Oncogenic K-Ras–driven T-cell disease is hypersensitive toward Notch1 inhibitor therapy. (A) Primary oncogenic K-Ras–expressing T-ALL cells and the human cell line CCRF-CEM were grown in RPMI media without cytokines and treated with the γ-secretase inhibitor XXI as indicated. Cell viability was measured after 48 hours, and the proportion of viable cells relative to the control (no inhibitor) was plotted. Experiments were performed in triplicate. Values are mean plus or minus SEM. (B) Inhibition of Notch1 signaling caused reduced expression of known Notch1 target genes. Primary cells were treated as indicated, and expression of Hes1, Hey1, and Dtx1 mRNA levels was determined by RQ-PCR. Each experiment was performed in duplicate. (C) Inhibition of Notch1 results in cleavage of PARP (top panel) and a decrease of ICN levels (middle panel). Equal loading was confirmed by measuring GAPDH protein levels. GSI indicates γ-secretase inhibitor. (D) The primary oncogenic K-Ras expressing T-ALL cell line 470 and the human cell line CCRF-CEM were grown in RPMI-media without cytokines and treated with rapamycin and PD98059 as indicated. Cell viability was measured after 48 hours, and the proportion of viable cells relative to the control (no inhibitor) was plotted. Experiments were performed in triplicate. Values are mean plus or minus SEM. (E) Primary 470 T-ALL cells were treated with GSI XXI or PD98059 for 6 hours as indicated. Protein lysates were extracted, and immunoblot analysis was performed using antiphosph-p42/44 and anti-ICN antibodies. Equal loading was confirmed by measuring p42/44 and GAPDH protein levels, respectively. GSI indicates γ-secretase inhibitor.