Co-targeting of Bcl-2 and mTOR pathway triggers synergistic apoptosis in BH3 mimetics resistant acute lymphoblastic leukemia.

Several chemo-resistance mechanisms including the Bcl-2 protein family overexpression and constitutive activation of the PI3K/Akt/mTOR signaling have been documented in acute lymphoblastic leukemia (ALL), encouraging targeted approaches to circumvent this clinical problem. Here we analyzed the activity of the BH3 mimetic ABT-737 in ALL, exploring the synergistic effects with the mTOR inhibitor CCI-779 on ABT-737 resistant cells. We showed that a low Mcl-1/Bcl-2 plus Bcl-xL protein ratio determined ABT-737 responsiveness. ABT-737 exposure further decreased Mcl-1, inducing apoptosis on sensitive models and primary samples, while not affecting resistant cells. Co-inhibition of Bcl-2 and the mTOR pathway resulted cytotoxic on ABT-737 resistant models, by downregulating mTORC1 activity and Mcl-1 in a proteasome-independent manner. Although Mcl-1 seemed to be critical, ectopic modulation did not correlate with apoptosis changes. Importantly, dual targeting proved effective on ABT-737 resistant samples, showing additive/synergistic effects. Together, our results show the efficacy of BH3 mimetics as single agent in the majority of the ALL samples and demonstrate that resistance to ABT-737 mostly correlated with Mcl-1 overexpression. Co-targeting of the Bcl-2 protein family and mTOR pathway enhanced drug-induced cytotoxicity by suppressing Mcl-1, providing a novel therapeutic approach to overcome BH3 mimetics resistance in ALL.


INTRODUCTION
Although treatment of pediatric and Philadelphiapositive ALL patients has shown continuous progress, adult patients, especially those characterized by unfavorable prognostic factors, still present high rates of relapse and develop chemo-resistance during the course of the disease, failing to achieve further remissions and ultimately dying [1,2].
Overexpression of the anti-apoptotic Bcl-2 family proteins is commonly observed in hematological malignancies, including ALL. By blocking apoptosis induction, Bcl-2 allows leukemic cell to survive and proliferate irrespective of death signals that physiologically avoid oncogenesis transformation [3][4][5][6]. Anti-apoptotic members perform their activity by binding and sequestering Bax and Bak, thus preventing mitochondria interaction [7,8]. An imbalanced expression of pro-and anti-apoptotic proteins has been documented in ALL [9] and Bax/Bcl-2 ratio has been inversely correlated to prognosis as it becomes significantly lower at relapse [10]. Moreover, increased expression of Bcl-2 related proteins, frequently observed at diagnosis, is associated with decreased responsiveness to chemotherapy induction and treatment refractoriness [11][12][13][14]. Therefore, strategies to inhibit Bcl-2 family proteins have been extensively investigated over the years [15][16][17] showing high in vitro and in vivo efficacy.
In the present study we analyzed the effects of the BH3 mimetic ABT-737 on ALL cell lines and primary samples, exploring the potential synergistic effects with the mTOR inhibitor CCI-779 to overcome ABT-737 acquired resistance. Moreover, we assessed the functional response of ALL cells to the agent combination prior and after Mcl-1 manipulation to establish its role in mediating ABT-737 resistance.

ABT-737 demonstrated anti-leukemic activity on ALL cells
To recapitulate ABT-737 effects on ALL, we initially assessed ABT-737 cytotoxicity as a single agent on 5 human ALL cell lines, looking for a potential correlation between drug sensitivity and Bcl-2 family members expression. A dose-dependent apoptosis induction was observed at increasing concentrations on the MOLT-4 and RS4;11 cell lines (IC 50 s: 198 and 2 nM, respectively) whereas JURKAT, CEM R and CEM S proved resistant (IC 50 s > 5 μM) (Fig. 1a). Western blot analysis revealed that Bcl-2, Bcl-xL and Mcl-1 were heterogeneously expressed among the cell models (Fig. 1b). Interestingly, resistant cell lines displayed a higher Mcl-1/Bcl-2 plus Bcl-xL protein ratio than sensitive models. Modulation of protein expression induced by ABT-737 treatment was then analyzed in a representative sensitive, MOLT-4, or resistant, CEM S, cell model. ABT-737 exposure caused the cleavage of Bcl-2 and the downregulation of Bcl-xL and Mcl-1 only in the MOLT-4 cell line, whereas their expression resulted not affect in the resistant CEM S model (Fig. 1c).
We next evaluated the efficacy of ABT-737 on cell growth and apoptosis induction in 9 ALL primary samples. Treatment with increasing concentrations of ABT-737 resulted in a significant decrease of cell counts and induced apoptosis in a dose-dependent manner. Following 24 h of exposure, subG 0 /G 1 positive cells increased from 17.4 ± 9.3% (vehicle) to 44.2 ± 24.6% (p < 0.01) and 73.8 ± 25.3% (p < 0.001) at 10 and 100 nM, respectively (Fig. 2a). No statistically significant correlation between ABT-737 sensitivity and clinical features of primary samples (age, WBC) was found. Analysis of Bcl-2, Bcl-xL and Mcl-1 protein levels on 7 evaluable samples showed an elevated expression of Bcl-2 in all primary samples with heterogeneous levels of Bcl-xL and Mcl-1 (Fig. 2a). Of note, pt #5 expressed Mcl-1 at high levels, similar to that of CEM S cell line. According to cell availability, we analyzed protein modulations in 3 primary specimens. Our results demonstrate that ABT-737 exposure caused the appearance of cleaved Bcl-2 and induced the downregulation of Bcl-xL and Mcl-1 in two sensitive ALL samples. Conversely, pt #6 showed unchanged levels of Bcl-2, Bcl-xL and of Mcl-1 (Fig. 2c), proving resistant to ABT-737 (subG 0 /G 1 cells = 6% at 100 nM). These data overlapped with the expression profiles previously observed on ALL cell lines, supporting the idea that Mcl-1 plays a role in the ABT-737 resistance also in ALL primary samples.
Finally, we examined the effect of ABT-737 on normal CD34 + cells isolated from the bone marrow of a healthy volunteer showing no significant increase of apoptosis (Fig. 2d).

Co-targeting of the Bcl-2 family proteins and of the mTOR pathway exerted synergistic activity on resistant ALL cell lines by downregulating Mcl-1 expression
The existence of ABT-737 resistant models led us to investigate whether combined treatment might enhance apoptosis sensitivity. We explored a novel therapeutic approach based on the simultaneous inhibition of the Bcl-2 family proteins and the mTOR pathway activity with ABT-737 plus CCI-779. CCI-779 concentrations were chosen based on previous studies demonstrating the response of ALL cell lines to this inhibitor (data not shown). As shown in Fig. 3, neither ABT-737 nor CCI-779 as single agent induced significant anti-proliferative or cytotoxic effects on the JURKAT, CEM R and CEM S cell lines. By contrast, the combination of ABT-737 and CCI-779 exhibited a synergistic activity (CI < 0.2) on the JURKAT and CEM R cell lines, causing a marked cell growth inhibition and a significant (p < 0.01) apoptosis induction. However, combined treatment did not affect the viability and apoptosis rates of the CEM S cell line (Fig. 3). Similar results were obtained with two additional mTOR inhibitors (RAD001 and INK128) (Fig. 4), thus confirming that there were not off-target effects implicated in our findings. Apoptosis induction (upper panels) and cell counts (lower panels) were assessed by AnnV/PI staining and Trypan blue exclusion method, respectively. Data are shown as mean ± SD of at least three independent experiments. IC 50 s were calculated after 72 h of exposure by using the CalcuSyn software. b. Basal expression of selected Bcl-2 family members was analyzed by western blotting in ALL cell lines (left) and quantified by densitometric analysis (right). HSP70 served as loading control. Relative amount of each protein was calculated by dividing the densitometric value of the protein for that of the corresponding HSP70 and then Mcl-1/Bcl-2 plus Bcl-xL ratio was assessed. c. Modulation of protein expression following 24 h of exposure to ABT-737 or vehicle (DMSO) in MOLT-4 and CEM S cell lines. Cleavage of Bcl-2 and levels of Bcl-xL and Mcl-1 were assessed by western blotting. GADPH was used as loading control. * for p < 0.05. www.impactjournals.com/oncotarget  CEM R and CEM S were exposed up to 72 h to ABT-737 and CCI-779, alone or in combination. Apoptosis induction (upper panels) and cell counts (lower panels) were assessed by AnnV/PI staining and Trypan blue exclusion method, respectively. Results are shown as mean ± SD of at least three independent experiments. CI was calculated by conservative isobologram analysis using the CalcuSyn software. CI values < 0.8 were considered synergistic, between 0.8 and 1.2 additive, > 1.2 antagonistic. * for p < 0.01 relative to control conditions. www.impactjournals.com/oncotarget To understand the molecular mechanisms underlying the synergistic interaction, we incubated JURKAT, CEM R and CEM S cells with ABT-737 and CCI-779, alone and in combination, and protein lysates were obtained at 3 and 24 h. Western blot analysis revealed that a 3 h CCI-779 exposure, alone and in combination with ABT-737, induced the downregulation of the mTORC1 downstream targets p-p70 S6K (T389) and p-4EBP1(T37/46) in JURKAT and CEM R cells, while no changes were observed in the CEM S cell line (Fig. 5a). Moreover, a prolonged exposure

Proteasome is not involved in decreasing Mcl-1 expression
To investigate whether Mcl-1 downregulation would be dependent on Gsk3-induced proteasomal degradation, we treated the JURKAT and CEM R cell lines with the proteasome inhibitor MG132 prior to the addition of ABT-737 and CCI-779 and the expression of Mcl-1 was monitored at 16 h. No increase in Mcl-1 protein level was observed in MG132-treated cells (Fig. 5c), thus indicating that proteasome is not involved in Mcl-1 degradation. To further support that Gsk3-induced Mcl-1 degradation is not involved in ABT-737 resistance, we transfected JURKAT and CEM R cells with a Mcl-1 protein mutant lacking the Gsk3 phosphorylation site S159A (Mcl-1 S159A ) which is known to regulate Mcl-1 stability. An efficient transfection was achieved (Fig. 6a) and both cell lines, as expected, showed a reduction in Mcl-1 downregulation after treatment with ABT-737 and CCI-779, both alone and in combination (Fig. 6b). Unexpectedly, however, cytotoxicity induced by single or dual treatment was not reduced in Mcl-1 S159A cells compared to control cell lines (Fig. 6c). Therefore, we hypothesized that inhibition of 4EBP1 and subsequently of protein translation in the JURKAT and CEM R cell lines led to the downregulation of Mcl-1 expression explaining the ability of the ABT-737/ CCI-779 combination to trigger apoptosis in JURKAT and CEM R cells but not in the CEM S cell line (Fig. 5b).

Combined treatment failed to show synergistic activity where Mcl-1 is not the main resistant factor
Since the combination of ABT-737 and CCI-779 resulted unable to reduce Mcl-1 expression in the CEM S cell line, we performed RNA interference of Mcl-1 (Mcl-1i). Efficient silencing was achieved without any changes in Bcl-2 and Bcl-xL protein levels (Fig. 7a). We then exposed wt and Mcl-1i cells to ABT-737 and CCI-779, alone and in combination, demonstrating neither changes in protein expression nor increase of apoptosis in Mcl-1i CEM S cells when compared to their controls (Fig. 7b). Only a slight reduction in cell growth was noticed. We thus concluded that in this model Mcl-1 is not the unique determinant of ABT-737 resistance.

ABT-737 synergized with CCI-779 enhancing apoptosis in resistant ALL primary samples
To evaluate the effectiveness of ABT-737 and CCI-779 as a potential therapeutic strategy in ALL, 18 additional primary samples were exposed to the drugs, alone and in combination. As before, basal expression levels of Bcl-2 family members was analyzed by western blot on 10 evaluable samples. Bcl-2 and Bcl-xL were heterogeneously expressed in the majority of samples (7/10 and 9/10, respectively) and almost all samples (9/10) shared the overexpression of Mcl-1 when compared to NPBLs (Fig. 8a). CCI-779 treatment as a single agent demonstrated a modest apoptosis activity (increase of subG 0 /G 1 peak from 19.9 ± 11.6% (vehicle) to 23.8 ± 15.6% at 5000 nM). By contrast, ABT-737 confirmed its cytotoxic effects also on these samples, inducing a significant (p < 0.001) apoptosis induction from 19.9% ± 11.6 (vehicle) to 69.9% ± 21.0 at 50 nM. Importantly, ABT-737 activity was observed in the majority of samples (15/18) while the remaining 3 specimens exhibited less than 20% of net apoptosis (Fig. 8b). Nevertheless, combined treatment was able to induce synergistic (pt #10, #26) or additive (pt #17) proapoptotic effects on these 3 samples (Fig. 8b, arrows). No significant differences in sensitivity to ABT-737 as single agent or in combination with CCI-779 were observed when data were sorted by age and/or WBC (data not shown). Further analysis of correlation between ABT-737/CCI-779 sensitivity and protein expression in these three resistant samples revealed that pt #17, which displayed additive effects in combination, was characterized by negligible levels of Bcl-2 and Bcl-xL with high expression of Mcl-1, thus supporting the lower ABT-737 activity as single agent and in combination. Conversely, pt #10 and #26, which displayed elevated protein levels of Bcl-2, Bcl-xL and Mcl-1, showed a synergistic apoptosis induction associated with a marked downregulation of p-4EBP1(T37/46) and Mcl-1 (Fig. 8c), thus resembling the molecular changes previously observed in the JURKAT and CEM R cell lines.

Discussion
Over the last years, attention in ALL has been focused on the molecular mechanisms underlying drug resistance, developing novel strategies to modulate aberrant signaling rising from acquired lesions [53]. Among these, therapeutic targeting of the deregulated apoptotic machinery represents an attractive area of investigation [54,55]. Anti-apoptotic Bcl-2 related proteins allow leukemic survival and cell proliferation by interfering with the intrinsic pathway of apoptosis. Thus, inhibition of Bcl-2 family members represents a highly potent stimulus to trigger apoptosis in leukemic cells. ABT-737 is a small-molecule inhibitor of Bcl-2, Bcl-xL and Bcl-w that has been investigated in several human cancers, including hematological neoplasias.
Here we first evaluated the activity of ABT-737 as single agent on ALL cell lines and primary samples, assessing the correlation between Bcl-2 family member expression and ABT-737 sensitivity. Previous reports showed that high Bcl-2 or low Mcl-1 expression correlate with in vitro sensitivity of several cancer cell lines to ABT-737 [24,27,56]. Consistently, Bcl-2 protein expression was associated with drug efficacy in acute myeloid leukemia (AML) [25]. However, in ALL, Bcl-2 dependence rather than basal expression seemed to have a greater impact on cellular response [14]. Indeed, Kang and colleagues [57] showed that levels of anti-apoptotic Bcl-2 family members are not associated with ALL susceptibility to ABT-737. More recently, it has been demonstrated that Mcl-1, Noxa and Bim protein levels significantly impact on in vitro resistance of ALL cell lines. By contrast, only Bim rather than Bax, Bak or increased Mcl-1 expression determined in vivo sensitivity to ABT-737 in a panel of ALL xenografts from pediatric patients [23]. Therefore, ABT-737 sensitivity in ALL seems to depend on several factors. We found that Mcl-1/Bcl-2 plus Bcl-xL expression ratios determined ALL cell lines in vitro response to ABT-737. Indeed, our sensitive cell models were characterized by lower Mcl-1 and upper Bcl-2 plus Bcl-xL protein expression than resistant cell lines. However, it is likely that different factors (e.g. Bim) other than the proteins assessed in this study may affect ABT-737 in vitro responsiveness. In agreement with published data [57,58], we demonstrated that increasing concentrations of ABT-737 further decreased Bcl-xL and Mcl-1 expression only in MOLT-4, a sensitive cell line,   Freshly isolated leukemic blasts from 18 ALL patients were seeded at a density of 1 × 10 6 /mL and treated for 24 h with ABT-737 (50 nM) and CCI-779 (5000 nM), alone or in combination. Apoptosis induction was determined by AO technique. Results are reported as percentage of subG 0 /G 1 positive cells for individual patient. Synergistic or additive interaction were indicated by black arrows. c. Leukemic blasts from sample #10 and #26 were exposed for 24 h to ABT-737, CCI-779 or their combination. Apoptosis induction and protein expression were assessed by AnnV/PI staining and western blot analysis, respectively. www.impactjournals.com/oncotarget inducing significant cell growth reduction and apoptosis at nanomolar concentration. By contrast, protein levels were not affected in CEM S cells, a resistant cell model, showing no significant changes of apoptosis induction even at micromolar doses. Cytotoxic efficacy of ABT-737 was then confirmed on ALL primary samples, with the majority of specimens demonstrating significant apoptosis induction when compared to their controls. By contrast, normal CD34 + cells were remarkably resistant to ABT-737, as already shown by Konopleva et al. [25], Interestingly, we found a resistant primary sample in which Mcl-1 expression was not affected by ABT-737 exposure. Conversely, analysis of two sensitive ALL samples revealed a marked Mcl-1 downregulation following ABT-737 exposure, likely due to apoptosis induction. These results, in accordance to published data [56], support the contribution of Mcl-1 downregulation in the ABT-737-induced cytotoxicity.
To overcome BH3 mimetics resistance, we explored a novel therapeutic approach by using the mTOR inhibitor CCI-779 in ALL cell lines and primary samples. The PI3K/mTOR pathway is frequently found constitutively activated in ALL [35,36,59] and targeted inhibition of mTOR, alone or in association with PI3K, has been investigated at multiple levels, generally displaying better results in vitro than in vivo. Recently, it has been demonstrated that activated Akt may decrease ABT-737 activity, suggesting a functional role of this signaling also in protecting leukemia cells from BH3 mimetic-induced apoptosis [60]. Consistently, simultaneous inhibition of the PI3K/mTOR pathway, by BEZ235, and Bcl-2/Bcl-xL, by ABT-737, strongly potentiated cytotoxicity of single agents and markedly reduced colony formation in multiple AML cell lines and primary blast specimens while exerted only modest toxicity toward normal hematopoietic progenitors [60]. Moreover, Jin and colleagues [61] showed that the combination of GDC-0941 and ABT-737 triggered mitochondrial apoptosis in AML cells co-cultured with stromal cells under hypoxic conditions, thus suggesting the efficacy of this approach even under the protective conditions afforded by the bone marrow microenvironment. It has also been demonstrated that PI3K/mTOR signaling contributed to ABT-737 resistance in Burkitt lymphoma cells, and their inhibition through dual or active site mTOR inhibitors induced synergistic caspase activation, rendering chemoresistant cells sensitive to apoptosis induction [62]. In addition, data obtained on different solid tumors [31,63,64] showed that blockade of mTOR signaling and Bcl-2 activity triggered massive in vitro apoptosis by loss of mitochondrial membrane potential, activation of caspases and Mcl-1 downregulation and was highly synergistic in vivo providing tumor regressions without notable hematologic suppression. Since numerous of the aforementioned inhibitors have shown promising results in different stages of clinical trials, alone or in combination with chemotherapy [clinicaltrials.gov], it has been proposed that the simultaneous inhibition of the mTOR pathway and Bcl-2 activity in cancer cells, even in those resistant to single agents, may have important clinical implications. We found that CCI-779, RAD001 and INK128, as single agents, modestly impaired resistant cell line proliferation, resulting in a no significant pro-apoptotic activity. By contrast, co-targeting of Bcl-2 and mTOR activity with all the above inhibitors strongly sensitized resistant JURKAT and CEM R cell lines to apoptosis, thus reverting the acquired ABT-737 resistance. Interestingly, since CEM R cells are known to be insensitive to vinblastine [65], we hypothesized that combined treatment can be effective also in other chemoresistant models even those MDR1related. However, the CEM S cell line failed to show any apoptotic increase after exposure to all tested combination, suggesting an analysis of protein expression to better understand the biological reasons underlying synergistic interaction between the aforementioned drugs. We observed that CCI-779, especially when in combination with ABT-737, inhibited the mTORC1 pathway activity by decreasing phosphorylation levels of p70 S6K and 4EBP1 only in JURKAT and CEM R, thus reinforcing the idea of a context-dependent role of mTORC1 in the increased ABT-737-mediated cell death [31]. Moreover, after 24 h of treatment, CCI-779 also affected the mTORC2 pathway, as demonstrated by the downregulation of p-Akt(S473) and p-Gsk3α/β(S21/9), indicating that prolonged exposure to rapamycin derivative may be effective, at least in part, also on PI3K downstream targets. Of note, combined treatment markedly decreased Mcl-1 protein levels in the JURKAT and CEM R cell lines, while not affecting its expression in CEM S cells. We excluded the involvement of proteasomal degradation in ABT-737-induced Mcl-1 downregulation since protein levels appeared not increased in cells treated with MG132, a well-known proteasome inhibitor, compared to their controls. We also transfected a mutant of Mcl-1 which lacks the S159 residue, a known Gsk3 phosphorylation site that marks Mcl-1 for proteasomal degradation, in the JURKAT and CEM R cell lines. Although an efficient transfection was achieved and both cell lines showed a reduction in Mcl-1 downregulation, we unexpectedly detected no reduction in apoptosis induction after single or combined treatment, further supporting the idea that proteasomal degradation is not involved in Mcl-1 degradation. These finding led us to hypothesize that inhibition of 4EBP1-dependent protein translation may be important for the synergistic interaction between ABT-737 and CCI-779. Further experiments are needed to clarify unequivocally the precise contribution of molecular mechanisms (e.g. caspase-dependent cleavage) in determining Mcl-1 levels on ALL cells.
However, our results demonstrated that in some model, as the CEM S cell line, Mcl-1 is not a unique determinant of ABT-737 resistance. Indeed, when we knocked down protein expression by RNA interference, we observed no changes in treatment cytotoxicity, even after exposure to the combination. Different groups have already reported that, in certain contexts, Mcl-1 downregulation although required is incapable itself of initiating apoptosis [66,67]. In addition, loss of Bim, combined absence of Bax and Bak or overexpression of Bcl-xL and Bcl-w have been shown to make lymphoma cells resistant to ABT-737 in vitro and in vivo, even in the absence of endogenous Mcl-1 [68], thus indicating that targeting of more than one antiapoptotic protein may be required for the full engagement of the apoptotic machinery. Therefore, we concluded that Mcl-1 downregulation likely cooperates with other independent mechanisms to support ABT-737 resistance in ALL.
The effectiveness of the ABT-737 and CCI-779 combination as a novel therapeutic strategy was further evaluated on additional primary ALL samples. CCI-779 treatment as monotherapy demonstrated only a modest apoptosis activity on these samples, whereas ABT-737 confirmed its cytotoxic effects inducing significant apoptosis increase on the large majority of samples. Importantly, combined treatment was able to sensitize ABT-737 resistant samples to apoptosis in an additive or synergistic fashion, thus overcoming the acquired resistance to ABT-737. Of note, analysis of protein expression revealed that samples that displayed additive effects in combination were characterized by negligible levels of Bcl-2 and Bcl-xL with high expression of Mcl-1, supporting lower drug activities. In conclusion, our study show the efficacy of BH3 mimetics as single agent in the majority of the ALL samples and demonstrate that Mcl-1 represents the main resistance factor to the BH3 mimetic ABT-737 in ALL cell lines and primary samples. Co-targeting of the Bcl-2 family proteins and mTOR pathway effectively enhanced single drug cytotoxicity by downregulating mTORC1 activity and Mcl-1 expression. Additional resistant mechanisms, however, should be taken into account since, in certain ALL models, Mcl-1 modulation failed to overcome by itself ABT-737 resistance, thus suggesting the existence of other mechanisms of resistance to BH3 mimetics.

Primary samples, cells cultures and in vitro treatments
Bone marrow or peripheral blood primary samples from 14 adult and 13 pediatric ALL patients (Table 1) were collected after informed consent in accordance with the Helsinki Declaration and approved by the Institutional Review Board of the Sapienza University of Rome and normal peripheral blood lymphocytes (NPBLs) were purified by Ficoll-Hypaque (Sigma-Aldrich, MO, USA) density-gradient centrifugation. Only specimens with blast counts > 70% were included in this study. For in vitro treatments, ABT-737 (kindly provided by Abbott Laboratories) and CCI-779 (Novartis, Basel, Switzerland) were prepared in DMSO and ethanol to a stock concentration of 10 and 20 mM, respectively. RAD001 (Sigma-Aldrich) and INK128 (Selleckchem, Germany) were both diluted in DMSO to a stock concentration of 10 mM. ALL cell lines (MOLT4, RS4;11, JURKAT, CEM R and CEM S) were cultured in RPMI 1640 medium (Euroclone, Milan, Italy) containing 10% heat inactivated fetal bovine serum, 2 mM l-glutamine and antibiotics at 37°C under 5% CO 2 -95% air and harvested in logphase growth. For each experiment, cell lines and primary samples were plated at concentration of 0.2 × 10 6 /mL and 1 × 10 6 /mL, respectively. Viability was assessed by triplicate Trypan blue exclusion counting. To establish the optimal concentration for drug treatment, MTT [3-4,5-dimethylthythiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays (Sigma-Aldrich) were performed. Cell lines and primary samples were incubated up to 72 h with ABT-737 (ranging from 10 to 5000 nM), CCI-779 (10 to 20000 nM), RAD001 (500 to 10000 nM) and INK128 (1 to 500 nM) alone and in combination. For proteasome inhibition, 3 μM of MG-132 (Sigma-Aldrich) was added to the medium 3 h before drugs administration.

Apoptosis and cell-cycle analysis
Cell cycle analysis was performed by Acridine Orange technique, as previously described [69]. To evaluate apoptosis levels, cells were examined over a culture period of 24-72 h using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining, as previously described [70]. Apoptosis induction was further measured by evaluating the subG 0 /G 1 peak using AO technique, as described [71]. Flow cytometric analyses were carried out using both FACSCan and Accuri C6 flow cytometer (BD Biosciences, CA, USA). Cytotoxic activity induced by in vitro treatments was evaluate by measuring the net apoptosis rate obtained from the difference between subG 0 /G 1 value of each condition and of that of controls.

Western blot analysis
Protein expression of Bcl-2 family and PI3K/ Akt/mTOR pathway key components of ALL cell lines and primary samples with available amount of cells was analyzed by western blotting. Cells were exposed to ABT-737 and CCI-779 and lysed in ice-solution containing 10 mM NaF, 1 mM Na 3 VO 4 , 150 mM NaCl, 1 mM MgCl 2 , 1 mM CaCl 2 , 0.1% NaN 3 , 10 mM www.impactjournals.com/oncotarget iodoacetamide, 3 mM PMSF and 1% Triton X-100 (Sigma-Aldrich) supplemented with protease inhibitor cocktail (Roche Diagnostics, Indianapolis, USA). Equal amounts of proteins were subjected to SDS-PAGE. After electrotransfer on nitrocellulose (Bio-Rad Laboratories, CA, USA), membranes were blocked in 5% w/v non-fat dry milk and incubated overnight with primary antibodies, according to manufacturer's instruction. Blots were then probed with HRP-conjugated secondary antibodies for 1 h at room temperature and developed using ECL reagent. Resulting signals were collected on x-ray films, digitally scanned and quantified using Image J software (NIH, Bethesda, MD, USA).
The antibodies used for Western blot were as follows: anti Bcl-2 (DAKO Corp, Carpinteria, CA, USA), anti Mcl-1 (Santa Cruz Biotechnology, CA, USA), all the others antibodies were purchased from Cell Signaling Technology (Beverly, Massachusetts, USA).

Plasmids and Infection
Each lentiviral shRNA construct, previously described [25], was transfected into 293T cells using Lipofectamine 2000 (Invitrogen). Following 72 h post-transfection, viruscontaining supernatants were collected and used to infect cells by the spin-inoculation method. Briefly, 2 × 10 6 cells were added with 2 ml of virus and 8 μg/mL polybrene, transferred in a multiwell plate and then centrifuged at 1500 g for 90 minutes at 30°C. Once the cells have entered log phase, selection was applied to obtain stable clones.

Statistical analysis
All data were obtained from at least three independent experiments and are presented as means ± SD (standard deviation). Significant differences between experimental values was determined by using the Student's t test. For combination experiments, analysis of synergism was assessed by using the Chou-Talalay method and the CalcuSyn software (Biosoft, Cambridge, MA, USA). CI values lower than 0.8 were considered synergistic, between 0.8 and 1.2 additive, and greater than 1.2 antagonistic.

ACKNOWLEDGMENTS
We thank to Davis RJ for the pcDNA3.1-hMcl-1(S159A) plasmid, Bossi G and Folgiero V for helping and supporting during the electroporation experiments. We also thank to Arlene Forte (Project Coordinator, Clinical Research, Oncology at AbbVie Inc) for supplying ABT-737, RomaAIL (Associazione Italiana contro le Leucemielinfomi e mieloma) for providing laboratory space and David Calef for editing the manuscript.

GRANT SUPPORT
This work was supported by Sapienza University (C26G13WSNF and C26A137XXS) and MIUR (PRIN 2010 AX2JX7_003) grants, and by Fondazione Frisiani-Santini.