Figure 2

Specific EZH2 inhibition is efficacious in vitro in both myeloma cell line and primary patient samples, even those with high-risk features. EZH2 inhibition induces cell cycle arrest followed by apoptosis. (a) Cell viability determined using the WST1 assay (normalised to DMSO control) in a panel of eight myeloma cell lines incubated with increasing concentrations of EZH2 inhibitor (EPZ005687) for 72 h. The GI50 for each cell line (calculated using the Graphpad Prism software) is shown. There was no statistically significant difference between the GI50s for each cell line (one-way analysis of variance (ANOVA) P>0.05). Graph shows mean and s.e.m. of at least three independent biological replicates. (b) CD138 selected plasma cells from six patients’ BM aspirate samples were co-cultured with the BM stromal cell line HS5 (GFP tagged) for 72 h in the presence of the indicated concentration of EPZ005687 or vehicle control (DMSO). Cells were then stained with Annexin V and DAPI prior to flow cytometric analysis. Results show the percentage of cell viability (of DMSO) measured as the percentage of cells that were Annexin V and PI-negative within the GFP-negative fraction. Raw data and mean value (horizontal line) are shown. One sample t-tests were performed to look for a significant reduction in viability at each concentration compared with 100%. Those with P-values<0.05 are indicated by an asterisk (*). (c) Cell viability determined using the WST1 assay (normalised to DMSO control) in a panel of eight myeloma cell lines incubated with increasing concentrations of EZH2 inhibitor (EPZ005687) for 6 days. Graph shows mean and s.e.m. of at least three independent replicates in each cell line. The cell line features of factors previously demonstrated to be relevant to EZH2 inhibition in myeloma and TP53 status are shown in the table below with further details in Supplementary Table S3. Of note, no cell lines used had EZH2 mutations (details from Broad CCLE, MMRF Myeloma Cell Line Characterization Data repository and van Haaften et al.¹⁷). HD=homozygous deletion, hom=homozygous mutation, het=heterozygous mutation. One sample t-tests were performed to look for a significant reduction in viability at 4 μM compared with 100%. Those with P-values<0.05 are indicated by an asterisk (*). (d) Cell cycle analysis with propidium iodide staining was performed following EZH2 inhibition with EPZ005687 for 3 days in KMS11 and KMM1 cell lines. The cells in each phase of the cell cycle are shown as a percentage of all cells in cycle. Results shown mean and s.e.m. of three independent replicate experiments. The mean percentage of cells in G1 was compared across conditions using a one-way ANOVA followed by multiple comparisons to DMSO control. There was a significant increase (adj. P<0.05) in G1% indicated by an asterisk (*). (e) Apoptosis was assessed after 6 days of incubation with increasing concentrations of EPZ005687 and compared with DMSO control by Annexin V/PI staining in the panel of eight cell lines. One sample t-tests were performed to look for a significant increase at each concentration compared with 1. Statistical significance (P<0.05) is indicated by an asterisk (*).