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

SS18-SSX translocation positive synovial sarcoma cell lines are sensitive to EZH2 inhibition <i>in vitro</i>.

<p>(A) Immunoblot analysis of human soft tissue sarcoma (RD and SW982), human synovial sarcoma (Fuji and HS-SY-II), human MRT (G401), and human embryonic kidney (293) cell lines to examine expression of EZH2, SMARCB1, SS18/SS18-SSX, and β-actin (loading control). Closed and open arrowheads represent SS18–SSX and SS18, respectively. (B) The synovial sarcoma cell lines were treated at increasing doses of tazemetostat for 14 days and proliferation was assessed at the indicated timepoints. Tazemetostat was refreshed on days 4 and 11. Data represent the geometric mean ± 95% confidence interval in triplicate from 3 independent experiments. IC₅₀, 50% inhibitory concentration value. (C) H3K27Me3 levels were assessed in HS-SY-II, Fuji and SW982 treated with tazemetostat for 96 hours. Levels were quantified using ELISA and data is represented as the ratio of H3K27Me3 to total H3 and are shown relative to the DMSO control of each concentration. Data are represented as mean values ± SEM (n = 3). (D, E) Apoptosis analysis (Annexin V-FITC assay) (<i>D</i>) or cell-cycle analysis (by flow cytometry) (<i>E)</i> of Fuji and HS-SY-II treated with the IC₅₀ for tazemetostat (0.15 μmol/L and 0.52 μmol/L, respectively). Data are represented as mean values ± SEM (n = 3). For panel D: * <i>P</i>< 0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, **** <i>P</i><0.0001, vs. untreated, one-way analysis of variance followed by the Dunnett’s multiple comparisons test.</p

EZH2 inhibition modulates expression of synovial sarcoma-related genes.

<p>(A) Quantitative real-time PCR (qPCR) analysis of the indicated cell lines treated with tazemetostat at the indicated doses for 2, 4 or 7 days. Data are normalized to <i>GAPDH</i> expression relative to the vehicle control. Data are represented as mean values ± SEM (n = 3). (B) qPCR analysis of HS-SY-II or Fuji xenograft samples treated with tazemetostat at the indicated doses, twice daily, for 7 days. Tumors were harvested 3 hours after the last dose on day 7. Data are normalized to <i>GAPDH</i> expression or <i>18s rRNA</i> expression relative to the vehicle control and represents the average of 5 independent animals per group ± SEM. For this whole figure: * <i>P</i>< 0.05, ** <i>P</i><0.01, *** <i>P</i><0.001, **** <i>P</i><0.0001, vs. vehicle control, one-way analysis of variance followed by the Dunnett’s multiple comparisons test.</p

Tazemetostat treatment inhibits growth of synovial sarcoma patient-derived xenograft (PDX) models.

<p>Three individual synovial sarcoma PDX models were dosed with tazemetostat on a BID schedule for 35 days at the indicated doses: CTG-0771 (A-C), CTG-0331 (D-F), and CTG-1169 (G-I). Arrowheads indicate the administration of doxorubicin, horizontal arrows indicate the dosing period for tazemetostat. For the CTG-0771 model tazemetostat was dosed at 500 mg/kg from days 1–17 followed by 400 mg/kg from days 19–35. A control group in each study was dosed with doxorubicin on days 1, 7, and 14. (A, D, G) Tazemetostat induced significant tumor growth inhibition in two of three synovial sarcoma PDX models. Data shown as mean values ±SEM; n = 12 per model. * <i>P</i><0.05 vs. vehicle group, two-tailed one-way analysis of variance followed by the Dunnett’s multiple comparisons test. (B, E, H) Mean tumor volumes ±SEM on at the end of dosing (day 35) are presented. * <i>P</i><0.05, ** <i>P</i><0.01, *** <i>P</i><0.001 vs. vehicle group, one-way analysis of variance followed by the Dunnett’s multiple comparisons test. (C, F, I) For the PDX, models 3 to 5 mice per group with the largest tumors were euthanized on day 35 for blood and tissue sampling, and the 7 remaining mice were followed for an additional 25 days with biweekly tumor measurements. Kaplan-Meier plots show that tazemetostat increased the survival of mice in a dose-dependent manner for CTG-0331 and CTG-0771, as assessed by a tumor volume endpoint of 1200 mm³. Note that in panel C the lines for the 250 and 400–500 mg/kg groups are overlapping. Kaplan-Meier plot for CTG-1169 was assessed by a tumor volume endpoint of 600 mm³ (a smaller endpoint was used due to the slow growing nature of this model).</p

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