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01.12.2016 | Research | Ausgabe 1/2016 Open Access

Journal of Hematology & Oncology 1/2016

Improving nelarabine efficacy in T cell acute lymphoblastic leukemia by targeting aberrant PI3K/AKT/mTOR signaling pathway

Zeitschrift:
Journal of Hematology & Oncology > Ausgabe 1/2016
Autoren:
Annalisa Lonetti, Alessandra Cappellini, Alice Bertaina, Franco Locatelli, Andrea Pession, Francesca Buontempo, Camilla Evangelisti, Cecilia Evangelisti, Ester Orsini, Laura Zambonin, Luca Maria Neri, Alberto Maria Martelli, Francesca Chiarini
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1186/​s13045-016-0344-4) contains supplementary material, which is available to authorized users.

Abstract

Background

Although in recent years, the introduction of novel chemotherapy protocols has improved the outcome of T cell acute lymphoblastic leukemia (T-ALL) patients, refractory and/or relapsing disease remains a foremost concern. In this context, a major contribution was provided by the introduction of the nucleoside analog nelarabine, approved for salvage treatment of T-ALL patients with refractory/relapsed disease. However, nelarabine could induce a life-threatening, dose-dependent neurotoxicity. To improve nelarabine efficacy, we have analyzed its molecular targets, testing selective inhibitors of such targets in combination with nelarabine.

Methods

The effectiveness of nelarabine as single agent or in combination with PI3K, Bcl2, and MEK inhibitors was evaluated on human T-ALL cell lines and primary T-ALL refractory/relapsed lymphoblasts. The efficacy of signal modulators in terms of cytotoxicity, induction of apoptosis, and changes in gene and protein expression was assessed by flow cytometry, western blotting, and quantitative real-time PCR in T-ALL settings.

Results

Treatment with nelarabine as a single agent identified two groups of T-ALL cell lines, one sensitive and one resistant to the drug. Whereas sensitive T-ALL cells showed a significant increase of apoptosis and a strong down-modulation of PI3K signaling, resistant T-ALL cells showed a hyperactivation of AKT and MEK/ERK1/2 signaling pathways, not caused by differences in the expression of nelarabine transporters or metabolic activators. We then studied the combination of nelarabine with the PI3K inhibitors (both pan and dual γ/δ inhibitors), with the Bcl2 specific inhibitor ABT199, and with the MEK inhibitor trametinib on both T-ALL cell lines and patient samples at relapse, which displayed constitutive activation of PI3K signaling and resistance to nelarabine alone. The combination with the pan PI3K inhibitor ZSTK-474 was the most effective in inhibiting the growth of T-ALL cells and was synergistic in decreasing cell survival and inducing apoptosis in nelarabine-resistant T-ALL cells. The drug combination caused AKT dephosphorylation and a downregulation of Bcl2, while nelarabine alone induced an increase in p-AKT and Bcl2 signaling in the resistant T-ALL cells and relapsed patient samples.

Conclusions

These findings indicate that nelarabine in combination with PI3K inhibitors may be a promising therapeutic strategy for the treatment of T-ALL relapsed patients.

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Zusatzmaterial
Additional file 1: Figure S1. DNA damage and ROS production. A. Flow cytometric analysis of γH2AX in MOLT-4 and Jurkat sensitive to nelarabine cells (upper panel). Percentages of γH2AX are shown in the table (lower panel). B. Reactive oxygen species (ROS) production was assessed in MOLT-4 and Jurkat sensitive T-ALL cell lines after 1 and 24 h treatment with nelarabine. Fluorescence values (DCF) were reported as the percentage of intracellular ROS in respect to controls. ROS increased significantly in both 1- and 24-h treatment compared to controls in MOLT-4 and Jurkat cells. Statistical analyses were performed with the Dunnett’s multiple comparison test.
13045_2016_344_MOESM1_ESM.pdf
Additional file 2: Figure S2. Expression of dCK and dGK in T-ALL cell lines. Western blotting analyses for the expression of dCK and dGK proteins in T-ALL cell lines. Fifty micrograms of protein was blotted to each lane. Antibody to β-actin served as a loading control. Molecular weights are indicated on the right. One representative of two different experiments is shown.
13045_2016_344_MOESM2_ESM.pdf
Additional file 3: Figure S3. Bcl2 expression in T-ALL cell lines. Western blotting analyses for the basal expression of Bcl2 in untreated T-ALL cell lines. Twenty micrograms of protein was blotted to each lane. Antibody to β-actin served as a loading control. Molecular weights are indicated on the right. Densitometric analysis was performed using a Chemidoc 810 Imager with the appropriate software (UVP, Upland, CA, USA), and for each cell line, Bcl2 protein expression is indicated relatively to β-actin expression.
13045_2016_344_MOESM3_ESM.pdf
Additional file 4: Figure S4. The combination of nelarabine and ZSTK-474 is synergistic in CEM-R cells, which overexpress P-gp. Cell viability assay of CEM-R cell line treated for 48 h with increasing concentrations of nelarabine alone or combined with the pan PI3K p110 inhibitor ZSTK-474. One representative of two different experiments is shown.
13045_2016_344_MOESM4_ESM.pdf
Additional file 5: Figure S5. Specific effects of nelarabine on PI3K/AKT and MEK/ERK1/2 pathways. Western blotting analyses for the expression of p-AKT and p-ERK in resistant T-ALL cell lines treated with the specific inhibitors LY294002 (PI3K inhibitor), CCI-779 (mTOR allosteric inhibitor) or trametinib (MEK1/2 inhibitor) alone or in combination with nelarabine. Thirty micrograms of protein was blotted to each lane. Antibody to β-actin served as a loading control. Molecular weights are indicated on the right. CTRL: untreated cells; Nela and N: nelarabine at 10 μM; LY: LY294002 at 10 μM; CCI: CCI-779 at 100 nM; Tram: trametinib at 1 μM. Cells were treated for 48 h.
13045_2016_344_MOESM5_ESM.pdf
Additional file 6: Figure S6. HS-5 stromal cells protect MOLT-4 cells from nelarabine cytotoxicity at least in part through CXCL12/CXCR4 interactions. A. MTT assays of MOLT-4 cells growing alone or in co-culture system with HS-5 cells and treated with nelarabine (2 μM for 48 h) in a Transwell@ system. Three hundred seventy-five thousand MOLT-4 cells were seeded in the upper chamber of the Transwell@ system. Results are the mean of three different experiments ± SD. B. Western blot analysis for p-ERK, p-CXCR4, and their total forms in MOLT-4 cells grown either in the absence or the presence of HS-5 stromal cells in a Transwell@ system. Nelarabine treatment (2 μM) was for 24 h. Fifty micrograms of protein was blotted to each lane. Antibody to β-actin served as a loading control. Molecular weights are indicated on the right. One representative of two different experiments is shown.
13045_2016_344_MOESM6_ESM.pdf
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