EPZ-5676 causes tumor regressions and demonstrates in vivo target inhibition in tumor and surrogate tissue in a rat model of MLL-rearranged leukemia. (A) Effect of EPZ-5676 administration on the growth of MV4-11 xenograft tumors implanted SC in immunocompromised rats. Rats were dosed continuously by IV infusion with vehicle (black line), with EPZ-5676 at 70 mg/kg/day (blue line), 35 mg/kg/day (red line), or for 8 hours daily at 67 mg/kg per day (green line) for 21 days. Median tumor sizes are plotted for each group (n = 10). Tumor growth in individual animals is shown in supplemental Figure 3. Significant P values of P ≤ .0005, calculated using a repeated-measures analysis of variance (ANOVA) and Dunnett posttest, were determined between the vehicle and all 3 treated groups. (B) Effect of additional EPZ-5676 dose and schedules on MV4-11 xenograft tumor growth. Significant tumor growth inhibition was observed in rats treated with 70 mg/kg per day EPZ-5676 by continuous IV infusion for 14 (orange line) or 21 days (blue line) (P = .0004, repeated-measures ANOVA with a Dunnett posttest). Treatment with 70 mg/kg per day continuously for 7 days followed by 14 days at 33.5 mg/kg per day infused for 8 hours daily (green line) led to weak, but statistically significant tumor growth inhibition (P = .04, repeated-measures ANOVA with a Dunnett posttest). A final group dosed for 21 days with 33.5 mg/kg per day infused for 8 hours daily (purple line) showed no tumor growth inhibition when compared with the vehicle control. Median tumor sizes are plotted for each group (vehicle [n = 9], 70 mg/kg per day, 21-day infusion [n = 9], 70 mg/kg per day, 14-day infusion [n = 10], 70 mg/kg per day, 7-day infusion, then 14 days at 33.5 mg/kg per day infused for 8 hours daily [n = 9], 33.5 mg/kg per day, 21 days infused for 8 hours daily [n = 10]). Tumor growth in individual animals is shown in supplemental Figure 4. (C) H3K79me2 levels in tumor tissue, PBMCs, and bone marrow cells harvested from nude rats dosed continuously by IV infusion for 14 days with vehicle or EPZ-5676 at 35 and 70 mg/kg per day. H3K79me2 levels were determined in acid extracted histones by ELISA (tumor, bone marrow) or quantitated immunoblot of whole cell lysates (PBMCs). H3K79me2 levels were normalized to those of total histone H3 in the same sample and are plotted as a percent of the mean H3K79me2 level in tissue from the vehicle-treated (control) group, which is set at 100%. Horizontal lines represent the mean percent H3K79me2 values for each group. (D) Reduced expression of MLL-fusion target genes HOXA9 and MEIS1 measured by qRT-PCR of RNA extracted from tumor tissue harvested from rats dosed continuously by IV infusion for 14 days with EPZ-5676 at 35 and 70 mg/kg per day. HOXA9 or MEIS1 transcript levels are plotted as a percent of the mean transcript level in tumors from the vehicle-treated (control) group, which is set at 100%. Horizontal lines represent the mean percent transcript level in each group.