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

<div><p>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 <i>EZH2</i> 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.</p></div

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

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

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

Combination benefit with CHOP components and EPZ-6438 in <i>EZH2</i> mutant germinal center B-cell lymphoma cell lines.

<p>Combination index (CI) graphs, generated in Calcusyn, of EPZ-6438 with Mafosfamide, Doxorubicin, or Vincristine in the <i>EZH2</i> Y646F mutant cell lines WSU-DLCL2 (A–C) or SUDHL10 (D–F). The 95% confidence interval is displayed in each graph (representative of 2 biological replicates for each cell line). The fractional effect (Fa) plotted is the fraction of cell growth (inhibition) resulting from a compound treatment, calculated from the DMSO control. A) Additivity was induced for the EPZ-6438/Mafosfamide combination at a 1∶20 constant ratio (doses were 16–125 nM for EPZ-6438 and 313–2500 nM for Mafosfamide). B) Synergy was induced for the EPZ-6438/Doxorubicin combination at a 50∶1 constant ratio (doses were 16–500 nM for EPZ-6438 and 0.3–10 nM for Doxorubicin). C) Additivity was induced for the EPZ-6438/Vincristine combination at a 400∶1 constant ratio (doses were 16–1000 nM for EPZ-6438 and 0.39–2.5 nM for Vincristine). D) Additivity was induced for the EPZ-6438/Mafosfamide combination at a 4∶25 constant ratio (doses were 12.5–200 nM for EPZ-6438 and 78–1250 nM for Mafosfamide). E) Additivity was induced for the EPZ-6438/Doxorubicin combination at a 10∶3 constant ratio (doses were 3–50 nM for EPZ-6438 and 0.94–15 nM for Doxorubicin). F) Additivity was shown for the EPZ-6438/Vincristine combination at an 800∶1 constant ratio (doses were 12.5–200 nM for EPZ-6438 and 15.6–250 pM for Vincristine).</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

EPZ-6438/CHOP combinations show enhanced anti-tumor activity compared to single agents in several <i>EZH2</i> mutant lymphoma xenograft models.

<p>A) WSU-DLCL2 (<i>EZH2</i> Y646F) xenograft-bearing mice were treated for 28 days as indicated. Mean tumor volumes ± SEM (n = 12) are plotted. Treatment with EPZ-6438 at 225 mg/kg BID plus CHOP induced the highest tumor growth inhibition (93%). B) SUDHL6 (<i>EZH2</i> Y646N) xenograft-bearing mice were treated for 28 days as indicated. Mean tumor volumes ± SEM (n = 12) are plotted in the top panel. * <i>p</i><0.05, *** <i>p</i><0.001 vs. vehicle, repeated measures ANOVA, Dunnett's post test. Kaplan-Meyer survival curves (bottom panel) out to 60 days demonstrate significant tumor growth delay in animals treated with EPZ-6438+CHOP (** <i>p</i><0.01). C) SUDHL10 (<i>EZH2</i> Y646F) xenograft-bearing mice were treated with EPZ-6438, COP (chemotherapy without the Doxorubicin component), or their combination for 28 days. Mean tumor volumes ± SEM (n = 8) are plotted in top panel. Percent survival out to 60 days is plotted in the bottom panel (note: 500 mg/kg and 250 mg/kg+COP survival curves overlap). D) SUDHL10 (<i>EZH2</i> Y646F) xenograft-bearing mice were treated for 28 days as indicated (Pred-1 = Prednisone at 0.15 mg/kg BID×5 on days 1–5 and 22–26; Pred-2 = Prednisone at 0.15 mg/kg BID×28). The scatter blot shows the tumor volumes on day 28, and the inset shows the mean tumor volumes ±SEM (n = 10) followed over 28 days (also presented in figure S6B). * <i>p</i><0.05, ** <i>p</i><0.01; two-tailed <i>t</i> test. All groups administered EPZ-6438 show statistically significant smaller tumor volumes on day 28 (<i>p</i><0.01 at least, vs. vehicle or Prednisone single agent at both schedules; two-tailed <i>t</i> test). CHOP: Cyclophosphamide, Hydroxyldaunorubicin (Doxorubine), Oncovin (Vincristine), Prednisone; COP: Cyclophosphamide, Oncovin (Vincristine), Prednisone; BID: two times a day every 12 hours; QD: once a day; TID: three times a day every 8 hours.</p

Glucocorticoid target genes are up-regulated by prednisolone/EPZ-6438 combination in <i>EZH2</i> mutant cell lines.

<p>Expression levels of Sestrin 1 (<i>SESN1</i>, A), <i>TNF</i> (B) and <i>GILZ</i> (C), normalized to DMSO controls, for each cell line treated with the indicated single agents or their combination (2 biological replicates, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111840#s4" target="_blank">materials and methods</a> section 5 for details). Fold change values were quantified using the ΔΔCt method, and <i>ACTB</i>, <i>B2M</i> and <i>GAPDH</i> as reference genes. Error bars represent SEM values. Statistical analysis of the data is presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0111840#pone-0111840-t002" target="_blank">table 2</a>. WT: wild-type.</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