Loss of Pten accelerates NOTCH1-induced leukemia. (A) Kaplan-Meier T-cell leukemia survival curves. Bone marrow from mice of indicated genotypes was transduced with mutated NOTCH1 (L1601P-ΔPEST) or empty retrovirus and transplanted into irradiated recipients. Median survivals are as follows: L1601P-ΔPEST retrovirus on Pten^wt Ink4a/Arf^wt (81.5 days, n = 6), Pten^f/f Ink4a/Arf^wt Mx1-Cre (35 days, n = 4), Pten^f/f Ink4a/Arf ^f/f Mx1-Cre (34 days, n = 12), and Pten^wt Ink4a/Arf^f/f Mx1-Cre (86.5 days, n = 2; 3 additional mice in cohort died of nonhematologic neoplasms) backgrounds; empty virus on Pten^f/f Ink4a/Arf^wt Mx1-Cre background (153.5 days, n = 4). Indicated pairwise comparisons are significantly different (*log-rank test, P < .007). (B-C) Flow cytometric analysis of Pten protein expression (B) and CD4/CD8 phenotype (C) in GFP⁺ splenic cells from L1601P-ΔPEST leukemias on Pten^f/f Ink4a/Arf^wt Mx1-Cre (7-x series) and Pten^wt Ink4a/Arf^wt (3-x series) backgrounds. Analysis of bone marrow cells showed similar results (data not shown). (D) Tumor burden in spleen and liver as measured by whole organ weight and GFP⁺ fraction is not significantly different between Pten-null and wild-type background leukemias (Student t test). (E) Clonality assessment by TCRβ rearrangement and proviral integration site analysis. Dominant single bands indicative of monoclonality are highlighted by asterisk (*). Normal tissue samples including wild-type thymus (WT Thy) and uninvolved bone marrow (uBM) represent “polyclonal” controls for comparison. ThyL indicates thymic lymphoma; MW, molecular weight marker; and GL, germline. DNA was prepared from either bone marrow or spleen for cases of disseminated leukemia. Each numbered sample represents a different individual transplant recipient mouse. Please note, L1601P-ΔPEST is abbreviated as L1601PdP in the figure labels. Ink4a-Arf is abbreviated as INK4A in the figure labels.