Sensitivity of Mll-Af4/N-Ras^G12DB-ALLs to Atr inhibition. (A) IC₅₀ summary of 72-hour treatment of Mll-Af4 B-ALLs vs Mll-Af9 AMLs. Mll-Af4/N-Ras^G12D preleukemic cells, Mll-Af9 primary leukemias from retroviral models (Retro), and Mll-Af9 knock-in mice (KI) were treated with AZ20, as previously described. One-way ANOVA analyses were conducted to calculate multivariate significance between MLL-r B-ALLs and MLL-r AMLs. (B) Correlation of mouse model line IC₅₀ values treated with AZ20 and fold expansion of cell lines at 72 hours. Fold expansion is expressed as a ratio of total cell count in dimethyl sulfoxide (DMSO) controls to initial plating. (C) Phospho- and total Chk1 (S345), Cdk1 (Y15), and Cdk2 (Y15) immunoblotting of Mll-Af4/N-Ras^G12D preleukemic cells treated for 24 hours with 0, 1, or 3 μM AZ20. (D) Representative cell cycle plots for representative primary murine Mll-Af9 AMLs generated by retroviral transformation (#45) and preleukemic Mll-Af4/Nras^G12D B-ALLs treated for 24 hours with DMSO and 3 μM AZ20. The plots are representative of ≥3 independent experiments. (E) Annexin V⁺Mll-Af9 AMLs and Mll-Af4 B-ALLs were treated for 24 hours with 0, 1, or 3 μM AZ20. (F) Treatment of secondary leukemias with 50 mg/kg AZ20 alone, 7 days after transplant (red arrow), extended the survival of treated mice. All in vitro data are representative of n ≥ 5 independent treatment experiments. For in vivo treatment experiments, n ≥ 4 mice per group were used. ***P < .001, **P < .01, *P < .05.