Figure 1.

Leukemia-initiating cells are responsible for therapeutic resistance in Pten null T-ALL. A, schematic diagram of strategy to investigate mechanisms underlying resistance to rapamycin. Pten null mice with T-ALL were treated for 7 days with rapamycin, and then bone marrow was harvested and transplanted into secondary and tertiary recipients in the absence of rapamycin. These recipients rapidly developed T-ALL and were re-treated for 2 days with rapamycin. After re-treatment, hematopoietic cells were harvested from Pten null T-ALL animals and analyzed for levels of phospho-S6, mutations in Notch1 and Fbxw7, and leukemia-initiating cell activity in blast and LIC compartments. B, quantitation of phospho-S6⁺ cells in Pten null TALL blast cells in bone marrow harvested from mice after 2 days of rapamycin treatment in vivo (4 mg/kg daily) using intracellular flow cytometric analysis. C, summary of mutational analysis of Notch1 exons 26, 27 and 34 and Fbxw7 exons 8, 9, and 10 using genomic DNA from splenocytes harvested from T-ALL mice after single or double rapamycin treatment. Ratios indicate number of T-ALL mice with mutations (Mut) over total number T-ALL mice screened. D, summary of transplantation assay with sorted fractions. Blast and LIC sorted populations were transplanted into NSG recipients at indicated doses and leukemia development was evaluated in recipients. Data in B are represented as mean ± SEM.