T cell acute lymphoblastic leukemia (T-ALL) is a hematologic malignancy resulting from the transformation of T cell progenitors that accounts for 15 % of pediatric and 25 % of adult ALL cases. Despite improvements in cure rates, the outcome of T-ALL patients with chemoresistant or relapsed leukemia is still poor [
1]. T-ALL requires aggressive chemotherapy. To minimize and overcome the detrimental effects of therapeutic regimens, it is essential to identify novel molecular targets in T-ALL and test selective inhibitors of such targets [
2]. Thus, major efforts are being made to develop targeted molecules against deregulated signaling pathways sustaining T-ALL cell growth/survival/drug resistance. Indeed, selective inhibitors of deregulated pathways could be used together with chemotherapy, allowing for a lower dosage of chemotherapeutic drugs which should minimize toxic side effects. The nucleoside analog nelarabine is a prodrug of the deoxyguanosine analog 9-β-
d-arabinofuranosylguanine (Ara-G) [
3,
4]. Nelarabine is rapidly metabolized in the plasma by an adenosine deaminase into the active metabolite Ara-G, the latter having a much longer plasma half-life and reaching higher plasma concentrations [
5]. Ara-G is taken up by leukemia cells via the nitrobenzylthioinosine-sensitive nucleoside membrane transporter equilibrative nucleoside transporter 1 (ENT1) [
6]. Ara-G is then phosphorylated to Ara-G monophosphate by the deoxycytidine and deoxyguanosine kinase (dCK and dGK, respectively). It is then phosphorylated to its triphosphate form Ara-GTP, which in turn competes with dGTP for DNA polymerase and is subsequently incorporated into DNA, resulting in termination of DNA synthesis [
7]. Since 2005, nelarabine has been approved for the treatment of both pediatric and adult T-ALL patients who have refractory or progressive disease after previous chemotherapy regimens [
5,
8]. Nelarabine is preferentially cytotoxic to T lymphoblasts through the accumulation of Ara-GTP, which occurs to a greater extent in T cells than in B cells, resulting in inhibition of ribonucleotide reductase and subsequent DNA synthesis [
9,
10]. Nelarabine as monotherapy was effective in inducing complete/partial responses in both children and adults with refractory or relapsed T-ALL [
3,
8,
11]. Given the impressive single-agent activity seen in refractory or relapsed T-ALL, substantial interest developed in evaluating nelarabine in the up-front treatment of T-ALL. Clinical trials have shown that nelarabine could be safely combined with intensive chemotherapy (BFM-86) in the frontline therapy of pediatric T-ALL, and in combination with hyper-CVAD in the frontline therapy for adults with T-ALL [
12‐
14]. Of 40 patients with T-ALL or T lymphoblastic lymphoma (T-LL), the CR rate was 89 % in T-ALL and 94 % in T-LL. Overall survival at 3 years was 63 % [
14]. However, in recent years, it is emerging that nelarabine could induce a significant neurotoxicity, particularly when given in conjunction with multi-agent chemotherapy regimens [
15,
16]. Patients who suffered from nelarabine neurotoxicity displayed myelopathy and severe necrotic changes in the nervous system [
17], as well as irreversible paresthesia or paraplegia [
17,
18]. Importantly, nelarabine-induced neurotoxicity depends on the dosage [
19]. Therefore, a combined therapy with signal transduction modulators could allow for the use of a lower dosage of nelarabine, resulting in a lower incidence and/or seriousness of neurotoxicity and resistance. However, there is no information regarding how aberrantly activated signaling pathways could influence T-ALL cell sensitivity to nelarabine. In contrast, in recent years, valuable information has been collected regarding aberrantly activated signaling pathways influencing leukemia cell sensitivity to fludarabine [
20], a nucleoside analog which is employed in the treatment of B cell chronic lymphocytic leukemia (CLL), or to clofarabine, a nucleoside analog used for treating acute myeloid leukemia (AML) patients [
21,
22]. The effects of phosphoinositide 3-kinase (PI3K) inhibition on fludarabine sensitivity could be related to changes in the expression of Bcl2 family proteins (Mcl-1 and Bim) [
23,
24] which are also involved in clofarabine sensitivity in AML cells [
22]. Importantly, PI3K/AKT/mammalian target of rapamycin (mTOR) signaling inhibition enhanced fludarabine-induced cell death in a T-ALL cell line (CEM-S) [
25]. Moreover, a combination of clofarabine and temsirolimus, an mTOR inhibitor, displayed synergistic cytotoxic effects in AML cell lines and primary samples, and showed an encouraging clinical activity [
21,
26]. A recent study underscored a potential role for Bcl2 family proteins in determining nelarabine resistance, in an Ara-G-resistant T-ALL cell line variant, established by serial incubation with Ara-G (the active metabolite of nelarabine) [
27]. In this study, anti-apoptotic Bcl-xL was augmented and pro-apoptotic Bax and Bad were reduced in CEM/Ara-G cells, suggesting refractoriness to Ara-G-induced apoptosis [
27].
Here, we demonstrate that in nelarabine-resistant T-ALL cell lines, there is a hyperactivation of PI3K/AKT/mTOR, ERK1/2 and Bcl2 signaling in response to nelarabine. Treatment with ZSTK-474 (a pan PI3K p110 inhibitor [
28]), IPI-145 (Duvelisib
@, a γ/δ PI3K p110 inhibitor [
29]), GSK1120212 (Trametinib
@, a MEK inhibitor [
30]), or ABT199 (Venetoclax
@, a Bcl2 inhibitor [
31]) synergized with nelarabine in reducing cell survival in nelarabine-resistant T-ALL cells. ZSTK-474 was the most potent drug in inducing cell death in combination with nelarabine, allowing for a lower dosage of nelarabine even in samples from T-ALL relapsed patients. Moreover, nelarabine combined with ZSTK-474 induced a dephosphorylation of AKT and ERK1/2 and induced an increase in the expression of Bax and Bak pro-apoptotic members of Bcl2 family in T-ALL cells resistant to nelarabine. The combination of nelarabine with ZSTK-474 was able to induce a marked cell death in MOLT-4 cells co-cultured with HS-5 human HS-5 stromal cells, which mimic bone marrow (BM) microenvironment. These observations suggest the possibility to combine nelarabine together with selective inhibitors of the PI3K signaling pathway, to improve the efficacy of T-ALL treatment of relapsed/refractory patients.