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

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

Effects of EZH2 inhibition in synovial sarcoma cell line xenograft models.

<p>(A) Tumor growth inhibition in Fuji xenograft induced by twice daily (BID) administration of tazemetostat for 35 days at the indicated dosage, with or without doxorubicin (10 mg/kg) treatment on days 1 and 22. An alternative EZH2 inhibitor, EPZ011989, was also included at 500 mg/kg BID. Treatment was stopped after 35 days and tumor regrowth was monitored. Data shown as mean values ±SEM; n = 7. Arrowheads indicate the administration of doxorubicin, lines indicate the dosing period for tazemetostat or EPZ011989. (B) EZH2 target inhibition in Fuji xenograft samples from mice treated with tazemetostat for seven days at the indicated doses in relationship to systemic C_trough levels of tazemetostat measured 5 minutes before the last dose on day 7. H3K27Me3 and H3 levels were measured in histones preparations by ELISA and data represents the ratio of H3K27Me3 to total H3. The horizontal line represents the mean. (C) Assessment of tumor growth in HS-SY-II xenograft model. Mice were treated with tazemetostat for 28 days at the indicated dosage, with or without doxorubicin (10 mg/kg) treatment on days 1 and 22. Data are shown as mean values ±SEM; n = 6 and representative of two independent experiments. Arrowheads indicate the administration of doxorubicin, horizontal arrows indicate the dosing period for tazemetostat. (D) EZH2 target inhibition in HS-SY-II xenograft samples from mice treated with tazemetostat for seven days at the indicated doses in relationship to systemic C_trough levels of tazemetostat measured 5 minutes before the last dose on day 7. H3K27Me3 and H3 levels were measured in histones preparations by ELISA and data represents the ratio of H3K27Me3 to total H3. The horizontal line represents the mean. * <i>P</i><0.05 vs. vehicle, # <i>P</i><0.05 vs. both 250 mg/kg tazemetostat and doxorubicin; one-way analysis of variance followed by Tukey’s multiple comparison test after logarithmic transformation.</p

Small molecule inhibitors and CRISPR/Cas9 mutagenesis demonstrate that SMYD2 and SMYD3 activity are dispensable for autonomous cancer cell proliferation.

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