Role of EZH2 mutations in conferring resistance to the EZH2 inhibitor. (A) SU-DHL-10-P or SU-DHL-10-R cells were treated with either DMSO or with GSK126 (1 μM) for 48 hours and analyzed for EZH2 and H3K27me3 expression. Histone H3 and ACTINB were used as loading controls. (B) The mRNA expression of the indicated EZH2 target genes in SU-DHL-10-P or SU-DHL-10-R cells that were treated with either DMSO or with GSK126 (1 μM) for 48 hours. (C) Gene and protein schematics for EZH2 and the mutations identified from the genomic DNA sequencing of the S-adenosyl-methionine domain are shown. (D) DNA-sequencing chromatograms reveal the presence of the C663Y, E720G, and Y726F mutations in EZH2 in GSK126-resistant DLBCL cell lines. (E) Table of previously reported EZH2 mutations found in sequenced cancer samples. Data were collected from the cBioPortal for the designated studies. (F) Mapping of the EZH2^C663Y, EZH2^E720G, and EZH2^Y726F mutations on to the structure of the EZH2 SET domain (Protein Data Bank [PDB] ID: 4MI5⁴² ) are shown. (G) A CETSA was performed for the EZH2^WT, EZH2^Y641F, EZH2^C663Y, EZH2^E720G, and EZH2^Y726F mutants in SU-DHL-10 cells. The samples were analyzed for EZH2 and ACTINB by immunoblotting. The densitometric quantitation for EZH2 at 52°C is presented below the respective blots and were normalized to ACTINB. The P values were calculated using a Student t test. Data are presented as mean ± SEM; *P < .05; **P < .01; ***P < .001. AA, amino acid; ID, identification; Mut, mutation; NA, not available.