KDM5 inhibition alters the expression of BCR signaling and apoptotic regulatory genes. (A) Expression of BCL2 protein was examined by western blot in 10 DLBCL cell lines exposed to DMSO or 1 μM KDM5-inh1 for 48 hours. (B) SU-DHL-6 and HT cells were treated with DMSO or 1 and 5 μM KDM5-inh1 for 5 days, with the cells reseeded in fresh drug/media after 48 hours. The expression of BCL2 family members was investigated by western blot, with HSC70 used as a loading control. Western blots are representative of 3 independent experiments, with the quantification relative to HSC70 displayed in panel C. Statistical significance was determined using an analysis of variance (ANOVA) with a Dunnett’s posttest vs untreated control: *P < .05; **P < .01; ****P < .0001. Viable cells were quantified in 10 lymphoma cell lines treated with increasing concentrations of KDM5-inh1 for 5 days, alongside increasing concentrations of S63845 (MCL1i) for 3 days. (D) Overall Loewe and highest single agent (HSA) synergy scores calculated for each cell line using Synergy Finder,42 with a score >10 indicating significant synergy. (E) Plots showing the degree of synergy between different concentrations of KDM5-inh1 and S63845 in OCI-LY-1 and WSU-DLCL2 cells, with the area of maximum synergy indicated by a blue box. (F-G) Global levels of H3K4me3 in tumors from mice treated with vehicle or 50 mg/kg KDM5-inh1 for 1 week (n = 3) were quantified by western blot and normalized to H3, whereas BCL2 and pERK levels were quantified and normalized to GAPDH and total ERK, respectively. (H) Activity of 50 mg/kg KDM5-inh1 on the growth of SU-DHL-6 xenografts in comparison with vehicle-treated mice. Data are the mean ± standard error of the mean of 10 individual mice, except in the vehicle group where one mouse was removed because of insufficient tumor growth (<300 mm3). Statistical significance was calculated using a 2-way ANOVA with a Dunnett’s posttest: ***P < .001; ****P < .0001.