Synergistic in vitro antileukemic effects of dual inhibition of MEK and ATR in Mll-Af4/N-Ras^G12DB-ALL. (A) Normalized isobologram for the nonconstant ratio combination design (Chou-Chou plot) of in vitro drug combinations. Mll-Af4/Nras^G12D preleukemic cells were treated with combinations of AZ20 and trametinib (GSK212) for 72 hours at doses ranging from 0 to 3 μM AZ20 and from 0 to 33 nM GSK212. Synergy was evaluated using the Chou-Talalay CI for Loewe additivity in R. The data are representative of n > 3 independent combination treatment experiments. (B) Annexin V⁺Mll-Af4 B-ALLs were treated for 24 hours with single-agent GSK212 (1 nM) or AZ20 (0.5 μM) or with the combination. One-way ANOVA analyses were conducted to calculate multivariate significance between dimethyl sulfoxide controls and single-agent treatments to combination treatment. (C) Viability of treatment of Mll-Af4/N-Ras^G12D preleukemic cells with GSK212 and AZ20 at concentrations well below the single-agent IC₅₀ values and represented as a percentage of DMSO controls. All experiments were performed in triplicate. The results are shown as an average. ***P < .001.