Improving nelarabine efficacy in refractory/relapsed T-cell Acute Lymphoblastic Leukemia by targeting aberrant PI3K/mTOR signaling

The introduction of novel chemotherapy protocols has improved the outcome of T-cell acute lymphoblastic leukemia (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 nucleoside analogs and in particular nelarabine, approved for salvage treatment of refractory/relapsed TALL patients. Aims: A serious nelarabine drawback is that it induces a dose-dependent neurotoxicity. To improve nelarabine 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 chemotherapeutic. Methods: Human T-ALL cell lines (HBP-ALL, DND41, Jurkat, MOLT-4, MOLT16, CEM, CEM-R drug-resistant, ALL-SIL, Loucy, HSB2, Peer, PF382, P12-ICHIKAWA) 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 and absolute cell counting by flow cytometry. Flow cytometry was also performed to analyze apoptosis and for phenotyping analyses. Protein expression was evaluated by Western Blot. ENT1/ENT2 gene expression was measured by quantitative real time PCR in T-ALL settings. Results: We examined cell viability of cell lines and primary T-ALL refractory/relapsed cells. Cell viability assays indicated the presence of T-ALL cell lines sensitive to nelarabine (IC50<5μM) and others which displayed higher IC50 (>15μM). The most resistant cell line (IC50>300μM) was Loucy, which is representative of ETP-ALL, a T-ALL subtype with a very poor prognosis. Nelarabine sensitive cells showed a significant increase of apoptotic cells after 48h treatment with 2-5 μM nelarabine, as demonstrated by flow cytometric analyses of stained with AnnexinV FITC/PI cells and western blot analyses of caspases 8, 9, 3, and PARP. In contrast, resistant T-ALL cells were not perturbated. It has been reported that the levels of expression of ENT 1/2 nucleoside transporters were related to in vitro nelarabine sensitivity of T-ALL cell lines and primary samples. The expression of ENT1/2 transporters 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 cells HS-5, which mimic the bone marrow microenvironment, partially supported cell survival. Upregulated phosphoinositide 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) signaling is a common feature of T-ALL, where it portends a poorer prognosis by influencing leukemic cell proliferation, survival, and drugresistance. We analyzed the effects of nelarabine on the phosphorylation status of key components of the PI3K/mTOR pathway in T-ALL settings. Sensitive TALL cells showed a strong decrease in the phosphorylation of Ser473 p-Akt and Ser235/236 p-S6 ribosomal protein (S6RP). In contrast, resistant cells treated with nelarabine alone, showed a hyperactivation of PI3K/mTOR and MEK/ERK signaling pathways. The combination of nelarabine with the pan PI3K inhibitors ZSTK474 or BKM120 was synergistic in reducing cell viability and in inducing strong apoptosis in all the resistant cell lines and in relapsed T-ALL patient samples with upregulated PI3K/mTOR signaling. Summary/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.