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. 2012 Aug;3(8):811-23.
doi: 10.18632/oncotarget.579.

Harnessing the PI3K/Akt/mTOR pathway in T-cell acute lymphoblastic leukemia: eliminating activity by targeting at different levels

Daniela Bressanin  1 , Camilla Evangelisti, Francesca Ricci, Giovanna Tabellini, Francesca Chiarini, Pier Luigi Tazzari, Fraia Melchionda, Francesca Buontempo, Pasqualepaolo Pagliaro, Andrea Pession, James A McCubrey, Alberto M Martelli
Affiliations

Harnessing the PI3K/Akt/mTOR pathway in T-cell acute lymphoblastic leukemia: eliminating activity by targeting at different levels

Daniela Bressanin et al. Oncotarget. 2012 Aug.

Abstract

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignant hematological disorder arising in the thymus from T-cell progenitors. T-ALL mainly affects children and young adults, and remains fatal in 20% of adolescents and 50% of adults, despite progress in polychemotherapy protocols. Therefore, innovative targeted therapies are desperately needed for patients with a dismal prognosis. Aberrant activation of PI3K/Akt/mTOR signaling is a common event in T-ALL patients and portends a poor prognosis. Preclinical studies have highlighted that modulators of PI3K/Akt/mTOR signaling could have a therapeutic relevance in T-ALL. However, the best strategy for inhibiting this highly complex signal transduction pathway is still unclear, as the pharmaceutical companies have disclosed an impressive array of small molecules targeting this signaling network at different levels. Here, we demonstrate that a dual PI3K/PDK1 inhibitor, NVP-BAG956, displayed the most powerful cytotoxic affects against T-ALL cell lines and primary patients samples, when compared with a pan class I PI3K inhibitor (GDC-0941), an allosteric Akt inhibitor (MK-2206), an mTORC1 allosteric inhibitor (RAD-001), or an ATP-competitive mTORC1/mTORC2 inhibitor (KU63794). Moreover, we also document that combinations of some of the aforementioned drugs strongly synergized against T-ALL cells at concentrations well below their respective IC50. This observation indicates that vertical inhibition at different levels of the PI3K/Akt/mTOR network could be considered as a future innovative strategy for treating T-ALL patients.

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Figures

Figure 1
Figure 1. Inhibitors of PI3K/Akt/mTOR signaling affect viability of T-ALL cell lines
MTT assays on T-ALL cell lines treated with GDC-0941, MK-2206, NVP-BAG956, KU-63794, RAD-001 for 24 h. The results are the mean of three different experiments ± s.d.
Figure 2
Figure 2. PI3K/Akt/mTOR signaling modulators affect cycle progression and induce apoptosis in MOLT-4 cells
(A) MOLT-4 cell line was treated for 24 h with increasing concentrations of GDC-0941, MK-2206, NVP-BAG956, RAD-001 and KU-63794. Then, cell cycle analysis was performed by flow cytometry. One representative of three separate experiments ± s.d. is shown that yielded similar results. CTRL: control cells. Asterisks indicate significant differences (p <0.05) relative to control cells. (B) MOLT-4 cell line was treated for 24 h with GDC-0941, MK-2206, NVP-BAG956, RAD-001 and KU-63794 (1 μM). Then, cells were stained with Annexin V-FITC/PI and analyzed by flow cytometry. One representative of three separate experiments is shown that yielded similar results. CTRL: control cells.
Figure 3
Figure 3. Effect of the signal transduction modulators on the phosphorylation status of PI3K/Akt/mTOR signaling components
MOLT-4, Jurkat, CEM-R, and CEM-S cell lines were treated with increasing concentrations of GDC-0941, MK-2206, NVP-BAG956, RAD-001, and KU-63794 for 24 h. Then, cells were collected, lysed, and analyzed by western blotting. An antibody to β-actin documented equal lane loading (50 μg of protein/lane).
Figure 4
Figure 4. Time course of the phosphorylation status of PI3K/Akt/mTOR signaling components in response to drug treatment
MOLT-4 (A) and CEM-R (B) cell lines were treated with 1 μM of GDC-0941, MK-2206, or NVP-BAG956, for 6, 16, 24 and 48 h, then they were collected, lysed, and analyzed by western blot. An antibody to β-actin documented equal lane loading (50 μg of protein/lane).
Figure 5
Figure 5. Vertical inhibition of the PI3K/Akt/mTOR pathway is synergistic in CEM-S cells
(A) CEM-S cell lines were treated for 24 h with either drug alone or the combination of the two drugs at a fixed ratio. The combination index (CI) value for each data point was calculated with the appropriate software for dose effect analysis (CalcuSyn). Results are the mean of three different experiments ± s.d. (B) CEM-S cell line was treated for 24 h either RAD-001or KU-63794 alone, and with a combination of the two drugs at the same concentration (1 μM). Then, cell cycle analysis was performed by flow cytometry. One representative of three separate experiments is shown that yielded similar results. CTRL: control cells.
Figure 6
Figure 6. Inhibitors of PI3K/Akt/mTOR signaling are cytotoxic to T-ALL primary cells
(A) MTT assay on four representative patient samples treated for 96 h with 10 ng/ml interleukin-7 and increasing concentrations of GDC-0941, MK-2206, NVP-BAG956, KU-63794, or RAD-001. Results are the mean of at three different experiments ± s.d. (B) Western blot analysis of a representative patient samples treated for 48 h with 2 μM MK-2206 or NVP-BAG956. Fifty μg of each lysate were electrophoresed on SDS-PAGE, transferred to a nitrocellulose membrane and probed with the appropriate antibodies. One representative of two different experiments is shown. CTRL: control cells.
Figure 7
Figure 7. ModeInhibitors of PI3K/Akt/mTOR signaling induce apoptosis in T-ALL lymphoblasts and putative LICs
(A) Flow cytometric analysis of patient samples treated for 24 h with 2 μM MK-2206 or NVP-BAG956. Cells were stained with AlexaFluor® 488-conjugated anti-cleaved caspase-3 or with Annexin V-FITC/PI. The percentages of positive cells are indicated. The plots are representative of three separate experiments which were performed in duplicate. (B) Flow cytometric analysis of patient samples enriched for LICs, treated for 48 h with 2 μM MK-2206 or NVP-BAG956. The CD34+/CD7−/CD4− subset was stained with Annexin-FITC, to analyze apoptosis induction by the drugs (2 μM). In A and B one representative of two different experiments is shown. CTRL: control cells.
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