The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in EZH2 were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) EZH2. Here, we demonstrate that cell lines bearing EZH2 mutations show a cytotoxic response, while cell lines with WT-EZH2 show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT-EZH2 models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT-EZH2 backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, PRDM1/BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased Bcell maturation and a greater dependence on B-cell activation signaling.

<div>Abstract<p>The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in <i>EZH2</i> were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) <i>EZH2</i>. Here, we demonstrate that cell lines bearing <i>EZH2</i> mutations show a cytotoxic response, while cell lines with WT-<i>EZH2</i> show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT-<i>EZH2</i> models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT-<i>EZH2</i> backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, <i>PRDM1</i>/BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased B-cell maturation and a greater dependence on B-cell activation signaling. <i>Mol Cancer Ther; 16(11); 2586–97. ©2017 AACR</i>.</p></div>

<p>Table S1: Conditions and seeding densities of all cell lines used in studies; Table S2. Single agent IC50 values for agents tested for 3 days in 96-well plates as described in Materials and Methods; Figure S1. Tazemetostat dosing reduced H3K27me3 in tumor xenografts and is tolerated by SCID mice; Figure S2: CD40L has little effect on growth of DLBCL cell lines; Figure S3. SU-DHL-5 cells were treated for 4 days with TAZ (0.1 to 1 µM) followed by addition of 500 ng/mL CD40L at the indicated time points (1-60 minutes; Figure S4. An ABC-DLBCL gene signature (1) is up-regulated following tazemetostat treatment in KARPAS-422 (EZH2 mutant GCB), Farage (EZH2 WT GCB), SU-DHL-5 (EZH2 WT GCB), TMD8 (EZH2 WT ABC) and ABC vs. GCB gene signature was applied to RNAseq data from each cell line with and without tazemetostat treatment.; Figure S5: CD40L, but not IL-21 induces PRDM1 in combination with tazemetostat (TAZ); Figure S6. A CD40 responsive gene set (2) is up-regulated following tazemetostat treatment in KARPAS-422 (EZH2 mutant GCB), Farage (EZH2 WT GCB), SU-DHL-5 (EZH2 WT GCB), TMD8 (EZH2 WT ABC) and Basso_CD40¬_SIGNALING_UP gene signature was applied to RNAseq data from each cell line with and without tazemetostat treatment; Figure S7. Model for tazametostat mechanism of action in DLBCL</p>

<p>Table S1: Conditions and seeding densities of all cell lines used in studies; Table S2. Single agent IC50 values for agents tested for 3 days in 96-well plates as described in Materials and Methods; Figure S1. Tazemetostat dosing reduced H3K27me3 in tumor xenografts and is tolerated by SCID mice; Figure S2: CD40L has little effect on growth of DLBCL cell lines; Figure S3. SU-DHL-5 cells were treated for 4 days with TAZ (0.1 to 1 µM) followed by addition of 500 ng/mL CD40L at the indicated time points (1-60 minutes; Figure S4. An ABC-DLBCL gene signature (1) is up-regulated following tazemetostat treatment in KARPAS-422 (EZH2 mutant GCB), Farage (EZH2 WT GCB), SU-DHL-5 (EZH2 WT GCB), TMD8 (EZH2 WT ABC) and ABC vs. GCB gene signature was applied to RNAseq data from each cell line with and without tazemetostat treatment.; Figure S5: CD40L, but not IL-21 induces PRDM1 in combination with tazemetostat (TAZ); Figure S6. A CD40 responsive gene set (2) is up-regulated following tazemetostat treatment in KARPAS-422 (EZH2 mutant GCB), Farage (EZH2 WT GCB), SU-DHL-5 (EZH2 WT GCB), TMD8 (EZH2 WT ABC) and Basso_CD40¬_SIGNALING_UP gene signature was applied to RNAseq data from each cell line with and without tazemetostat treatment; Figure S7. Model for tazametostat mechanism of action in DLBCL</p>

<div>Abstract<p>The EZH2 small-molecule inhibitor tazemetostat (EPZ-6438) is currently being evaluated in phase II clinical trials for the treatment of non-Hodgkin lymphoma (NHL). We have previously shown that EZH2 inhibitors display an antiproliferative effect in multiple preclinical models of NHL, and that models bearing gain-of-function mutations in <i>EZH2</i> were consistently more sensitive to EZH2 inhibition than lymphomas with wild-type (WT) <i>EZH2</i>. Here, we demonstrate that cell lines bearing <i>EZH2</i> mutations show a cytotoxic response, while cell lines with WT-<i>EZH2</i> show a cytostatic response and only tumor growth inhibition without regression in a xenograft model. Previous work has demonstrated that cotreatment with tazemetostat and glucocorticoid receptor agonists lead to a synergistic antiproliferative effect in both mutant and wild-type backgrounds, which may provide clues to the mechanism of action of EZH2 inhibition in WT-<i>EZH2</i> models. Multiple agents that inhibit the B-cell receptor pathway (e.g., ibrutinib) were found to have synergistic benefit when combined with tazemetostat in both mutant and WT-<i>EZH2</i> backgrounds of diffuse large B-cell lymphomas (DLBCL). The relationship between B-cell activation and EZH2 inhibition is consistent with the proposed role of EZH2 in B-cell maturation. To further support this, we observe that cell lines treated with tazemetostat show an increase in the B-cell maturation regulator, <i>PRDM1</i>/BLIMP1, and gene signatures corresponding to more advanced stages of maturation. These findings suggest that EZH2 inhibition in both mutant and wild-type backgrounds leads to increased B-cell maturation and a greater dependence on B-cell activation signaling. <i>Mol Cancer Ther; 16(11); 2586–97. ©2017 AACR</i>.</p></div>