PTEN posttranslational inactivation and hyperactivation of the PI3K/Akt pathway sustain primary T cell leukemia viability
Ana Silva, J. Andrés Yunes, Bruno A. Cardoso, Leila R. Martins, Patrícia Y. Jotta, Miguel Abecasis, Alexandre E. Nowill, Nick R. Leslie, Angelo A. Cardoso, Joao T. Barata
Ana Silva, J. Andrés Yunes, Bruno A. Cardoso, Leila R. Martins, Patrícia Y. Jotta, Miguel Abecasis, Alexandre E. Nowill, Nick R. Leslie, Angelo A. Cardoso, Joao T. Barata
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Research Article Hematology

PTEN posttranslational inactivation and hyperactivation of the PI3K/Akt pathway sustain primary T cell leukemia viability

Abstract

Mutations in the phosphatase and tensin homolog (PTEN) gene leading to PTEN protein deletion and subsequent activation of the PI3K/Akt signaling pathway are common in cancer. Here we show that PTEN inactivation in human T cell acute lymphoblastic leukemia (T-ALL) cells is not always synonymous with PTEN gene lesions and diminished protein expression. Samples taken from patients with T-ALL at the time of diagnosis very frequently showed constitutive hyperactivation of the PI3K/Akt pathway. In contrast to immortalized cell lines, most primary T-ALL cells did not harbor PTEN gene alterations, displayed normal PTEN mRNA levels, and expressed higher PTEN protein levels than normal T cell precursors. However, PTEN overexpression was associated with decreased PTEN lipid phosphatase activity, resulting from casein kinase 2 (CK2) overexpression and hyperactivation. In addition, T-ALL cells had constitutively high levels of ROS, which can also downmodulate PTEN activity. Accordingly, both CK2 inhibitors and ROS scavengers restored PTEN activity and impaired PI3K/Akt signaling in T-ALL cells. Strikingly, inhibition of PI3K and/or CK2 promoted T-ALL cell death without affecting normal T cell precursors. Overall, our data indicate that T-ALL cells inactivate PTEN mostly in a nondeletional, posttranslational manner. Pharmacological manipulation of these mechanisms may open new avenues for T-ALL treatment.

Authors

Ana Silva, J. Andrés Yunes, Bruno A. Cardoso, Leila R. Martins, Patrícia Y. Jotta, Miguel Abecasis, Alexandre E. Nowill, Nick R. Leslie, Angelo A. Cardoso, Joao T. Barata

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Figure 4

PTEN activity is downregulated by ROS in T-ALL.

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PTEN activity is downregulated by ROS in T-ALL. (A) Intracellular H2O2 l...
(A) Intracellular H2O2 levels were assessed by flow cytometry after incubation with the redox-sensitive fluorescent dye DCF-DA. Shown is 1 representative patient sample and 1 representative normal control. (B) Mean intensity of DCF-DA fluorescence was compared between normal thymocytes (n = 7) and T-ALL (n = 5). Data are representative of most T-ALL samples (see Table 1) analyzed in 4 independent experiments. (C) TAIL7 cells previously treated with or without 1 mM H2O2 for 30 min were lysed in nonreducing conditions and analyzed for expression of PTEN oxidized and reduced bands. DTT was added where indicated to demonstrate the specificity of the lower, oxidized band. (D and E) PTEN-positive TAIL7, HPB-ALL, or primary T-ALL cells and PTEN-null Jurkat cells were treated for 2 h with 0.5 mM β-ME (D) or for 30 min with 1 mM H2O2 (E), and levels of Akt and GSK-3β phosphorylation were determined by immunoblot. (F) TAIL7 and HPB-ALL cells were pretreated for 1 h with 25 μm LY294002 (LY) or with DMSO vehicle control, stimulated with 1 mM H2O2 for 30 min, and analyzed for Akt phosphorylation by immunoblot. (G and H) Effects of β-ME (G) or H2O2 (H) on PTEN activity were measured by using the indirect PTEN redox assay, as described previously (19). More relative PTEN activity in the assay reflects less PTEN activity in the cell. Data from 2 independent experiments were normalized to the highest value in the control conditions. Values in B, G, and H are mean ± SEM.