Figure 2. Oncogenic mutations correct aging-associated functional defects in B progenitors, leading to increased leukemogenesis.
(A) Ba/F3 cells expressing vector (V) or oncogenes (BCR-ABL, NRASV12, Myc) were grown in various concentrations of IL-3 for 24 hours, and STAT5 activation in these cells was determined by flow cytometry. (B) Ba/F3 cells were grown overnight in media containing or lacking IL-3, and expression levels of Myc and Hprt in these cells were determined by qPCR. Values in A and B represent the mean ± SEM of 3 independent experiments (9 total samples). (C–F) c-KIT+ BM cells were isolated from young (2-month-old) or old (24-month-old) mice, retrovirally transduced to express vector or oncogenic BCR-ABL, NRASV12,, or Myc (each with coexpressed GFP), and transplanted into sublethally irradiated young BALB/c mice. Three weeks after transplantation, mice were sacrificed, and STAT5 activity (C) and mRNA levels of Hprt, Myc, and Ebf (D–F) were determined in vector-expressing or oncogene-expressing pro–B cell progenitors. Values in C–F represent the mean ± SEM for more than 5 mice per group. (G and H) Young mice were lethally irradiated and transplanted with either 2 × 106 young or old whole BM cells. Four days later, mice reconstituted with young or old BM cells were transplanted, respectively, with young or old c-KIT+ cells expressing oncogenic NRASV12 or Myc. Leukemia-free survival is plotted using Kaplan-Meier graphs. Most mice developed B220+CD43+ pro–B cell–like acute lymphoblastic leukemia (ALL) (87% and 80% for NRAS- and Myc-driven leukemias, respectively, on the old backgrounds). (I) Young or old mice were treated with busulfan and transplanted with young or old c-KIT+ cells expressing oncogenic NRASV12. Leukemia-free survival is plotted using Kaplan-Meier graphs. Values in G and H represent the mean ± SEM of 2 independent experiments, with more than 15 mice per group in total, and values in I represent 5 mice per treatment group. (A–F) **P < 0.01 and #P < 0.001, by Student’s t test . In C–F, oncogene-bearing samples were compared with vector-expressing controls (old to old). (G–I) **P < 0.01 and #P < 0.001, by Cox proportional hazards test. BMT, BM transplantation.