Publications by authors named "Roy M Pollock"

27 Publications

  • Page 1 of 1

High-Throughput Quantitative Assay Technologies for Accelerating the Discovery and Optimization of Targeted Protein Degradation Therapeutics.

SLAS Discov 2021 Jan 11:2472555220985049. Epub 2021 Jan 11.

C4 Therapeutics, Inc., Watertown, MA, USA.

The aberrant regulation of protein expression and function can drastically alter cellular physiology and lead to numerous pathophysiological conditions such as cancer, inflammatory diseases, and neurodegeneration. The steady-state expression levels of endogenous proteins are controlled by a balance of de novo synthesis rates and degradation rates. Moreover, the levels of activated proteins in signaling cascades can be further modulated by a variety of posttranslational modifications and protein-protein interactions. The field of targeted protein degradation is an emerging area for drug discovery in which small molecules are used to recruit E3 ubiquitin ligases to catalyze the ubiquitination and subsequent degradation of disease-causing target proteins by the proteasome in both a dose- and time-dependent manner. Traditional approaches for quantifying protein level changes in cells, such as Western blots, are typically low throughput with limited quantification, making it hard to drive the rapid development of therapeutics that induce selective, rapid, and sustained protein degradation. In the last decade, a number of techniques and technologies have emerged that have helped to accelerate targeted protein degradation drug discovery efforts, including the use of fluorescent protein fusions and reporter tags, flow cytometry, time-resolved fluorescence energy transfer (TR-FRET), and split luciferase systems. Here we discuss the advantages and disadvantages associated with these technologies and their application to the development and optimization of degraders as therapeutics.
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http://dx.doi.org/10.1177/2472555220985049DOI Listing
January 2021

Genomic discovery of an evolutionarily programmed modality for small-molecule targeting of an intractable protein surface.

Proc Natl Acad Sci U S A 2020 07 30;117(29):17195-17203. Epub 2020 Jun 30.

Warp Drive Bio, Inc., Redwood City, CA 94063;

The vast majority of intracellular protein targets are refractory toward small-molecule therapeutic engagement, and additional therapeutic modalities are needed to overcome this deficiency. Here, the identification and characterization of a natural product, WDB002, reveals a therapeutic modality that dramatically expands the currently accepted limits of druggability. WDB002, in complex with the FK506-binding protein (FKBP12), potently and selectively binds the human centrosomal protein 250 (CEP250), resulting in disruption of CEP250 function in cells. The recognition mode is unprecedented in that the targeted domain of CEP250 is a coiled coil and is topologically featureless, embodying both a structural motif and surface topology previously considered on the extreme limits of "undruggability" for an intracellular target. Structural studies reveal extensive protein-WDB002 and protein-protein contacts, with the latter being distinct from those seen in FKBP12 ternary complexes formed by FK506 and rapamycin. Outward-facing structural changes in a bound small molecule can thus reprogram FKBP12 to engage diverse, otherwise "undruggable" targets. The flat-targeting modality demonstrated here has the potential to expand the druggable target range of small-molecule therapeutics. As CEP250 was recently found to be an interaction partner with the Nsp13 protein of the SARS-CoV-2 virus that causes COVID-19 disease, it is possible that WDB002 or an analog may exert useful antiviral activity through its ability to form high-affinity ternary complexes containing CEP250 and FKBP12.
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http://dx.doi.org/10.1073/pnas.2006560117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7382241PMC
July 2020

Mechanisms of Pinometostat (EPZ-5676) Treatment-Emergent Resistance in -Rearranged Leukemia.

Mol Cancer Ther 2017 08 20;16(8):1669-1679. Epub 2017 Apr 20.

Epizyme Inc., Cambridge, Massachusetts.

DOT1L is a protein methyltransferase involved in the development and maintenance of -rearranged (-r) leukemia through its ectopic methylation of histones associated with well-characterized leukemic genes. Pinometostat (EPZ-5676), a selective inhibitor of DOT1L, is in clinical development in relapsed/refractory acute leukemia patients harboring rearrangements of the gene. The observation of responses and subsequent relapses in the adult trial treating -r patients motivated preclinical investigations into potential mechanisms of pinometostat treatment-emergent resistance (TER) in cell lines confirmed to have -r. TER was achieved in five -r cell lines, KOPN-8, MOLM-13, MV4-11, NOMO-1, and SEM. Two of the cell lines, KOPN-8 and NOMO-1, were thoroughly characterized to understand the mechanisms involved in pinometostat resistance. Unlike many other targeted therapies, resistance does not appear to be achieved through drug-induced selection of mutations of the target itself. Instead, we identified both drug efflux transporter dependent and independent mechanisms of resistance to pinometostat. In KOPN-8 TER cells, increased expression of the drug efflux transporter ABCB1 (P-glycoprotein, MDR1) was the primary mechanism of drug resistance. In contrast, resistance in NOMO-1 cells occurs through a mechanism other than upregulation of a specific efflux pump. RNA-seq analysis performed on both parental and resistant KOPN-8 and NOMO-1 cell lines supported two unique candidate pathway mechanisms that may explain the pinometostat resistance observed in these cell lines. These results are the first demonstration of TER models of the DOT1L inhibitor pinometostat and may provide useful tools for investigating clinical resistance. .
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http://dx.doi.org/10.1158/1535-7163.MCT-16-0693DOI Listing
August 2017

Preclinical Evidence of Anti-Tumor Activity Induced by EZH2 Inhibition in Human Models of Synovial Sarcoma.

PLoS One 2016 8;11(7):e0158888. Epub 2016 Jul 8.

Epizyme Inc., Cambridge, Massachusetts, United States of America.

The catalytic activities of covalent and ATP-dependent chromatin remodeling are central to regulating the conformational state of chromatin and the resultant transcriptional output. The enzymes that catalyze these activities are often contained within multiprotein complexes in nature. Two such multiprotein complexes, the polycomb repressive complex 2 (PRC2) methyltransferase and the SWItch/Sucrose Non-Fermentable (SWI/SNF) chromatin remodeler have been reported to act in opposition to each other during development and homeostasis. An imbalance in their activities induced by mutations/deletions in complex members (e.g. SMARCB1) has been suggested to be a pathogenic mechanism in certain human cancers. Here we show that preclinical models of synovial sarcoma-a cancer characterized by functional SMARCB1 loss via its displacement from the SWI/SNF complex through the pathognomonic SS18-SSX fusion protein-display sensitivity to pharmacologic inhibition of EZH2, the catalytic subunit of PRC2. Treatment with tazemetostat, a clinical-stage, selective and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity reverses a subset of synovial sarcoma gene expression and results in concentration-dependent cell growth inhibition and cell death specifically in SS18-SSX fusion-positive cells in vitro. Treatment of mice bearing either a cell line or two patient-derived xenograft models of synovial sarcoma leads to dose-dependent tumor growth inhibition with correlative inhibition of trimethylation levels of the EZH2-specific substrate, lysine 27 on histone H3. These data demonstrate a dependency of SS18-SSX-positive, SMARCB1-deficient synovial sarcomas on EZH2 enzymatic activity and suggests the potential utility of EZH2-targeted drugs in these genetically defined cancers.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0158888PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4938529PMC
July 2017

Structure and Property Guided Design in the Identification of PRMT5 Tool Compound EPZ015666.

ACS Med Chem Lett 2016 Feb 2;7(2):162-6. Epub 2015 Dec 2.

Epizyme, Inc. , 400 Technology Square, Cambridge, Massachusetts 02139, United States.

The recent publication of a potent and selective inhibitor of protein methyltransferase 5 (PRMT5) provides the scientific community with in vivo-active tool compound EPZ015666 (GSK3235025) to probe the underlying pharmacology of this key enzyme. Herein, we report the design and optimization strategies employed on an initial hit compound with poor in vitro clearance to yield in vivo tool compound EPZ015666 and an additional potent in vitro tool molecule EPZ015866 (GSK3203591).
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http://dx.doi.org/10.1021/acsmedchemlett.5b00380DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4753547PMC
February 2016

MLL1 and DOT1L cooperate with meningioma-1 to induce acute myeloid leukemia.

J Clin Invest 2016 Apr 29;126(4):1438-50. Epub 2016 Feb 29.

Meningioma-1 (MN1) overexpression is frequently observed in patients with acute myeloid leukemia (AML) and is predictive of poor prognosis. In murine models, forced expression of MN1 in hematopoietic progenitors induces an aggressive myeloid leukemia that is strictly dependent on a defined gene expression program in the cell of origin, which includes the homeobox genes Hoxa9 and Meis1 as key components. Here, we have shown that this program is controlled by two histone methyltransferases, MLL1 and DOT1L, as deletion of either Mll1 or Dot1l in MN1-expressing cells abrogated the cell of origin-derived gene expression program, including the expression of Hoxa cluster genes. In murine models, genetic inactivation of either Mll1 or Dot1l impaired MN1-mediated leukemogenesis. We determined that HOXA9 and MEIS1 are coexpressed with MN1 in a subset of clinical MN1hi leukemia, and human MN1hi/HOXA9hi leukemias were sensitive to pharmacologic inhibition of DOT1L. Together, these data point to DOT1L as a potential therapeutic target in MN1hi AML. In addition, our findings suggest that epigenetic modulation of the interplay between an oncogenic lesion and its cooperating developmental program has therapeutic potential in AML.
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http://dx.doi.org/10.1172/JCI80825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4811120PMC
April 2016

The Importance of Being Me: Magic Methyls, Methyltransferase Inhibitors, and the Discovery of Tazemetostat.

J Med Chem 2016 Feb 27;59(4):1556-64. Epub 2016 Jan 27.

Epizyme , 400 Technology Square, Fourth Floor, Cambridge, Massachusetts 02139, United States.

Posttranslational methylation of histones plays a critical role in gene regulation. Misregulation of histone methylation can lead to oncogenic transformation. Enhancer of Zeste homologue 2 (EZH2) methylates histone 3 at lysine 27 (H3K27) and abnormal methylation of this site is found in many cancers. Tazemetostat, an EHZ2 inhibitor in clinical development, has shown activity in both preclinical models of cancer as well as in patients with lymphoma or INI1-deficient solid tumors. Herein we report the structure-activity relationships from identification of an initial hit in a high-throughput screen through selection of tazemetostat for clinical development. The importance of several methyl groups to the potency of the inhibitors is highlighted as well as the importance of balancing pharmacokinetic properties with potency.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01501DOI Listing
February 2016

Metabolism and disposition of the DOT1L inhibitor, pinometostat (EPZ-5676), in rat, dog and human.

Cancer Chemother Pharmacol 2016 Jan 8;77(1):43-62. Epub 2015 Dec 8.

Epizyme, Inc., 400 Technology Square, Cambridge, MA, 02139, USA.

Purpose: The metabolism and disposition of the first-in-class DOT1L inhibitor, EPZ-5676 (pinometostat), was investigated in rat and dog. Metabolite profiles were compared with those from adult patients in the first-in-man phase 1 study as well as the cross-species metabolism observed in vitro.

Methods: EPZ-5676 was administered to rat and dog as a 24-h IV infusion of [(14)C]-EPZ-5676 for determination of pharmacokinetics, mass balance, metabolite profiling and biodistribution by quantitative whole-body autoradiography (QWBA). Metabolite profiling and identification was performed by radiometric and LC-MS/MS analysis.

Results: Fecal excretion was the major route of elimination, representing 79 and 81% of the total dose in and rat and dog, respectively. QWBA in rats showed that the radioactivity was well distributed in the body, except for the central nervous system, and the majority of radioactivity was eliminated from most tissues by 168 h. Fecal recovery of dose-related material in bile duct-cannulated animals as well as higher radioactivity concentrations in the wall of the large intestine relative to liver implicated intestinal secretion as well as biliary elimination. EPZ-5676 underwent extensive oxidative metabolism with the major metabolic pathways being hydroxylation of the t-butyl group (EPZ007769) and N-dealkylation of the central nitrogen. Loss of adenine from parent EPZ-5676 (M7) was observed only in rat and dog feces, suggesting the involvement of gut microbiota. In rat and dog, steady-state plasma levels of total radioactivity and parent EPZ-5676 were attained rapidly and maintained through the infusion period before declining rapidly on cessation of dosing. Unchanged EPZ-5676 was the predominant circulating species in rat, dog and man.

Conclusions: The excretory and metabolic pathways for EPZ-5676 were very similar across species. Renal excretion of both parent EPZ-5676 and EPZ-5676-related material was low, and in preclinical species fecal excretion of parent EPZ-5676 and EPZ007769 accounted for the majority of drug-related elimination.
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http://dx.doi.org/10.1007/s00280-015-2929-yDOI Listing
January 2016

Exploring drug delivery for the DOT1L inhibitor pinometostat (EPZ-5676): Subcutaneous administration as an alternative to continuous IV infusion, in the pursuit of an epigenetic target.

J Control Release 2015 Dec 15;220(Pt B):758-65. Epub 2015 Sep 15.

Epizyme, Inc., 400 Technology Square, Cambridge, MA 02139, United States.

Protein methyltransferases are emerging as promising drug targets for therapeutic intervention in human cancers. Pinometostat (EPZ-5676) is a small molecule inhibitor of the DOT1L enzyme, a histone methyltransferase that methylates lysine 79 of histone H3. DOT1L activity is dysregulated in the pathophysiology of rearranged mixed lineage leukemia (MLL-r). Pinometostat is currently in Phase 1 clinical trials in relapsed refractory acute leukemia patients and is administered as a continuous IV infusion (CIV). The studies herein investigated alternatives to CIV administration of pinometostat to improve patient convenience. Various sustained release technologies were considered, and based on the required dose size as well as practical considerations, subcutaneous (SC) bolus administration of a solution formulation was selected for further evaluation in preclinical studies. SC administration offered improved exposure and complete bioavailability of pinometostat relative to CIV and oral administration. These findings warranted further evaluation in rat xenograft models of MLL-r leukemia. SC dosing in xenograft models demonstrated inhibition of MLL-r tumor growth and inhibition of pharmacodynamic markers of DOT1L activity. However, a dosing frequency of thrice daily (t.i.d) was required in these studies to elicit optimal inhibition of DOT1L target genes and tumor growth inhibition. Development of an extended release formulation may prove useful in the further optimization of the SC delivery of pinometostat, moving towards a more convenient dosing paradigm for patients.
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http://dx.doi.org/10.1016/j.jconrel.2015.09.023DOI Listing
December 2015

A selective inhibitor of PRMT5 with in vivo and in vitro potency in MCL models.

Nat Chem Biol 2015 Jun 27;11(6):432-7. Epub 2015 Apr 27.

Departments of Biology and Molecular Discovery, Epizyme, Inc., Cambridge, Massachusetts, USA.

Protein arginine methyltransferase-5 (PRMT5) is reported to have a role in diverse cellular processes, including tumorigenesis, and its overexpression is observed in cell lines and primary patient samples derived from lymphomas, particularly mantle cell lymphoma (MCL). Here we describe the identification and characterization of a potent and selective inhibitor of PRMT5 with antiproliferative effects in both in vitro and in vivo models of MCL. EPZ015666 (GSK3235025) is an orally available inhibitor of PRMT5 enzymatic activity in biochemical assays with a half-maximal inhibitory concentration (IC50) of 22 nM and broad selectivity against a panel of other histone methyltransferases. Treatment of MCL cell lines with EPZ015666 led to inhibition of SmD3 methylation and cell death, with IC50 values in the nanomolar range. Oral dosing with EPZ015666 demonstrated dose-dependent antitumor activity in multiple MCL xenograft models. EPZ015666 represents a validated chemical probe for further study of PRMT5 biology and arginine methylation in cancer and other diseases.
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http://dx.doi.org/10.1038/nchembio.1810DOI Listing
June 2015

MLL partial tandem duplication leukemia cells are sensitive to small molecule DOT1L inhibition.

Haematologica 2015 May 16;100(5):e190-3. Epub 2015 Jan 16.

Human Oncology and Pathogenesis Program, Memorial Sloan Kettering Cancer Center, New York, NY, USA Department of Pediatrics, Memorial Sloan Kettering Cancer Center, New York, NY, USA

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http://dx.doi.org/10.3324/haematol.2014.115337DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4420229PMC
May 2015

Synergistic Anti-Tumor Activity of EZH2 Inhibitors and Glucocorticoid Receptor Agonists in Models of Germinal Center Non-Hodgkin Lymphomas.

PLoS One 2014 10;9(12):e111840. Epub 2014 Dec 10.

Research and Development, Epizyme Inc., Cambridge, Massachusetts, United States of America.

Patients with non-Hodgkin lymphoma (NHL) are treated today with a cocktail of drugs referred to as CHOP (Cyclophosphamide, Hydroxyldaunorubicin, Oncovin, and Prednisone). Subsets of patients with NHL of germinal center origin bear oncogenic mutations in the EZH2 histone methyltransferase. Clinical testing of the EZH2 inhibitor EPZ-6438 has recently begun in patients. We report here that combining EPZ-6438 with CHOP in preclinical cell culture and mouse models results in dramatic synergy for cell killing in EZH2 mutant germinal center NHL cells. Surprisingly, we observe that much of this synergy is due to Prednisolone - a glucocorticoid receptor agonist (GRag) component of CHOP. Dramatic synergy was observed when EPZ-6438 is combined with Prednisolone alone, and a similar effect was observed with Dexamethasone, another GRag. Remarkably, the anti-proliferative effect of the EPZ-6438+GRag combination extends beyond EZH2 mutant-bearing cells to more generally impact germinal center NHL. These preclinical data reveal an unanticipated biological intersection between GR-mediated gene regulation and EZH2-mediated chromatin remodeling. The data also suggest the possibility of a significant and practical benefit of combining EZH2 inhibitors and GRag that warrants further investigation in a clinical setting.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0111840PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4262195PMC
October 2017

Reaction coupling between wild-type and disease-associated mutant EZH2.

ACS Chem Biol 2014 Nov 28;9(11):2459-64. Epub 2014 Aug 28.

Epizyme, Inc. 400 Technology Square, Fourth Floor, Cambridge, Massachusetts 02139, United States.

EZH2 and EZH1 are protein methyltransferases (PMTs) responsible for histone H3, lysine 27 (H3K27) methylation. Trimethylation of H3K27 (H3K27me3) is a hallmark of many cancers, including non-Hodgkin lymphoma (NHL). Heterozygous EZH2 point mutations at Tyr641, Ala677, and Ala687 have been observed in NHL. The Tyr641 mutations enhance activity on H3K27me2 but have weak or no activity on unmethylated H3K27, whereas the Ala677 and Ala687 mutations use substrates of all methylation states effectively. It has been proposed that enzymatic coupling of the wild-type and mutant enzymes leads to the oncogenic H3K27me3 mark in mutant-bearing NHL. We show that coupling with the wild-type enzyme is needed to achieve H3K27me3 for several mutants, but that others are capable of achieving H3K27me3 on their own. All forms of PRC2 (wild-type and mutants) display kinetic signatures that are consistent with a distributive mechanism of catalysis.
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http://dx.doi.org/10.1021/cb500548bDOI Listing
November 2014

DOT1L inhibitor EPZ-5676 displays synergistic antiproliferative activity in combination with standard of care drugs and hypomethylating agents in MLL-rearranged leukemia cells.

J Pharmacol Exp Ther 2014 Sep 3;350(3):646-56. Epub 2014 Jul 3.

Epizyme Inc., Cambridge, Massachusetts

EPZ-5676 [(2R,3R,4S,5R)-2-(6-amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol], a small-molecule inhibitor of the protein methyltransferase DOT1L, is currently under clinical investigation for acute leukemias bearing MLL-rearrangements (MLL-r). In this study, we evaluated EPZ-5676 in combination with standard of care (SOC) agents for acute leukemias as well as other chromatin-modifying drugs in cellular assays with three human acute leukemia cell lines: MOLM-13 (MLL-AF9), MV4-11 (MLL-AF4), and SKM-1 (non-MLL-r). Studies were performed to evaluate the antiproliferative effects of EPZ-5676 combinations in a cotreatment model in which the second agent was added simultaneously with EPZ-5676 at the beginning of the assay, or in a pretreatment model in which cells were incubated for several days in the presence of EPZ-5676 prior to the addition of the second agent. EPZ-5676 was found to act synergistically with the acute myeloid leukemia (AML) SOC agents cytarabine or daunorubicin in MOLM-13 and MV4-11 MLL-r cell lines. EPZ-5676 is selective for MLL-r cell lines as demonstrated by its lack of effect either alone or in combination in the nonrearranged SKM-1 cell line. In MLL-r cells, the combination benefit was observed even when EPZ-5676 was washed out prior to the addition of the chemotherapeutic agents, suggesting that EPZ-5676 sets up a durable, altered chromatin state that enhances the chemotherapeutic effects. Our evaluation of EPZ-5676 in conjunction with other chromatin-modifying drugs also revealed a consistent combination benefit, including synergy with DNA hypomethylating agents. These results indicate that EPZ-5676 is highly efficacious as a single agent and synergistically acts with other chemotherapeutics, including AML SOC drugs and DNA hypomethylating agents in MLL-r cells.
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http://dx.doi.org/10.1124/jpet.114.214577DOI Listing
September 2014

Selective inhibition of EZH2 by EPZ-6438 leads to potent antitumor activity in EZH2-mutant non-Hodgkin lymphoma.

Mol Cancer Ther 2014 Apr 21;13(4):842-54. Epub 2014 Feb 21.

Authors' Affiliations: Epizyme Inc., Cambridge; Eisai Inc., Andover, Massachusetts; and Eisai Co. Ltd., Tsukuba-shi, Ibaraki, Japan.

Mutations within the catalytic domain of the histone methyltransferase EZH2 have been identified in subsets of patients with non-Hodgkin lymphoma (NHL). These genetic alterations are hypothesized to confer an oncogenic dependency on EZH2 enzymatic activity in these cancers. We have previously reported the discovery of EPZ005678 and EPZ-6438, potent and selective S-adenosyl-methionine-competitive small molecule inhibitors of EZH2. Although both compounds are similar with respect to their mechanism of action and selectivity, EPZ-6438 possesses superior potency and drug-like properties, including good oral bioavailability in animals. Here, we characterize the activity of EPZ-6438 in preclinical models of NHL. EPZ-6438 selectively inhibits intracellular lysine 27 of histone H3 (H3K27) methylation in a concentration- and time-dependent manner in both EZH2 wild-type and mutant lymphoma cells. Inhibition of H3K27 trimethylation (H3K27Me3) leads to selective cell killing of human lymphoma cell lines bearing EZH2 catalytic domain point mutations. Treatment of EZH2-mutant NHL xenograft-bearing mice with EPZ-6438 causes dose-dependent tumor growth inhibition, including complete and sustained tumor regressions with correlative diminution of H3K27Me3 levels in tumors and selected normal tissues. Mice dosed orally with EPZ-6438 for 28 days remained tumor free for up to 63 days after stopping compound treatment in two EZH2-mutant xenograft models. These data confirm the dependency of EZH2-mutant NHL on EZH2 activity and portend the utility of EPZ-6438 as a potential treatment for these genetically defined cancers.
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http://dx.doi.org/10.1158/1535-7163.MCT-13-0773DOI Listing
April 2014

Nonclinical pharmacokinetics and metabolism of EPZ-5676, a novel DOT1L histone methyltransferase inhibitor.

Biopharm Drug Dispos 2014 May 14;35(4):237-52. Epub 2014 Feb 14.

Epizyme Inc., 400 Technology Square, Cambridge, MA, USA.

(2R,3R,4S,5R)-2-(6-Amino-9H-purin-9-yl)-5-((((1r,3S)-3-(2-(5-(tert-butyl)-1H-benzo[d]imidazol-2-yl)ethyl)cyclobutyl)(isopropyl)amino)methyl)tetrahydrofuran-3,4-diol (EPZ-5676) is a novel DOT1L histone methyltransferase inhibitor currently in clinical development for the treatment of MLL-rearranged leukemias. This report describes the preclinical pharmacokinetics and metabolism of EPZ-5676, an aminonucleoside analog with exquisite target potency and selectivity that has shown robust and durable tumor growth inhibition in preclinical models. The in vivo pharmacokinetics in mouse, rat and dog were characterized following i.v. and p.o. administration; EPZ-5676 had moderate to high clearance, low oral bioavailability with a steady-state volume of distribution 2-3 fold higher than total body water. EPZ-5676 showed biexponential kinetics following i.v. administration, giving rise to a terminal elimination half-life (t1/2 ) of 1.1, 3.7 and 13.6 h in mouse, rat and dog, respectively. The corresponding in vitro ADME parameters were also studied and utilized for in vitro-in vivo extrapolation purposes. There was good agreement between the microsomal clearance and the in vivo clearance implicating hepatic oxidative metabolism as the predominant elimination route in preclinical species. Furthermore, low renal clearance was observed in mouse, approximating to fu -corrected glomerular filtration rate (GFR) and thus passive glomerular filtration. The metabolic pathways across species were studied in liver microsomes in which EPZ-5676 was metabolized to three monohydroxylated metabolites (M1, M3 and M5), one N-dealkylated product (M4) as well as an N-oxide (M6).
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http://dx.doi.org/10.1002/bdd.1889DOI Listing
May 2014

Potent inhibition of DOT1L as treatment of MLL-fusion leukemia.

Blood 2013 Aug 25;122(6):1017-25. Epub 2013 Jun 25.

Epizyme, Inc., 400 Technology Square, Cambridge, MA 02139, USA.

Rearrangements of the MLL gene define a genetically distinct subset of acute leukemias with poor prognosis. Current treatment options are of limited effectiveness; thus, there is a pressing need for new therapies for this disease. Genetic and small molecule inhibitor studies have demonstrated that the histone methyltransferase DOT1L is required for the development and maintenance of MLL-rearranged leukemia in model systems. Here we describe the characterization of EPZ-5676, a potent and selective aminonucleoside inhibitor of DOT1L histone methyltransferase activity. The compound has an inhibition constant value of 80 pM, and demonstrates 37 000-fold selectivity over all other methyltransferases tested. In cellular studies, EPZ-5676 inhibited H3K79 methylation and MLL-fusion target gene expression and demonstrated potent cell killing that was selective for acute leukemia lines bearing MLL translocations. Continuous IV infusion of EPZ-5676 in a rat xenograft model of MLL-rearranged leukemia caused complete tumor regressions that were sustained well beyond the compound infusion period with no significant weight loss or signs of toxicity. EPZ-5676 is therefore a potential treatment of MLL-rearranged leukemia and is under clinical investigation.
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http://dx.doi.org/10.1182/blood-2013-04-497644DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3739029PMC
August 2013

Durable tumor regression in genetically altered malignant rhabdoid tumors by inhibition of methyltransferase EZH2.

Proc Natl Acad Sci U S A 2013 May 25;110(19):7922-7. Epub 2013 Apr 25.

Epizyme, Inc, Cambridge, MA 02139, USA.

Inactivation of the switch/sucrose nonfermentable complex component SMARCB1 is extremely prevalent in pediatric malignant rhabdoid tumors (MRTs) or atypical teratoid rhabdoid tumors. This alteration is hypothesized to confer oncogenic dependency on EZH2 in these cancers. We report the discovery of a potent, selective, and orally bioavailable small-molecule inhibitor of EZH2 enzymatic activity, (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide). The compound induces apoptosis and differentiation specifically in SMARCB1-deleted MRT cells. Treatment of xenograft-bearing mice with (N-((4,6-dimethyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-5-(ethyl(tetrahydro-2H-pyran-4-yl)amino)-4-methyl-4'-(morpholinomethyl)-[1,1'-biphenyl]-3-carboxamide) leads to dose-dependent regression of MRTs with correlative diminution of intratumoral trimethylation levels of lysine 27 on histone H3, and prevention of tumor regrowth after dosing cessation. These data demonstrate the dependency of SMARCB1 mutant MRTs on EZH2 enzymatic activity and portend the utility of EZH2-targeted drugs for the treatment of these genetically defined cancers.
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http://dx.doi.org/10.1073/pnas.1303800110DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3651445PMC
May 2013

Leukemic transformation by the MLL-AF6 fusion oncogene requires the H3K79 methyltransferase Dot1l.

Blood 2013 Mar 29;121(13):2533-41. Epub 2013 Jan 29.

Division of Hematology/Oncology, Children's Hospital, Boston, MA, USA.

The t(6;11)(q27;q23) is a recurrent chromosomal rearrangement that encodes the MLLAF6 fusion oncoprotein and is observed in patients with diverse hematologic malignancies. The presence of the t(6;11)(q27;q23) has been linked to poor overall survival in patients with AML. In this study, we demonstrate that MLL-AF6 requires continued activity of the histone-methyltransferase DOT1L to maintain expression of the MLL-AF6-driven oncogenic gene-expression program. Using gene-expression analysis and genome-wide chromatin immunoprecipitation studies followed by next generation sequencing, we found that MLL-fusion target genes display markedly high levels of histone 3 at lysine 79 (H3K79) dimethylation in murine MLL-AF6 leukemias as well as in ML2, a human myelomonocytic leukemia cell line bearing the t(6;11)(q27;q23) translocation. Targeted disruption of Dot1l using a conditional knockout mouse model inhibited leukemogenesis mediated by the MLL-AF6 fusion oncogene. Moreover, both murine MLL-AF6-transformed cells as well as the human MLL-AF6-positive ML2 leukemia cell line displayed specific sensitivity to EPZ0004777, a recently described, selective, small-molecule inhibitor of Dot1l. Dot1l inhibition resulted in significantly decreased proliferation, decreased expression of MLL-AF6 target genes, and cell cycle arrest of MLL-AF6-transformed cells. These results indicate that patients bearing the t(6;11)(q27;q23) translocation may benefit from therapeutic agents targeting aberrant H3K79 methylation.
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http://dx.doi.org/10.1182/blood-2012-11-465120DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3612861PMC
March 2013

A selective inhibitor of EZH2 blocks H3K27 methylation and kills mutant lymphoma cells.

Nat Chem Biol 2012 Nov 30;8(11):890-6. Epub 2012 Sep 30.

Epizyme, Inc., Cambridge, MA, USA.

EZH2 catalyzes trimethylation of histone H3 lysine 27 (H3K27). Point mutations of EZH2 at Tyr641 and Ala677 occur in subpopulations of non-Hodgkin's lymphoma, where they drive H3K27 hypertrimethylation. Here we report the discovery of EPZ005687, a potent inhibitor of EZH2 (K(i) of 24 nM). EPZ005687 has greater than 500-fold selectivity against 15 other protein methyltransferases and has 50-fold selectivity against the closely related enzyme EZH1. The compound reduces H3K27 methylation in various lymphoma cells; this translates into apoptotic cell killing in heterozygous Tyr641 or Ala677 mutant cells, with minimal effects on the proliferation of wild-type cells. These data suggest that genetic alteration of EZH2 (for example, mutations at Tyr641 or Ala677) results in a critical dependency on enzymatic activity for proliferation (that is, the equivalent of oncogene addiction), thus portending the clinical use of EZH2 inhibitors for cancers in which EZH2 is genetically altered.
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http://dx.doi.org/10.1038/nchembio.1084DOI Listing
November 2012

Conformational adaptation drives potent, selective and durable inhibition of the human protein methyltransferase DOT1L.

Chem Biol Drug Des 2012 Dec 9;80(6):971-80. Epub 2012 Oct 9.

Epizyme Inc., 325 Vassar Street, Cambridge, MA 02139, USA.

DOT1L is the human protein methyltransferase responsible for catalyzing the methylation of histone H3 on lysine 79 (H3K79). The ectopic activity of DOT1L, associated with the chromosomal translocation that is a universal hallmark of MLL-rearranged leukemia, is a required driver of leukemogenesis in this malignancy. Here, we present studies on the structure-activity relationship of aminonucleoside-based DOT1L inhibitors. Within this series, we find that improvements in target enzyme affinity and selectivity are driven entirely by diminution of the dissociation rate constant for the enzyme-inhibitor complex, leading to long residence times for the binary complex. The biochemical K(i) and residence times measured for these inhibitors correlate well with their effects on intracellular H3K79 methylation and MLL-rearranged leukemic cell killing. Crystallographic studies reveal a conformational adaptation mechanism associated with high-affinity inhibitor binding and prolonged residence time; these studies also suggest that conformational adaptation likewise plays a critical role in natural ligand interactions with the enzyme, hence, facilitating enzyme turnover. These results provide critical insights into the role of conformational adaptation in the enzymatic mechanism of catalysis and in pharmacologic intervention for DOT1L and other members of this enzyme class.
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http://dx.doi.org/10.1111/cbdd.12050DOI Listing
December 2012

The Y641C mutation of EZH2 alters substrate specificity for histone H3 lysine 27 methylation states.

FEBS Lett 2011 Oct 17;585(19):3011-4. Epub 2011 Aug 17.

Epizyme, Inc., 325 Vassar St., Cambridge, MA 02139, USA.

Mutations at tyrosine 641 (Y641F, Y641N, Y641S and Y641H) in the SET domain of EZH2 have been identified in patients with certain subtypes of non-Hodgkin lymphoma (NHL). These mutations were shown to change the substrate specificity of EZH2 for various methylation states of lysine 27 on histone H3 (H3K27). An additional mutation at EZH2 Y641 to cysteine (Y641C) was also found in one patient with NHL and in SKM-1 cells derived from a patient with myelodisplastic syndrome (MDS). The Y641C mutation has been reported to dramatically reduce enzymatic activity. Here, we demonstrate that while the Y641C mutation ablates enzymatic activity against unmethylated and monomethylated H3K27, it is superior to wild-type in catalyzing the formation of trimethylated H3K27 from the dimethylated precursor.
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http://dx.doi.org/10.1016/j.febslet.2011.08.018DOI Listing
October 2011

MLL-rearranged leukemia is dependent on aberrant H3K79 methylation by DOT1L.

Cancer Cell 2011 Jul;20(1):66-78

Division of Hematology/Oncology, Children's Hospital, Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA.

The histone 3 lysine 79 (H3K79) methyltransferase Dot1l has been implicated in the development of leukemias bearing translocations of the Mixed Lineage Leukemia (MLL) gene. We identified the MLL-fusion targets in an MLL-AF9 leukemia model, and conducted epigenetic profiling for H3K79me2, H3K4me3, H3K27me3, and H3K36me3 in hematopoietic progenitor and leukemia stem cells (LSCs). We found abnormal profiles only for H3K79me2 on MLL-AF9 fusion target loci in LSCs. Inactivation of Dot1l led to downregulation of direct MLL-AF9 targets and an MLL translocation-associated gene expression signature, whereas global gene expression remained largely unaffected. Suppression of MLL translocation-associated gene expression corresponded with dependence of MLL-AF9 leukemia on Dot1l in vivo. These data point to DOT1L as a potential therapeutic target in MLL-rearranged leukemia.
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http://dx.doi.org/10.1016/j.ccr.2011.06.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3329803PMC
July 2011

Selective killing of mixed lineage leukemia cells by a potent small-molecule DOT1L inhibitor.

Cancer Cell 2011 Jul;20(1):53-65

Epizyme, Inc., Cambridge, MA 02139, USA.

Mislocated enzymatic activity of DOT1L has been proposed as a driver of leukemogenesis in mixed lineage leukemia (MLL). The characterization of EPZ004777, a potent, selective inhibitor of DOT1L is reported. Treatment of MLL cells with the compound selectively inhibits H3K79 methylation and blocks expression of leukemogenic genes. Exposure of leukemic cells to EPZ004777 results in selective killing of those cells bearing the MLL gene translocation, with little effect on non-MLL-translocated cells. Finally, in vivo administration of EPZ004777 leads to extension of survival in a mouse MLL xenograft model. These results provide compelling support for DOT1L inhibition as a basis for targeted therapeutics against MLL.
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http://dx.doi.org/10.1016/j.ccr.2011.06.009DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4046888PMC
July 2011

Coordinated activities of wild-type plus mutant EZH2 drive tumor-associated hypertrimethylation of lysine 27 on histone H3 (H3K27) in human B-cell lymphomas.

Proc Natl Acad Sci U S A 2010 Dec 15;107(49):20980-5. Epub 2010 Nov 15.

Epizyme, Inc, 840 Memorial Drive, Cambridge, MA 02139, USA.

EZH2, the catalytic subunit of the PRC2 complex, catalyzes the mono- through trimethylation of lysine 27 on histone H3 (H3K27). Histone H3K27 trimethylation is a mechanism for suppressing transcription of specific genes that are proximal to the site of histone modification. Point mutations of the EZH2 gene (Tyr641) have been reported to be linked to subsets of human B-cell lymphoma. The mutant allele is always found associated with a wild-type allele (heterozygous) in disease cells, and the mutations were reported to ablate the enzymatic activity of the PRC2 complex for methylating an unmodified peptide substrate. Here we demonstrate that the WT enzyme displays greatest catalytic efficiency (k(cat)/K) for the zero to monomethylation reaction of H3K27 and diminished efficiency for subsequent (mono- to di- and di- to trimethylation) reactions. In stark contrast, the disease-associated Y641 mutations display very limited ability to perform the first methylation reaction, but have enhanced catalytic efficiency for the subsequent reactions, relative to the WT enzyme. These results imply that the malignant phenotype of disease requires the combined activities of a H3K27 monomethylating enzyme (PRC2 containing WT EZH2 or EZH1) together with the mutant PRC2s for augmented conversion of H3K27 to the trimethylated form. To our knowledge, this is the first example of a human disease that is dependent on the coordinated activities of normal and disease-associated mutant enzymatic function.
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http://dx.doi.org/10.1073/pnas.1012525107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3000297PMC
December 2010

Quantitative analysis of histone deacetylase-1 selective histone modifications by differential mass spectrometry.

J Proteome Res 2008 Dec;7(12):5177-86

Department of Proteomics, Merck Research Laboratories, Merck & Co. Inc., Rahway, New Jersey 07065, USA.

Inhibitors of class 1 and class 2 histone deacetylase (HDAC) enzymes have shown antitumor activity in human clinical trials. More recently, there has been interest in developing subtype-selective HDAC inhibitors designed to retain anticancer activity while reducing potential side effects. Efforts have been initiated to selectively target HDAC1 given its role in tumor proliferation and survival. The development of HDAC1-specific inhibitors will require the identification of HDAC1-selective pharmacodynamic markers that correlate closely with HDAC1-inhibition in vitro and in vivo. Existing histone markers of HDAC target engagement were developed using pan-HDAC inhibitors and do not necessarily represent robust readouts for isoform-specific inhibitors. Therefore, we have initiated a proteomic approach to identify readouts for HDAC1 inhibition. This approach involves the use of differential mass spectrometry (dMS) to identify post-translational changes in histones by profiling histone-enriched cellular fractions treated with various HDAC inhibitors. In this study, we profiled histones isolated from the HCT116 human colon cancer cell line that have been treated with compounds from multiple chemical classes that are specific for HDAC1; HDAC1 and 3; and HDAC1, 3, and 6 enzymes. In two independent experiments, we identified 24 features that correlated with HDAC1-inhibition. Among the peptides modulated by HDAC1-selective inhibitors were Ac-H2B-K5 from histone H2B, and Ac-H3-K18 from histone H3. Commercially available antibodies to specific histone acetyl-lysine residues were used to confirm that these peptides also provide pharmacodynamic readouts for HDAC1-selective inhibitors in vivo and in vitro. These results show the utility of dMS in guiding the identification of specific readouts to aid in the development of HDAC-selective inhibitors.
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http://dx.doi.org/10.1021/pr800510pDOI Listing
December 2008