PI3K isoform inhibition associated with anti Bcr-Abl drugs shows in vitro increased anti-leukemic activity in Philadelphia chromosome-positive B-acute lymphoblastic leukemia cell lines

B-acute lymphoblastic leukemia (B-ALL) is a malignant disorder characterized by the abnormal proliferation of B-cell progenitors. Philadelphia chromosome-positive (Ph+) B-ALL is a subtype that expresses the Bcr-Abl fusion protein which represents a negative prognostic factor. Constitutive activation of the phosphatidylinositol 3-kinase/Akt/mammalian target of rapamycin (PI3K/Akt/mTOR) network is a common feature of B-ALL, influencing cell growth and survival. In the present study, we aimed to investigate the efficacy of PI3K isoform inhibition in B-ALL cell lines harboring the Bcr-Abl fusion protein. We studied the effects of anti Bcr-Abl drugs Imatinib, Nilotinib and GZD824 associated with PI3K isoform inhibitors. We used a panel of six compounds which specifically target PI3K isoforms including the pan-PI3K inhibitor ZSTK474, p110α BYL719 inhibitor and the dual p110γ/p110δ inhibitor IPI145. The effects of single drugs and of several drug combinations were analyzed to assess cytotoxicity by MTS assays, apoptosis and autophagy by flow cytometry and Western blot, as well as the phosphorylation status of the pathway. ZSTK474, BYL719 and IPI145 administered in combination with imatinib, nilotinib and GZD824 for 48 h, decreased cell viability, induced apoptosis and autophagy in a marked synergistic manner. These findings suggest that selected PI3K isoform inhibitors used in combination with anti Bcr-Abl drugs may be an attractive novel therapeutic intervention in Ph+ B-ALL.


INTRODUCTION
The over-production of immature white blood cells is a relevant feature of ALL, a heterogeneous disease characterized by multiple, prognostically relevant genetic aberrations.
The selective Bcr-Abl tyrosine kinase inhibitor Imatinib deeply changed the pharmacological treatment Research Paper of another form of Ph + cancer, i.e. chronic myeloid leukemia (CML), by inducing long lasting remissions [5]. Unfortunately, Imatinib showed much less efficiency in treating Ph + ALL and although unrelated to the Bcr-Abl kinase domain alterations, the underlying mechanisms are largely unknown [1]. The second generation tyrosine kinase inhibitor (TKI) Nilotinib shows both a stronger potency than Imatinib and also acts against most Imatinibunresponsive Bcr-Abl mutation variants [6][7][8]. A new, third generation orally bioavailable Bcr-Abl inhibitor, GZD824, has been recently developed, with potency against a wide range of Bcr-Abl mutants [9]. In general, Ph + B-ALL is less sensitive to TKIs that CML. Therefore, novel drugs are needed to improve response rates and to circumvent TKI-resistance in Ph + B-ALL [10].
Class I phosphatidylinositol 3-kinases (PI3Ks) comprises members of a conserved family of heterodimeric intracellular lipid kinases capable to activate Akt which in turn phosphorylates target proteins thus affecting cell growth, cell cycle progression, and cell survival [11,12].
Class IA PI3Ks comprises a catalytic subunit (p110α, p110β, p110γ, p110δ) and a tightly bound regulatory subunit (p85α, p55α, p50α, p85β, or p55γ). The regulatory subunits maintain the integrity of the catalytic one and direct the heterodimer to membraneassociated signaling complexes [13]. The activation of the PI3K network is regulated by the 3'-phosphate lipid phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome 10) and loss of activity of this tumor suppressor gene induces an increased downstream activation [14][15][16]. Alterations in PTEN have been reported in several tumors, most notably endometrial, central nervous system, skin and prostate cancers [17].
Due to the crucial role of PI3Ks in regulating cell cycle, metabolism, and survival, the PI3K signaling cascade in human leukemias is one of the most often altered pathways [18,19] and different compounds targeting members of the PI3K network have been developed and entered clinical trials [20]. Currently several PI3K inhibitors are under development: ZSTK474, a specific pan-PI3K inhibitor [21,22], displays potent antitumor efficacy on various solid tumors [23,24]. PIK3CA, the gene which encodes for p110α PI3K, is mutated in a variety of tumor types [25][26][27] and BYL719 is a specific class-IA PI3K inhibitor, which acts by binding the ATPbinding domain of the p110α subunit [28]. BYL719 has recently been reported to have significant activity against tumors carrying mutations in the p110α subunit of PI3K [29,30].
Pharmacological blockade of both p110γ and p110δ reduced T-ALL proliferation and survival, indicating these isoforms as therapeutic targets for T-ALL treatment [31]. AS605240 has been previously described as an isoformselective ATP-competitive inhibitor of PI3Kγ [32] while CAL-101 is an oral p110δ inhibitor currently under clinical evaluation in patients with B-cell malignancies [33].
Aim of this study is to investigate the effects of a panel of PI3K isoform inhibitors in Ph + B-ALL cell lines by combining their anti-tumor activity with anti Bcr-Abl drugs. We decided to specifically target p110α, p110β, p110γ and p110δ PI3K catalytic subunits, along with dual p110γ/p110δ and pan-PI3K inhibitors, and we evaluated their effects on leukemic cell proliferation and survival. Our results demonstrated that the pan PI3K isoform inhibitor ZSTK474, the p110α inhibitor BYL719 and the IPI145 dual p110γ/p110δ inhibitor exerted the most powerful effects in all the Ph + B-ALL cell lines tested and their combined use with TKIs enhanced the treatment efficacy. Our findings support the concept that clinical trials examining PI3K inhibitors in combination with TKIs are warranted in patients with Ph + B-ALL.

PI3K inhibitors affect cell viability of Ph + B-ALL cell lines
In order to establish the role of the different PI3K catalytic subunits in sustaining leukemic cells proliferation and survival, we investigated by Western blot analysis the baseline expression of Bcr-Abl and its substrate CrkL, the PI3K catalytic subunits and the key enzymes of the PI3K/Akt/mTOR network in three B-ALL cell lines (TOM-1, BV-173 and SUP-B15). As shown in Figure 1, Bcr-Abl and CrkL, the p110α, -β, -γ, -δ, PI3K catalytic subunits and the downstream PI3K substrate Akt and the pathway negative regulator PTEN were expressed and/or phosphorylated in all cell lines.
For the inhibition of p110α, p110β, p110γ and p110δ, we employed ZSTK474, BYL719, TGX221, CAL101 and AS605240, whose selectivity has been reported [36]. Because of the prominent role of p110δ and p110γ isoforms in white blood cells, effects of the γ/δ dual inhibitor IPI145, as well as of a combination consisting of CAL101 and AS605240, were also evaluated. Cell lines were cultured with increasing concentrations of the drugs for 48 h followed by metabolic activity assessment by MTS assay (Figure 2A). In all cell lines, cell viability decreased after treatment with ZSTK474 with IC 50 values close to 0.5 µM. Selective inhibition of p110α, p110β, p110γ and p110δ isoforms had less efficacy, and IC 50 values were not attained up to a 10 µM concentration. Longer time points (72 h) did not further affect the metabolic activity of inhibitor-treated cell lines (data not shown).
We decided to focus our study on ZSTK474 and γ/δ dual inhibitor IPI145 as well as on the p110α inhibitor BYL719, whose role in this type of leukemia has not been thoroughly investigated yet [30,37,38]. www.impactjournals.com/oncotarget The PI3K/Akt/mTOR network, its expression and modulation status of key components were assessed to evaluate the efficacy of these inhibitors. Cells were treated with 0.25 µM ZSTK474, 2.5 µM BYL719 or IPI145 and Western blot analysis was performed. These concentrations were chosen as they produced the maximal effects on protein dephosphorylation at the lowest dose. All the PI3K isoform inhibitors decreased the phosphorylation level of the PI3K/Akt/mTOR axis key substrates Akt and S6 ribosomal protein ( Figure 2B).

Autophagy and apoptosis are induced by PI3K inhibitors in B-ALL cell lines
Autophagic process is an evolutionarily mechanism involved in the degradation of cellular components and is crucial for the maintenance of cell homeostasis [39]. Altered autophagy is related with cancer and has been implicated in the disease development and survival [40][41][42]. To assess whether the administration of PI3K inhibitors could induce autophagy, we analyzed LC3A/B I (non lipidated) and LC3A/B II (lipidated) levels by Western blot in TOM-1, BV-173 and SUP-B15 cell lines after 24 h ( Figure 3A). LC3A/B II expression increased with ZSTK474 pan-inhibitor treatment in all cell lines and to a lower extent with BYL719 and IPI145, indicating the induction of autophagy.
To analyze whether the reduced viability was apoptosis-related, cell lines were treated with the inhibitors for 24 h at the same doses used to detect autophagy. Poly (ADP-ribose) polymerase (PARP) cleavage in TOM-1, BV-173 and SUP-B15 cells demonstrated the pro-apoptotic effect induced by the PI3K inhibitors. As demonstrated for the autophagy marker LC3A/B, the expression levels of cleaved PARP increased when cell lines were treated with ZSTK474 pan-inhibitor, or the p110α-selective inhibitor BYL719 and to a lower extent with IPI145 ( Figure 3A). The role of autophagy was further investigated using the lysosomal inhibitor chloroquine at 1 µM. In all cases, chloroquine increased the cytotoxic effects of the PI3K inhibitors, suggesting that autophagy is a protective mechanism against these drugs ( Figure 3B). Moreover, caspase 3/7 activation was quantified by measuring the enzymatic cleavage of the profluorescent component rhodamine 110, bis-N-CBZ-L-aspartyl-L-glutamyl-L-valyl-L-aspartic acid amide (Z-DEVD-R110), with release of the strongly fluorescent rhodamine 110-cleaving group. The data on caspase 3/7 activity were in agreement with those of Western blot on PARP cleavage ( Figure 3C).

Bcr-Abl inhibitors decreased cell viability in Ph + B-ALL cell lines
We examined by MTS assay the IC 50 Table 1).

Expression levels of pCrkL protein and PI3K/ Akt/mTOR signaling pathway substrates
Requirement for CrkL kinase activity has been demonstrated in a recent report with Bcr-Abl transformation and oncogenic signal transduction [43]. As shown in Figure  5A, cells were exposed for 48 h to Imatinib, Nilotinib or GZD824, and lysates were analyzed by Western blot with an antibody directed to the phosphorylated forms of CrkL and S6 ribosomal protein. All anti Bcr-Abl drugs decreased the levels of phosphorylated CrkL with little or no effects on S6 ribosomal protein. In contrast, cell treatment with 0.25 µM ZSTK474 or 2.5 µM BYL719 or IPI145 for 48 h did not affect the phosphorylation levels of pCrkL, while the expression of phosphorylated S6 ribosomal protein was abolished ( Figure 5B).

Synergistic cytotoxic effects combining selected PI3K isoform inhibitors with anti Bcr-Abl drugs
We analyzed whether the combination of ZSTK474, BYL719 or IPI145 with Imatinib, Nilotinib or GZD824 could increase cytotoxic effects on Ph + B-ALL cells. Therefore, MTS assays were performed in SUP-B15 cells treated with the different drugs administered together for 48 h. As shown in Figure 6, SUP-B15 were responsive and showed a good, synergistic cytotoxicity as demonstrated by the CIs.
As representative of the combined treatments, MTS assays on TOM-1 and BV-173 cell lines were also performed with ZSTK474/Imatinib,BYL719/Nilotinibor IPI145/GZD824. As expected, the combination of the drugs resulted in a potent synergism also at low concentrations of each inhibitor in these cell lines ( Figure 7A and 7B).

PI3K isoforms inhibition combined with anti Bcr-Abl drugs increased apoptotic effects in Ph + B-ALL cells
The apoptotic effects of the single and combined drug administration compared to the control in the three cell lines was then evaluated (Figure 8A-8C). The analysis was performed by Annexin-V-FITC staining and PI in flow cytometry after 24 h of drug treatments. The drug concentrations used were 0.25 μM for ZSTK474 and GZD824, and 2.5 μM for the other inhibitors.
The combined administration of the PI3K inhibitors with anti Bcr-Abl drugs showed a significant increase in the percentage of apoptotic cells.

PI3K isoforms inhibition combined with anti Bcr-Abl drugs induced increased autophagy in Ph + B-ALL cells
To better quantify autophagy, the detection of LC3A/B was performed in all cell lines by flow cytometry. After a 24 h incubation, a statistically relevant increase in autophagy was induced by combined treatment of TKIs and PI3K inhibitors when compared to single administrations ( Figure 9A-9C).

DISCUSSION
It has been reported that targeted cancer therapy could be more effective and less harmful than  conventional chemotherapy [44]. PI3K plays a key role in regulating cell cycle, survival and metabolism, and the PI3K signaling cascade is a very frequently altered axis in human tumors [45]. Different compounds targeting members of the PI3K have been synthesized and are now in clinical trials [36]. Better understanding of the PI3K pathway modulation have led to a rational development and utilization of PI3K inhibitors in patients with leukemias. The pan-PI3K ZSTK474 inhibitor and the dual γ/δ IPI145 inhibitor are undergoing analysis and are already demonstrating increasing preclinical and clinical results [23,46]. For example, ZSTK474 inhibitor displayed a potent anticancer activity in human tumor xenografts [47] while BYL719 has shown synergistic antineoplastic efficacy when used in endocrine cure against ER + /PIK3CA mutated breast tumor cells [48].
In Ph + B-ALL cells, relapses are very frequent, especially in adults, with a very poor prognosis, highlighting the need for new therapeutic strategies [49]. Moreover, PI3K network has been strongly involved either in malignant transformation and in the development of TKI-resistance in Ph + B-ALL [50].
We explored the therapeutic potential of the PI3K pan-inhibitor ZSTK474, the p110α inhibitor BYL719 and dual γ/δ inhibitor IPI145 in a set of Ph + B-ALL cells. It is worth noting that these cell lines showed an hyperphosphorylation of PTEN, that results in its inactivation and hyperactivation of PI3K/Akt/mTOR signaling network [51].
We reported that ZSTK474 reduced cell viability and caused both autophagy and apoptosis in Ph + B-ALL cells. ZSTK474, when administered in the micromolar range, synergized in all cell lines with either Imatinib, Nilotinib or GZD824. In this study, we analyzed for the first time the efficacy of PI3K isoform-selective inhibitors in Ph + B-ALL. BYL719 and IPI145 were cytotoxic in the micromolar range to Ph + B-ALL cell lines, as shown by MTS and Annexin V-stained samples analysis. However, ZSTK474 was more cytotoxic than BYL719 and IPI145, as it displayed an IC 50 value of 0.5 μM. The phosphorylation level of the key components of the PI3K/ Akt/mTOR axis, analyzed by Western blot, showed the same sensitivity to ZSTK474 inhibition in all cell lines. It should be highlighted that another recent study has demonstrated the efficacy of the pan-PI3K inhibitor, NVP-BKM120, in pre-clinical models of Ph + B-ALL [52]. Interestingly, this inhibitor displayed an IC 50 of 0.5 µM which is the same as that of ZSTK474.
However, it should be considered that NVP-BKM120 displays considerable off-target effects [53] which so far have not been reported for ZSTK474.
It is also important to emphasize that a previous study from our group has documented how ZSTK474 was not cytotoxic to healthy T-cells [54].
A greater efficacy was also evident in case of GZD824, a third generation anti Bcr-Abl drug, which showed an increased anti-leukemic activity in comparison to first and second generation inhibitors in Ph + B-ALL cells.
When cell lines were treated with anti Bcr-Abl drugs the phosphorylated form of CrkL protein was abolished, whereas the phosphorylated S6 ribosomal protein was minimally affected. Interestingly, these findings are in agreement with those of others who documented in their study that Akt activity was surprisingly not inhibited by Imatinib administration of primary CML-cells [55].
This demonstrated the specificity of these inhibitors against the Bcr-Abl fusion protein. In contrast, cell samples treated with selected PI3K isoform inhibitors showed an unchanged expression level of phosphorylated CrkL protein and a strong decrease in the phosphorylated S6 ribosomal protein.
Overall, the PI3K isoform inhibitors used in this study displayed increased potency when combined with anti Bcr-Abl drugs in terms of cell viability reduction as well as, apoptosis and autophagy induction. Future studies should address the issue of how these drug combinations increase apoptosis and autophagy.
In conclusion, our data show that investigational PI3K inhibitors can block the growth of Ph + B-ALL cell lines but are likely to be most effective when used in combination with Bcr-Abl inhibitors and provide an important preclinical rationale for future clinical applications.    (Milan, Italy). Annexin V/7-ADD detection kit was from Merck-Millipore (Darmstadt, Germany). Western blot antibodies for total Akt-1, Ser473 p-Akt-1 and Thr308 p-Akt-1 were from Santa Cruz Biotechnology (Santa Cruz, CA, USA), while all the other antibodies were from Cell Signaling Technology (Danvers, MA, USA), including the rabbit secondary antibody. Chloroquine, the mouse secondary antibody and the monoclonal β-Actin antibody were purchased from Sigma Aldrich (Milan, Italy). Signals were detected using ECL Plus reagent from Perkin Elmer (Boston, MA, USA).

Cell cultures
The SUP-B15 Ph + B-ALL cell line was obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (Braunschweig, Germany). BV-173 and TOM-1 Ph + B acute lymphoblastic leukemia (B-ALL) cell lines were obtained from Dr. Fabrizio Pane's laboratory at University of Naples, Italy. BV-173 and TOM-1 were maintained in RPMI-1640 medium supplemented with 20% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin and 100 mg/ml streptomycin at a density of 0.5 to 2 × 10 6 cells/ml and were incubated at 37°C with 5% CO 2 .

PI/Annexin V assay
Apoptosis analysis was performed by staining with Annexin V/7-AAD, using the Muse ™ Cell Analyzer (Merck Millipore, Milan, Italy) in according to the manufacturer's instructions. In brief, a 100 µl treated cell suspension was labeled for 20 min in the dark with the same volume of the Muse TM Annexin-V & Dead Cell reagent (Merck Millipore). Subsequently, quantitative detection of Annexin-V/7-AAD positive cells was performed using the Muse ™ Cell Analyzer.

Autophagy analysis and detection of endogenous LC3
Autophagy analysis was performed using the Muse ™ Cell Analyzer (Merck Millipore, Milan, Italy). In brief, 8 × 10 4 cells were plated in 96 well plates and treated with the different drugs for 24 h. Then, cells were harvested, treated with Autophagy Reagent A for 2-6 h, washed with Assay Buffer, incubated for 30 minutes in the dark with Anti-LC3 Alexa Fluor ® 555 Antibody and acquired by Muse. Samples were then analyzed according to the instrument protocol.

Caspase 3/7 activity assay
Caspase activity was measured with the Apo-One Homogeneous Caspase 3/7 assay kit (Promega Corporation, Madison, WI, USA), according to the manufacturer's instructions. The induction of apoptosis and associated activation of caspases 3 and 7 were measured by enzymatic cleavage of the profluorescent substrate rhodamine 110, bis-N-CBZ-L-aspartyl-Lglutaml-L-valyl-L-aspartic acid amide (Z-DEVD-R110), which releases the intensely fluorescent rhodamine 110-cleaving group. Cells were seeded at a density of 1 × 105/ml and incubated in a 96-well plate in the presence or absence of drug for 48 h. 100 μl of the homogeneous caspase-3/-7 reagent was added to each well and the reaction mixture was incubated for 2 h at room temperature. Fluorescence was measured at an excitation wavelength of 485 nm and an emission wavelength of 538 nm. Results are expressed as relative fluorescence units (RFU), as previously described [54].

Combined drug effect analysis
The effects and the potential synergy of drug combinations were evaluated from quantitative analysis of dose-effect relationship, as described previously [58].
For each experiment, a combination index (CI) number was calculated using the Biosoft CalcuSyn software (Biosoft, Cambridge, UK). This method of analysis generally defines CI values from 0.9 to 1.1 as additive, from 0.3 to 0.9 as synergistic and below 0.3 as strongly synergistic, whereas values over 1.1 are considered as antagonistic.