PRECLINICAL EVALUATION OF EZH2 INHIBITORS IN MODELS OF HUMAN SYNOVIAL SARCOMA Heike Keilhack1, Satoshi Kawano2, Sarah K. Knutson1, Natalie M. Warholic1, Jamie Kubica1, Galina Kuznetsov3, Shanqin Xu3, Yonghong Xiao1, Roy M. Pollock1, Jesse S. Smith1, Kevin K. Kuntz1, Yukinori Minoshima2, Masumi Tsuda4, Shinya Tanaka4 and Robert A. Copeland1 1Epizyme Inc., Cambridge, MA, USA; 2Eisai Co., Ltd., Tsukuba, Japan; 3Eisai, Inc., Andover MA, USA; 4Hokkaido University, Sapporo, Japan Abstract Results

Objective: Posttranslational modification of histone proteins and ATP‐dependent chromatin remodeling are crucial processes for the regulation of the fidelity of normal gene expression. Proteins involved in these processes, such as the PRC2 and SWI/SNF complexes are often genetically altered in cancer. Importantly, these two complexes normally compete with each other in binding to and affecting chromatin; genetic alterations can create an imbalance in this antagonism. For instance, INI1 (SMARCB1, SNF5, BAF47) is a subunit of the SWI/SNF complex that is lost in nearly all rhabdoid tumors, which creates an oncogenic dependency on the PRC2‐EZH2 methyltransferase, and rhabdoid tumor models are sensitive to EZH2 small molecule inhibitors. In synovial sarcoma, another INI1‐deficient tumor type, a recurrent chromosomal translocation fuses the SS18 gene (a subunit of the SWI/SNF chromatin remodeling complex) on chromosome 18 to one of three related genes on the X chromosome, SSX1, SSX2 and rarely SSX4. This results in the Three synovial sarcoma cell lines and one malignant rhabdoid tumor (MRT) cell line were Athymic nude mice bearing Fuji synovial sarcoma xenografts were dosed with either A) AthymicnudemicebearingHS‐SY‐II synovial sarcoma xenografts were dosed with expression of an oncogenic SS18‐SSX fusion protein that binds to the SWI/SNF grownineither2Dor3DcellculturesandwereincubatedwitheitherDMSOorEPZ‐6438 vehicle, EPZ‐6438, Doxorubicin or an EPZ‐6438/Doxorubicin combination at the indicated either vehicle (oral or iv), EPZ‐6438(oral),Doxorubicin(iv)oranEPZ‐6438/Doxorubicin complex evicting both the wild‐type SS18 and the tumor suppressor INI1, which at the indicated concentrations. Cell viability was assessed by measuring ATP on days 4, 7, doses, routes and durations. Dosing was stopped on day 35 (day 29 for the combination combination at the indicated doses for 28 days. Two independent studies were 11 and 14, and cells were split an re‐plated at the original seeding density on day 7. Data group) and animals were observed until day 64. When the tumor volume of a given performed. B) Tumors from animals of the second study were harvested on day 28 (3 h are subsequently degraded. This results in aberrant gene expression and are expressed as the fold growth compared to day 0 for each day. animal exceeded 2000 mm3 mice were euthanized. after the last dose) and subjected H3K27 trimethylation analysis by ELISA or IHC for the proliferation marker Ki67. Ki67 positive cells (one entire section) were quantified by ultimately the development of cancer. Aperio imaging and analysis. Methods: Synovial sarcoma cell lines and patient‐derived xenograft (PDX) models were evaluated for their sensitivity to EZH2 inhibition in vitro and in vivo. In addition, histone methylation, changes in gene expression and histology endpoints were assessed. Results: Here we show that EPZ‐6438 (E7438), an early clinical‐stage, selective and orally bioavailable small‐molecule inhibitor of EZH2 enzymatic activity induces anti‐proliferative activity in preclinical models of synovial sarcoma both as a single agent and in combination with chemotherapy. The compound induces dose‐ dependent cell growth inhibition and cell death specifically in SS18‐SSX fusion‐ positive cells in vitro. Treatment of mice bearing either a cell line xenograft or two PDX models leads to dose‐dependent tumor growth inhibition with correlative inhibition of tri‐methylation levels of the EZH2‐specific substrate, lysine 27 on histone H3, except for one SS18‐SSX fusion‐positive cell line.

Conclusion: These data demonstrate a dependency of SS18‐SSX‐positive synovial Three synovial sarcoma cell lines were incubated with either DMSO or EPZ‐6438 at the Athymic nude mice bearing 2 different patient derived xenografts (PDX) of synovial Synovial sarcoma PDX tumors from subsets of mice were harvested on day 35 and sarcomas on EZH2 enzymatic activity in xenograft models which warrants further indicated durations and concentrations. Gene expression was determined by qPCR (n = 3) sarcoma were dosed with either vehicle (oral), EPZ‐6438 (oral) or Doxorubicin (iv) at the analyzed by RNA‐seq analysis, and gene set enrichment analysis was performed. The KEGG andisexpressedrelativetotheDMSOcontrolofeachtimepoint.Theinvivotumor indicated doses for 35 days. Dosing was stopped on day 35 and animals were observed genelist Graft_versus_host_disease was one of the top hits. For instance, expression of investigations in the clinical setting. samples from 7‐day dosing studies in 2 models were also subjected to the analysis. The until day 60. When the tumor volume of a given animal exceeded 2000 mm3 mice were HLA genes in tumors from mice treated with either with vehicle or at 400 mg/kg EPZ‐6438 data were normalized by measuring the relative level of GAPDH mRNA. euthanized (analyzed in the Kaplan‐Meyer plots). BID are shown. Background Conclusions • EPZ‐6438 inhibits cell proliferation in SS18‐SSX fusion positive synovial sarcoma cells in vitro. • EPZ‐6438 increases expression of genes that are aberrantly suppressed in synovial sarcoma. • Antitumor activity was observed in 3 out of 4 synovial sarcoma in vivo models, including cell line and patient derived xenografts grown in mice. • In the Fuji xenograft model EPZ‐6438/Doxorubicin combination shows a superior antitumor effect when compared to each monotherapy; the combination dosage amounts need further optimization to improve tolerability. • Although sensitive to EPZ‐6438 in 2D cell culture, HS‐SY‐II cell line xenografts exhibits dose‐related tumor volume increase in a mouse xenograft model, which is being further investigated. • EPZ‐6438 combined with Doxorubicin does not diminish the antitumor effect of Doxorubicin in the HS‐SY‐II model. • E7438 (EPZ‐6438) has transitioned into clinical development for evaluation in genetically defined cancers. www.epizyme.com We would like to thank the employees of Epizyme, Eisai and Hokkaido University for their contribution to these studies.