(A) Heterozygous LSL–K-ras^G12D mice were bred to homozygous LCK-Cre transgenic mice to generate mice expressing K-ras^G12D in the T cell lineage (LCR) and littermate control mice not expressing K-ras^G12D (LC). (B) Kaplan-Meier graph showing that LCR mice spontaneously develop T-ALL with 100% penetrance and a median latency of approximately 180 days, while LC control mice do not. (C) Lethally irradiated LC recipient mice were reconstituted with 5-FU–treated donor LCR BM cells after transduction with empty MigR1 (negative control), L1601P, L1601PΔP, or N1ΔP. Mice were bled 6 weeks after transduction and evaluated for circulating DP cells. Numbers within scatter plots refer to the percentages of live gated cells. (D) Kaplan-Meier graph shows the fraction of mice developing T-ALL over time. K-ras/L1601P: P = 0.005, K-ras/L1601PΔP: P < 0.0001, K-ras/N1ΔP: P = 0.802 versus K-ras/MigR1. (E) Western blot for activated Notch1 antibody (anti-V1744) shows activated, truncated forms of Notch1 in both K-ras/N1ΔP mice and K-ras/MigR1 mice. Extract from 293T cells transiently transfected with N1ΔP is shown as a negative control. Extract from the T-ALL cell line KOPT-K1, which contains both an HD mutation and a PEST deletion (Δ2518), is shown as a positive control. (F) Primary cells cultured from tumors that developed in mice reconstituted with K-ras/L1601P or K-ras/L1601PΔP BM cells were treated with 1 μM JC19 (GSI) or DMSO carrier in triplicate wells and measured for fold cell growth (relative to initial cell number) after 6 days. Cell counts were extrapolated. In experiments involving mice, at least 5 mice were present in each test group, and each experiment was performed twice. Error bars represent single SDs of the mean.