Ablation of PI3K blocks BCR-ABL leukemogenesis in mice, and a dual PI3K/mTOR inhibitor prevents expansion of human BCR-ABL+ leukemia cells
Michael G. Kharas, … , Kevan M. Shokat, David A. Fruman
Michael G. Kharas, … , Kevan M. Shokat, David A. Fruman
Published August 14, 2008
Citation Information: J Clin Invest. 2008;118(9):3038-3050. https://doi.org/10.1172/JCI33337.
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Research Article

Ablation of PI3K blocks BCR-ABL leukemogenesis in mice, and a dual PI3K/mTOR inhibitor prevents expansion of human BCR-ABL+ leukemia cells

Abstract

Some cases of pre–B cell acute lymphoblastic leukemia (pre–B-ALL) are caused by the Philadelphia (Ph) chromosome–encoded BCR-ABL oncogene, and these tend to have a poor prognosis. Inhibitors of the PI3K/AKT pathway reduce BCR-ABL–mediated transformation in vitro; however, the specific PI3K isoforms involved are poorly defined. Using a murine model of Ph+ pre–B-ALL, we found that deletion of both Pik3r1 and Pik3r2, genes encoding class IA PI3K regulatory isoforms, severely impaired transformation. BCR-ABL–dependent pre/pro-B cell lines could be established at low frequency from progenitors that lacked these genes, but the cells were smaller, proliferated more slowly, and failed to cause leukemia in vivo. These cell lines displayed nearly undetectable PI3K signaling function and were resistant to the PI3K inhibitor wortmannin. However, they maintained activation of mammalian target of rapamycin (mTOR) and were more sensitive to rapamycin. Treatment with rapamycin caused feedback activation of AKT in WT cell lines but not PI3K-deficient lines. A dual inhibitor of PI3K and mTOR, PI-103, was more effective than rapamycin at suppressing proliferation of mouse pre–B-ALL and human CD19+CD34+ Ph+ ALL leukemia cells treated with the ABL kinase inhibitor imatinib. Our findings provide mechanistic insights into PI3K dependency in oncogenic networks and provide a rationale for targeting class IA PI3K, alone or together with mTOR, in the treatment of Ph+ ALL.

Authors

Michael G. Kharas, Matthew R. Janes, Vanessa M. Scarfone, Michael B. Lilly, Zachary A. Knight, Kevan M. Shokat, David A. Fruman

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Figure 3

Lack of PI3K/AKT/FOXO functional signaling output in α/β-null L-CFCs.

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Lack of PI3K/AKT/FOXO functional signaling output in α/β-null L-CFCs. (A...
(A) β-null and α/β-null cells were fixed, permeabilized, and immunolabeled with anti-PIP3 antibodies (orange). Nuclei were stained by DAPI (blue). Indicated cells were pretreated with 50 nM wortmannin 30 minutes prior to fixation. Immunoreactive cells were quantitated by counting 100 cells in images acquired with a 40× objective (represented in the graph) and visualized with a 100× objective (represented in the images). Typical examples of n = 4 clones per genotype, 2 independent experiments (see also Supplemental Figure 2). Scale bars: 10.0 μm. **P < 0.001 versus untreated β-null cells; 1-way ANOVA. (B) Multiple clones of L-CFCs of the indicated genotypes were immunoblotted for class IA PI3K isoforms and assessed for phosphorylation of AKT (p-AKT S473) and its substrates FOXO1 and FOXO3a (p-FOXO1 T24/p-FOXO3a T32, right panel; or p-FOXO1 S256, left panel. Note: Anti–p-FOXO1 (S256) can detect p-FOXO4 (S193), but antibodies are not available to confirm mouse FOXO4 expression. Asterisk indicates representative α/β-null L-CFC clone that upregulated p55γ, with a concomitant increase in AKT activity. (C) p190 L-CFCs of the indicated genotypes were treated for 15 minutes with LY294002 (10 μM) and subsequently immunoblotted for p-AKT (S473) and p-FOXO1/4 (S256/S193; note the overexposure with p-FOXO for detection of low signal). n = 3 experiments using 2 different clones. (D) p190 L-CFCs were infected with retroviruses MSCV-IRES-Thy1.1 (Ctrl), MSCV-FOXO3a-IRES-Thy1.1, or MSCV-FOXO3a.A3-IRES-Thy1.1. Apoptosis was assessed in Thy1.1+ cells by annexin V/7AAD staining 48 hours after infection. Data are representative of 3 independent experiments using 2 different clones.