• EPZ-5676 is a potent DOT1Linhibitor that causes tumor regressions in a rat xenograft model of MLL-rearranged leukemia.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. (Blood. 2013;122(6):1017-1025 Introduction Rearrangements in the MLL gene at position 11q23 occur in 5% to 10% of acute leukemias of lymphoid, myeloid, or mixed/ indeterminant lineage and are especially common in infant acute leukemias and in secondary acute myeloid leukemias arising in patients following treatment of other malignancies with topoisomerase II inhibitors. [1][2][3][4] Acute leukemias bearing MLL rearrangements are aggressive diseases. Current treatment options are limited to chemotherapy and allogeneic hematopoietic stem cell transplantation; however, these have significant side effects and outcomes remain poor. As a result, there is intense interest in developing novel therapeutic strategies for this disease. The MLL gene encodes a large multidomain protein (MLL) that regulates transcription of developmental genes including the HOX genes. 1 The amino terminal portion of the protein contains regions that target MLL to DNA directly, whereas the carboxyl terminal portion of the protein contains a Su(Var)3-9, Enhancer of zeste and Trithorax domain with methyltransferase activity specific for lysine 4 of histone H3 (H3K4).5-9 MLL rearrangements result in the loss of the carboxyterminal methyltransferase domain and an in-frame fusion of the amino-terminal region of MLL to 1 of more than 60 potential fusion partners. [1][2][3] The vast majority of translocations result in oncogenic fusion proteins in which the native methyltransferase domain is replaced by sequences derived from AF4, AF9, AF10, and ENL, which interact with DOT1L directly or indirectly in complexes that promote transcriptional elongation.10-18 DOT1L is a histone methyltransferase enz...

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.

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 activ-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.epigenetic cancer therapy | EZH2 inhibitor

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. Mol Cancer Ther; 13(4); 842-54. Ó2014 AACR.

Inhibitors of the protein methyltransferase Enhancer of Zeste Homolog 2 (EZH2) may have significant therapeutic potential for the treatment of B cell lymphomas and other cancer indications. The ability of the scientific community to explore fully the spectrum of EZH2-associated pathobiology has been hampered by the lack of in vivo-active tool compounds for this enzyme. Here we report the discovery and characterization of EPZ011989, a potent, selective, orally bioavailable inhibitor of EZH2 with useful pharmacokinetic properties. EPZ011989 demonstrates significant tumor growth inhibition in a mouse xenograft model of human B cell lymphoma. Hence, this compound represents a powerful tool for the expanded exploration of EZH2 activity in biology.

SMYD3 has been implicated in a range of cancers; however, until now no potent selective small molecule inhibitors have been available for target validation studies. A novel oxindole series of SMYD3 inhibitors was identified through screening of the Epizyme proprietary histone methyltransferase-biased library. Potency optimization afforded two tool compounds, sulfonamide EPZ031686 and sulfamide EPZ030456, with cellular potency at a level sufficient to probe the in vitro biology of SMYD3 inhibition. EPZ031686 shows good bioavailability following oral dosing in mice making it a suitable tool for potential in vivo target validation studies. KEYWORDS: SMYD3, oxindole, methyltransferase, KMT, oncology, tool compound S et and Mynd Domain containing 3 (SMYD3) is a lysine methyltransferase (KMT) expressed at high levels in a number of different cancer histologies and is associated with a poor clinical prognosis. 1−10 While no single mechanism has emerged to explain this correlation, a number of studies have implicated SMYD3 in the regulation of gene transcription and signal transduction pathways critical for cell survival in breast, liver, prostate, pancreatic, and lung cancer models. 4,7−9 In addition, considerable evidence has been reported in the literature showing that genetic knockdown of SMYD3 leads to a decrease in proliferation of a variety of cancer cell lines. 4,[7][8][9]11 Two studies, employing RNAi-based technologies, have shown that ablation of SMYD3 in hepatocellular carcinoma cell lines greatly reduces cell viability and that its pro-oncogenic role is dependent on its catalytic activity. 7,9 Moreover, SMYD3 has also been shown to be a critical mediator of transformation induced by a KRAS gain-of-function mutation in both pancreatic and lung adenocarcinoma mouse models; these models were likewise dependent on the catalytic activity of SMYD3. 11 The biological function of SMYD3 is still poorly understood. Early studies of SMYD3 suggested that its primary function is to methylate histones. Indeed, several reports have indicated that SMYD3 modifies histone H3 on lysine 4, 9,12 but have also identified a novel modification of histone H4 on lysine 5. 7 The results of these studies have not yet yielded a clear picture of how SMYD3 might be regulating chromatin, but a recent study has strongly implicated SMYD3 as a direct regulator of MAPK pathways in the cytoplasm and not as a regulator of transcription. MAP3K2 (MEKK2) was shown to be trimethylated at lysine 260 by SMYD3. Modification of this residue leads to enhanced downstream MAPK activation and appears to be critical for mutant KRAS driven oncogenesis. 11 SMYD3's role in cancer cell line proliferation, its effect on known oncogenic signal transduction pathways, and the association of SMYD3 mRNA expression with aggressive transformed phenotypes make SMYD3 an attractive target for therapeutic intervention. We report here the first potent and selective small molecule inhibitors suitable for target validation studies.Compound 1 was identified as a mi...

A key challenge in the development of precision medicine is defining the phenotypic consequences of pharmacological modulation of specific target macromolecules. To address this issue, a variety of genetic, molecular and chemical tools can be used. All of these approaches can produce misleading results if the specificity of the tools is not well understood and the proper controls are not performed. In this paper we illustrate these general themes by providing detailed studies of small molecule inhibitors of the enzymatic activity of two members of the SMYD branch of the protein lysine methyltransferases, SMYD2 and SMYD3. We show that tool compounds as well as CRISPR/Cas9 fail to reproduce many of the cell proliferation findings associated with SMYD2 and SMYD3 inhibition previously obtained with RNAi based approaches and with early stage chemical probes.

The microglial triggering receptor expressed on myeloid cells 2 (TREM2) signals via the activatory membrane adaptor molecule TYROBP. Genetic variants or mutations of TREM2 or TYROBP have been linked to inflammatory neurodegenerative diseases associated with aging. The typical aging process goes along with microglial changes and mild neuronal loss, but the exact contribution of TREM2 is still unclear. Aged TREM2 knock‐out mice showed decreased age‐related neuronal loss in the substantia nigra and the hippocampus. Transcriptomic analysis of the brains of 24 months old TREM2 knock‐out mice revealed 211 differentially expressed genes mostly downregulated and associated with complement activation and oxidative stress response pathways. Consistently, 24 months old TREM2 knock‐out mice showed lower transcription of microglial ( Aif1 and Tmem119 ), oxidative stress markers ( Inos, Cyba, and Cybb ) and complement components ( C1qa, C1qb, C1qc, C3, C4b, Itgam, and Itgb2 ), decreased microglial numbers and expression of the microglial activation marker Cd68, as well as accumulation of oxidized lipids. Cultured microglia of TREM2 knock‐out mice showed reduced phagocytosis and oxidative burst. Thus, microglial TREM2 contributes to age‐related microglial changes, phagocytic oxidative burst, and loss of neurons with possible detrimental effects during physiological aging.