alter the interaction of cancer cells with their microenvironment and provide a selective advantage during tumorigenesis.

4:55 Kinetics and Assay Condition-Dependence of Activity Enhancement by the NSD2 E1099K and T1150A Mutations

Konrad T. Howitz, Ph.D., Director, Epigenetics, Reaction Biology Corporation

Over expression of the histone H3K36 methyltransferase NSD2 (MMSET/WHSC1), for example due to the t(4;14) translocation in multiple myelomas, can help drive oncogenesis. This effect may be phenocopied, particularly in certain leukemias and lymphomas, by what are presumed to be hyperactivating NSD2 mutations. We have investigated the effects of two such mutations, E1099K and T1150A, using full-length, insect cell-expressed, wild-type and mutant enzymes to perform in vitro kinetic studies with nucleosomal substrates.

5:25 Coffee Break in the Foyer

5:40 Critical Roles of Histone Methyltransferases and Demethylases in Human Carcinogenesis

Ryuji Hamamoto, Ph.D., Associate Professor, Hematology & Oncology, The University of Chicago

Recent development of basic cancer research indicated that deregulation of histone methylation plays a critical role in human carcinogenesis. We have comprehensively conducted expression profile and functional analyses of histone methyltransferases and demethylases, and identified several ideal candidates for anti-cancer therapy. Furthermore, we have also been developing anti-cancer drugs targeting these enzymes. I will introduce the current progress of our research.

6:10 JARID1/KDM5 Demethylases as Cancer Targets

Qin Yan, Ph.D., Assistant Professor, Pathology, School of Medicine, Yale University

Epigenetic aberrations often lead to cancer and other human diseases. My laboratory focuses on the roles and regulatory mechanisms of the JARID1/KDM5 histone demethylases. The JARID1A/B demethylases play critical roles in tumor formation, metastasis and drug resistance, and therefore are novel targets for cancer treatment. We have identified novel mechanisms by which the JARID1 enzymes regulate gene expression and promote tumorigenesis. The implications of these results in cancer treatment will be discussed.

6:40 Close of Day

7:00 Dinner Short Courses*

*Separate registration required

Friday, October 10

7:30 am Registration

8:00 Interactive Breakfast Breakout Discussion Groups

Enjoy breakfast while joining a discussion group. These are moderated discussions with brainstorming and interactive problem solving, allowing conference participants from diverse backgrounds to exchange ideas, experiences, and develop future collaborations around a focused topic.

Rational Combination Therapies  

Robert Rollins, Ph.D., Principal Scientist, Oncology, Pfizer

  • What are appropriate clinical endpoints for epigenetic therapies?
  • Certain epigenetic drugs change the fate of the cell to induce differentiation rather than cell death, which means that markers of response as well as the kinetics of response may be different than standard cytotoxic drugs.
  • This also has implications for combination strategies, as differentiating agents may actually be antagonistic to chemotherapy (as cells are no longer dividing as quickly).

Gain-of-Function Mutations Affecting Chromatin Methylation 

Konrad T. Howitz, Ph.D., Director, Epigenetics, Reaction Biology Corporation

  • Well-known examples include histone H3 alterations, EZH2 mutations that enhance H3K27 trimethylation and IDH1/2 mutant forms that generate 2-hydroxyglutarate. Are there lesser known, new or novel examples that you would add to the list?
  • Which might hold the most promise for therapeutic targeting?
  • Is it always necessary and/or desirable to selectively target mutant enzymes?



9:00 Chairperson’s Remarks

Peter Staller, Ph.D., Director, Oncology Research, EpiTherapeutics ApS

9:10 FEATURED PRESENTATION: EZH2 as a Target for Genetically Defined Cancers

RoyPollockRoy M. Pollock, Ph.D., Senior Director, Biological Sciences, Epizyme

Epizyme has discovered potent and selective small molecule inhibitors of EZH2 as targeted therapeutics for subsets of human cancers bearing defined genetic lesions. These include non-Hodgkin’s lymphomas with EZH2 gain-of-function mutations and other tumor types with loss of INI1 function. The properties of these inhibitors, including their ability to selectively kill tumor cells bearing specific genetic alterations in cell culture and animal models will be discussed as well as an update on the early clinical experience with EPZ-6438 (E7438) – an EZH2 inhibitor that recently entered Phase I clinical testing.

9:40 EZH2 Inhibitors and Their Application in Cancer

Bill Bradley, Ph.D., Scientist, Constellation Pharmaceuticals

Constellation has identified potent, selective small molecule inhibitors of the histone H3 lysine 27 (H3K27)-specific methyltransferase Enhancer of Zeste Homolog 2 (EZH2). These compounds cause selective cell killing of Non Hodgkin Lymphoma cell lines and regression in subcutaneous NHL models in vivo. The impact on tumor growth is correlated with global reduction of H3K27me3 levels and the induction of EZH2 target gene expression. We have identified Multiple Myeloma as an additional potential application for EZH2 inhibitors. Moreover, we have identified drug combinations that expand the application repertoire of EZH2 inhibitors.

10:10 Coffee Break in the Exhibit Hall with Poster Viewing

10:55 Development of Histone Demethylase Inhibitors for Oncological and Neurodegenerative Disease

Tamara Maes, Ph.D., Co-Founder, Vice President & CSO, Oryzon Genomics

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. ORY-1001 is a potent, selective LSD1 inhibitor, 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 is currently in Phase I studies in UK and Spain and has been partnered with Roche. ORY-2001 is a dual LSD1/MAO-B inhibitor with near equipotent activity in both targets but selective over MAO-A and other FAD dependent aminooxidases. The compound effectively protects mice from MPTP insult, demonstrating its brain MAOBi capacity, and restores the memory loss of SAMP-8 mice, a non transgenic model for accelerated aging and Alzheimer disease. The mechanisms by which ORY-2001 acts on the mouse hippocampus will be discussed.

11:25 Inhibition of LSD1 as a Therapeutic Strategy for the Treatment of AML and SCLC

Ryan Kruger, Ph.D., Director, Discovery Biology, GlaxoSmithKline

Lysine specific demethylase 1 (LSD1) is a H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. Pre-clinical data demonstrate that pharmacological inhibition of LSD1 causes differentiation of AML cells in vitro and in vivo. In SCLC cell line and primary sample xenograft studies LSD1 inhibition resulted in potent tumor growth inhibition. The current study describes the anti-tumor effects of GSK2879552, a novel, potent, selective, irreversible LSD1 inhibitor currently in clinical development

11:55 Therapeutic Inhibitors of the KDM5 Histone Demethylases

Peter Staller, Ph.D., Director, Oncology Research, EpiTherapeutics ApS

The histone demethylases KDM5A and KDM5B target methylation of histone H3 at lysine 4 and contribute to cancer cell proliferation and to the induction of drug tolerance. EpiTherapeutics has developed specific and potent inhibitors of KDM5. The pharmacological properties of selected compounds and their in vivo activity as well as potential therapeutic applications will be discussed.

12:25 pm Label-Free Biochemical Assays for Epigenetics Targets

Michael D. Scholle, Co-Founder, Director, Technology and Operations, SAMDI Tech, Inc

SAMDI is a unique label-free assay platform for quantitative measurement of biochemical activity of epigenetics targets: including methyltranferases, demethylases, deacetylases and many others. This pioneering technology uses high-throughput mass spectrometry in 1536 format for rapid assay development, high-throughput screening, and peptide substrate discovery efforts.

Late-Breaking Research: Selected Poster Presentation*

12:40 pm Pharmacological Inhibitors of LSD1 Promote Differentiation of Myeloid Leukemia Cells Through a Mechanism Independent of Histone Demethylation*

Tim Somervaille, Ph.D., Group Leader, Leukemia Biology, Cancer Research UK Manchester Institute

Lysine Specific Demethylase 1 (LSD1 or KDM1A) is one of a number of epigenetic regulators which have recently emerged as candidate therapeutic targets in acute myeloid leukaemia (AML). It is a flavin adenine dinucleotide (FAD) dependent homolog of the amine oxidase family with an ability to demethylate monomethyl or dimethyl lysine 4 (K4) of histone H3, in addition to other substrates. Pharmacological inhibitors of LSD1 such as the tranylcypromine derivatives have already commenced evaluation in early phase clinical trials. While it has been widely assumed that these compounds promote differentiation of AML cells through inhibition of the demethylase activity of LSD1, the precise mechanisms by which LSD1 inhibitors function has not yet been determined. If changes in histone methylation are a central and critical mediator of the effects of LSD1 inhibitors in promoting AML cell differentiation, it would be expected that global changes in transcription would be tightly linked temporally to changes in histone methylation following drug treatment of cells. Through RNA sequencing and ChIP sequencing experiments performed in human THP1 AML cells treated for 24 hours with a potent and specific tranylcypromine-derivative LSD1 inhibitor (ORY86, trans-N-((2-methoxypyridin-3-yl)methyl)-2-phenylcyclopropan-1-amine), we have established that wholescale up regulation of a myeloid differentiation transcription programme occurs in the absence of any significant genome-wide changes in mono- and di-methyl H3K4 and H3K9 (which are key enzymatic targets of LSD1). Thus LSD1 inhibitor-induced up regulation of myeloid differentiation gene expression is not downstream of changes in histone methylation. We further demonstrated that non-enzymatic functions of LSD1 are essential in AML cells by expressing either wild-type (WT) or catalytically inactive LSD1 (K661A) in LSD1 knockdown (KD) THP1 cells. While LSD1 KD cells exhibit myeloid differentiation and loss of clonogenic potential, both the WT and mutant versions of LSD1 were able to rescue the in vitroclonogenic potential of KD cells to an equivalent extent. Thus the histone demethylase activity of LSD1 is not required to sustain AML blasts in an undifferentiated state. Comparison of the transcriptional consequences of LSD1 inhibition with the transcriptional consequences of transcription factor knockdown in THP1 AML cells using GSEA revealed that pharmacological inhibition of LSD1 mimics depletion of GFI1. Immunoprecipitation experiments confirmed the previously described physical association of GFI1 with LSD1. Critically, the physical interactions of LSD1 with GFI1 was reversed by pharmacological inhibition of LSD1 with ORY86. Furthermore, in ChIP sequencing experiments drug treatment led to dissociation of LSD1 from promoters and enhancers. By contrast, there was no disruption of the endogenous level interaction of LSD1 with RCOR1, HDAC1 and HDAC2 (i.e. the CoREST complex) following drug treatment. To determine whether the inhibitor-induced separation of LSD1 from GFI1 is required for induction of myeloid differentiation by ORY86, we performed experiments using a GFI1-LSD1 fusion construct expressed in THP1 cells under the control of a doxycycline-regulated promoter. This construct tethers LSD1 directly to the transcription factor and circumvents any drug induced physical separation. THP1 cells expressing GFI1-LSD1 were drug resistant (as determined by immunophenotyping and clonogenic potential), in contrast to control cells expressing GFI1, LSD1 or an empty vector in the same inducible system. Thus, drug-induced physical separation of GFI1 from LSD1 is required for THP1 AML cells to undergo differentiation. Our data support a model whereby the physical association of LSD1 with transcription factors such as GFI1 is essential to maintain the differentiation block in AML. Unexpectedly, tranylcypromine-derivative inhibitors target this novel scaffolding function of LSD1, rather than its histone demethylase activity, to promote differentiation of AML cells.

12:55 Session Break

1:05 Enjoy Lunch on Your Own

1:45 Session Break

Discovery and Development of Novel Bioactive Chemical Matter

1:55 Chairperson’s Remarks

Qin Yan, Ph.D., Assistant Professor, Pathology, School of Medicine, Yale University

2:00 Targeting the MLL1-WDR5 Protein-Protein Interaction as a Novel Therapeutic Strategy for Acute Leukemia Harboring MLL1 Fusion Protein

Shaomeng Wang, Ph.D., Director, Center for Discovery of New Medicines; Warner-Lambert/Parke-Davis Professor, Medicine, Pharmacology and Medicinal Chemistry, University of Michigan Comprehensive Cancer Center

I will present our structure-based design of highly potent and specific small-molecule inhibitors of the MLL1-WDR5 protein-protein interaction as a new therapeutic strategy for the treatment of acute leukemia harboring MLL1 fusion protein. Their mode of action and therapeutic potential will be discussed.

2:30 Targeting Arginine Methyltransferases

Masoud Vedadi, Ph.D., Principal Investigator, Molecular Biophysics, Structural Genomics Consortium; Assistant Professor, Department of Pharmacology and Toxicology, University of Toronto

Epigenetic regulation of gene expression through histone methylation has been implicated in a variety of diseases. Protein arginine methyltransferases (PRMTs) is an emerging class of therapeutic targets. We previously reported the first allosteric inhibitor of PRMT3 with an IC50 value of 2.5 µM, and a significantly more potent next generation inhibitor with an IC50 value of 230 nM. Here we will report on further optimization of this series of inhibitors (less than 100 nM), discuss their target engagement in cells, and possible allosteric inhibition of other PRMTs. We also report on our progress in targeting active sites of all PRMTs.

3:00 Refreshment Break in the Exhibit Hall with Poster Viewing

3:30 A Dual Approach to Develop Inhibitors of Protein Methyltransferases

Minkui Luo, Ph.D., Assistant Member & Assistant Professor, Molecular Pharmacology and Chemistry, Memorial Sloan-Kettering Cancer Center

Epigenetic regulations are involved in establishing cell-lineage diversity and the errors in these processes have been linked to many diseases including cancer. Among the key biochemical modifications in epigenetics is protein methylation, a process orchestrated by over 60 human protein methyltransferases (PMTs). Although a dozen of PMTs have been linked to cancer proliferation, few target-specific PMT inhibitors have been reported so far. It is of great challenge to develop PMT inhibitors aiming at potency and specificity, given highly conserved SAM-binding pockets and floppy substrate-binding sites of PMTs. We therefore combined rational design and high throughput screening (HTS) to develop PMT inhibitors within the scope of our interest in chemical probes and cancer therapy. We designed sinefungin analogues against a collection of human PMTs and identified structurally-distinct sinefungin scaffolds that can selectively bind a subset of PMTs such as SETD2, CARM1 and SET7/9. Through a scintillation proximity assay (SPA) HTS, we also identified novel scaffolds to inhibit SET8. The mechanisms of the action and implication of these inhibitors will be discussed as well.

4:00 Small Molecule Epigenetic Modulators for the Treatment of Cardiovascular Disorders

Patrick M Woster, Ph.D., Endowed Chair, Drug Discovery; Professor, Pharmaceutical and Biomedical Sciences, College of Pharmacy, Medical University of South Carolina

We have identified a new series of small molecules that act as potent and selective LSD1 inhibitors. Because of their relatively low toxicity, we have explored the use of these molecules in diseases other than cancer, where cytotoxicity is not a desirable endpoint. In this presentation, we will describe the optimization of this new series of LSD1 inhibitors, and present evidence that LSD1 inhibitor-mediated correction of aberrant gene silencing can have therapeutic potential in cardiovascular disease.

4:30 Strategies for Developing Histone Demethylase Inhibitors

Akane Kawamura, Ph.D., Senior Investigator, Department of Chemistry, University of Oxford

Histone demethylases are involved in the epigenetic regulation of gene transcription via controlling the methylation status on histone tails. Chemical probes against these enzymes are needed to understand their complex biology in development and disease. Approximately 20 JmjC-domain containing KDMs have been identified, which forms a cluster within theFe(II) and 2OG-dependent oxygenase superfamily of proteins. The talk will focus on our recent efforts and different strategies employed for JmjC-KDM inhibitor development.

5:00 Close of Conference

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