Improving nelarabine efficacy in refractory/relapsed T-cell acute lymphoblastic leukemia (T-ALL) by targeting aberrant PI3K/mTOR signaling

Background: The introduction of novel chemotherapy protocols has improved the outcome of T-ALL patients, but refractory and/or relapsing disease remains a major concern. In this context, a major contribution was provided by the introduction of the nelarabine, approved for salvage treatment of refractory/relapsed T-ALL patients. Aims: Nelarabine could induce a dose-dependent neurotoxicity. To improve its efficacy, it is essential to study its molecular targets, testing selective inhibitors of such targets, to be administered in combination with nelarabine, allowing for a lower dosage of the drug. Methods: Human T-ALL cell lines and primary T-ALL refractory/relapsed lymphoblasts from patients were incubated with increasing concentrations of nelarabine alone or combinated with PI3K/mTOR inhibitors for cell viability assays. Apoptosis and phenotyping analyses were performed by flow cytometry. Protein expression was evaluated by Western Blot. ENT1/2 gene expression was measured by quantitative real time PCR in T-ALL settings. Results: Cell viability assays indicated the presence of T-ALL cell lines sensitive to nelarabine (IC50 <5 μM) and others which displayed higher IC50 (>15 μM). Nelarabine sensitive cells showed a significant increase of apoptotic cells after 48 h treatment with 2-5 μM nelarabine, as demonstrated by caspases and PARP cleavage. In contrast, resistant T-ALL cells were not perturbated. Levels of expression of ENT1/2 nucleoside transporters could be related to in vitro nelarabine sensitivity of T-ALL cells, and could be also dependent on interactions between leukemic cells and tumor microenvironment. No significant differences in ENT1/2 mRNA levels between samples sensitive or resistant to nelarabine were seen. Modulation of ENT1/2 gene expression was not related to nelarabine treatment, even if in some cases the co-culture with human stromal HS-5 cells supported cell survival. Upregulated PI3K/mTOR signaling is a common feature of T-ALL, where it portends a poorer prognosis by influencing leukemic cell proliferation/survival/drug-resistance. Sensitive T-ALL cells treated with nelarabine showed a strong decrease in the phosphorylation of Ser473 p-Akt and Ser235/236 p-S6 ribosomal protein. In contrast, resistant cells, showed a hyperactivation of PI3K/mTOR signaling pathway. The combination of nelarabine with the pan PI3K inhibitors ZSTK474 or BKM120 was synergistic in reducing proliferation and in inducing strong apoptosis in all the resistant cell lines and in relapsed T-ALL patient samples with upregulated PI3K/mTOR. Conclusions: Nelarabine combined with PI3K inhibitors efficiently reduced cell viability and induced apoptosis in T-ALL settings, allowing for a lower dosage of nelarabine and therefore, synergizing with conventional therapies in relapsed/refractory T-ALL patients with upregulation of PI3K signaling.