The mRNA and protein samples of PBMC from healthy donors were obtained from cells separated by Ficoll-Paque centrifugation, while healthy B-cells (mainly CD19⁺) were obtained by cell sorting of bone marrows from healthy volunteers. Diagnostic RNA samples of bone marrow (BM) aspirates of B-leukaemic patients with a blast count of 80–95% were kindly allowed from the Cell Bank of the Dipartimento di Salute della Donna e del Bambino, University of Padova, Italy. B-ALL patient samples were obtained after informed consent following the tenets of the Declaration of Helsinki. The study was approved by the Italian Association of Pediatric Onco-Hematology (AIEOP). Written consent was obtained from participants. All analyzed B-ALL samples were obtained at the time of diagnosis before treatment, after Ficoll-Hypaque (Pharmacia, Uppsala, Sweden) separation of mononuclear cells as described previously (22). The percentage of CD19⁺ cells ranged from 80 to 95%. Human B-leukemia cell lines, REH, SEM, MHH-CALL2, RS4;11 and NALM-6, were grown in RPMI-1640 medium (Gibco, Milan, Italy) all supplemented with 115 U/ml penicillin G (Gibco), 115 μg/ml streptomycin (Invitrogen), 10% fetal bovine serum (Invitrogen), and maintained at 37°C in a humidified atmosphere with 5% CO₂.

Total RNA was isolated from frozen cell pellets using the RNeasy Mini kit (Qiagen, UK) according to the manufacturer's instructions and RNA purity and concentration were determined by measuring the spectrophotometric absorption at 260 nm and 280 nm on NanoDrop ND-1000. Total RNA (1 μg) was reverse transcribed into first strand cDNA using Superscript III first stand cDNA synthesis (Life Technologies, UK) Briefly, 1 μl of 50 μM oligo(dT)20 and 1 μl of 10 mM dNTPs mix were added to the RNA before the volume was adjusted to 11 μl using RNase-free water. Samples were denaturated at 65°C for 5 min and then quickly chilled on ice for 1 min. Subsequently, 1 μl of the reverse transcriptase Superscript III (200 U/μl) was added, along with 1 μl 0.1 M DTT, 1 μl RNaseOUT Recombinase Inhibitor and 1X first stand buffer. The solution was incubated at 25°C for 5 min then heated at 50°C for 50 min. The reaction was inactivated by heating at 70°C for 15 min. For real-time quantitative PCR, 1 μl of cDNA was used as template in a 24-μl reaction carried out with Power SYBR Green kit (Applied Biosystems, UK) with ABI 7800 system (Applied Biosystems). The mRNA levels of target genes were calculated relative to the expression of L19 mRNA levels using the ΔCt method. Primers used: FOXM1-fwd, 5′-TGCAGCTAGGGATGTGAATCTTC-3′; FOXM1-rv, 5′-G GAG CCCAGTCCATCAGA ACT-3′; CCNB1-fwd, 5′-CAGTTATGCAGCACCTGGCTAAG-3′; CCNB1-rv, 5′-TGTGGTAGAGTGCTGATCTTAGCAT-3′; AURKB-fwd, 5′-AGTGGGACACCCGACATC-3′; AURKB-rv, 5′-G CCCA ATCTCA A AGTCATCA AT T-3′; L19-fwd, 5′-GCGGAAGGGTACAGCCAAT-3′; L19-rv, 5′-GCAGCCGGCGCAAA-3′.

REH, SEM, MHH-CALL2, RS4;11 and NALM-6, after experimental conditions, were collected, centrifuged, and washed two times with ice cold phosphate-buffered saline (PBS). For western blot analysis cells were lysed as described previously (23). Proteins were resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE) (using 7 or 10% acrylamide gels), transferred to PVDF Hybond-P membrane (GE Healthcare) and immunoblotted with primary antibodies against, FOXM1 (Santa Cruz, C-20), β-tubulin (Santa Cruz), Aurora B (Cell Signaling), Cyclin B1 (Santa Cruz). The membranes were washed four times with Tris-buffered saline and Tween-20 (TBS-T) for 15 min prior to incubation with the respective peroxidise (HRP)-conjugated secondary antibody (Dako, Ely, UK), at 1:2,000 dilution for 30 min at room temperature and again washed four times with TBST-T for 20 min. Protein were visualised using enhanced chemiluminescence (ECL) detection system (Perkin-Elmer, Seer Green, UK) with Amsterdam Hyperfilm ECL (GE Helthcare, Little Chalfont, UK) and signal was detected using the SRX-101A X-ray developer (Konica Minolta, Tokyo, Japan).

For FOXM1 silencing, REH or NALM-6 cells were transiently transfected with siRNA SMARTpool reagents purchased from Thermo Scientific Dharmacon (Lafayette, CO, USA) using the transfection reagent Oligofectamine (Life Technologies, UK) according to the manufacturer's instructions. SMARTpool siRNAs used were: siRNA FOXM1 (l-009762-00) and the non-specific (NS) control siRNA, ON-TARGETplus Non-Targeting pool (D-001810-10) confirmed to have minimal targeting of known genes. All siRNA pools were resuspended to 20 μM in 1X siRNA buffer.

Surface exposure of phosphatidylserine on apoptotic cells was measured by flow cytometry with a Coulter Cytomics FC500 (Beckman Coulter) by adding Annexin V conjugated to fluorescein isothiocyanate (FITC) to cells according to the manufacturer's instructions (Annexin V Fluos, Roche Diagnostic). Simultaneously, the cells were stained with PI. Excitation was set at 488 nm, and the emission filters were at 525 and 585 nm, respectively, for FITC and PI.

For flow cytometric analysis of DNA content, 5×10⁵ of REH, and NALM-6 cells were either treated with 0.5 and 1 μM of thiostrepton or knocked-down for FOXM1 and after 24, 48 and 72 h of treatment or knockdown, cell cycle analysis was performed as previously described (24).

Cell proliferation was assessed by MTT (3-4,5-dimethylthiazol-2-yl-2,5-diphenyl tetrazolium bromide) assay after treatment. Equal concentrations of cells were plated in triplicate in a 96-well plate and incubated with 10 μl of MTT (Sigma-Aldrich, St. Louis, MO, USA) for 4 h. Absorbance was measured at 562 nm using Victor3™ 1420 Multilabel Counter (Perkin-Elmer, Waltham, MA, USA). Cells were treated for 48 h using scalar dilutions of thiostrepton (Sigma-Aldrich), combined with cytarabine (Aractyn, Pfizer), daunorubicin (Pfizer), vincristine, dexamethasone (Sigma-Aldrich). Thiostrepton was also added to drug solutions at fixed combination ratios. The effectiveness of various drug combinations was analyzed by Calcusyn Version 2.1 software (Biosoft). The combination index (CI) was calculated according to the Chou-Talalay method (25). A combination index of 1 indicates an additive effect of the 2 drugs. Combination index values <1 indicate synergy, and combination index values >1 indicate antagonism.

Results are presented as the mean ± SEM. The differences between different conditions were analyzed using the two-sided Student's t-test. P-values <0.05 were considered statistically significant.

To investigate whether FOXM1 regulates B lymphoblastic leukemia (B-ALL) proliferation, we analyzed FOXM1 mRNA levels in ten B-ALL pediatric patients comparing them to peripheral blood mononuclear cells (PBMC) from healthy donors. For these experiments, the mRNA was extracted from cell pellets of patients recruited at the time of diagnosis. The first two selected patients were characterized by chromosomal translocations at chromosome 12 and 21 [t(12;21)]; patients 3, 4 and 5 carried the translocation between the chromosome 9 and 22 [t(9;22)], whereas the other five patients were without translocations (Table I). As shown in Fig. 1A, FOXM1 mRNA levels were significantly higher in patients when compared to the samples of healthy donors. Interestingly, a significant increment on Cyclin B1 and Aurora B mRNA levels, two genes whose expression is directly regulated by FOXM1 was also observed (Fig. 1B and C). In addition to mRNA levels, protein expression was also examined by western blot analysis. As shown in Fig. 1D, the expression of FOXM1 and of its targets Aurora B and Cyclin B1 was generally higher in patients compared to healthy cells, confirming the upregulated FOXM1 activity. Similar results were obtained when the mRNA or protein levels of B-ALL patients were compared to those of CD19-positive cells isolated from bone marrows of healthy donors (data not shown). Taken together, these data showed that the expression levels and activity of FOXM1 are increased in B-ALL patient samples when compared to healthy lymphocytes and normal B-cells, suggesting that FOXM1 could plays a key function in supporting the cell proliferation of B-ALL.

Next, we analyzed FOXM1 expression levels in five different B-ALL cell lines. More specifically, we quantified FOXM1 mRNA levels, at basal conditions, comparing five different B-ALL cell lines (REH, MHH-CALL2, SEM, NALM6, RS4;11) with mRNA samples of PBMC obtained from healthy donors. RT-qPCR analysis showed in Fig. 2A reveals that FOXM1 mRNA levels were significantly higher in B-ALL cell lines when compared to normal lymphocytes. This pattern was again accompanied by increased transcriptional levels of Cyclin B1 and Aurora B (Fig. 2B and C). In agreement with the mRNA expression patterns, western blot analysis revealed that FOXM1 expression was generally higher in leukaemic cells compared to normal lymphocytes. This was again associated with Cyclin B1 and Aurora B upregulation in leukaemic cells (Fig. 2D). Similar results were obtained comparing the FOXM1 mRNA and protein expression of B-ALL cell lines with healthy CD19⁺ cells (data not shown).

To test if FOXM1 has a role in promoting B-ALL proliferation, we knocked down its expression in two B-ALL cell lines REH and NALM-6 using a specific FOXM1 siRNA pool and assessed the cell viability at 24, 48, and 72 h post-transfection. FOXM1 silencing was confirmed by western blot and RT-qPCR analysis (Fig. 3A–D). Aurora B and Cyclin B1 protein levels were clearly downregulated in FOXM1-depleted NALM-6 and REH cells compared to cells transfected with non-specific (NSC) control siRNA pool (Fig. 3A and B). Furthermore, in both REH and NALM-6 cell lines, RT-qPCR analysis revealed a significant decrease in Aurora B mRNA levels, although Cyclin B1 mRNA levels appeared significantly decreased only in REH and not in NALM-6 cells (Fig. 3C and D).

Cell proliferation analysis assessed by trypan blue exclusion assay, revealed that upon FOXM1 depletion, there is a significant decrease in cell proliferation after 72 h of transfection in NALM-6 (Fig. 3E) and after 48 h in REH cells (Fig. 3F), suggesting that FOXM1 plays an important role in B-ALL cell proliferation. Trypan blue-negative population (live cells) was strongly decreased in cells that were silenced for FOXM1, with no significant changes in the levels of dead cells (trypan blue-positive) in NALM-6, and only with a slight increase in the percentage of dead cells in REH. These results pointed out that FOXM1 plays an important role in B-ALL cell proliferation.

To further analyze the role of FOXM1 on cell cycle progression in B-ALL, we analyzed the cell cycle phase distribution in NALM-6 and REH cells following FOXM1 knockdown. As shown in Fig. 4A and B, there was a significant increase on the percentage of cells in G2/M in both cell lines, and a corresponding decrease of cells in the S phase compared with non-specific (NSC) control cells, only in NALM-6 cells at 72 h. The G2/M cell cycle arrest following FOXM1 silencing by siRNA is confirmed by the consistent downregulation of the expression of the G2/M regulators, Cyclin B1 and Aurora B (Fig. 4C and D). This further supports the role of FOXM1 in cell cycle progression in B-ALL, particularly at the G2/M transition.

We next evaluated if FOXM1 knockdown could modulate the expression of proteins involved in late phase cell cycle regulation. As depicted in Fig. 4, the FOXM1 silencing in both NALM-6 (Fig. 4C) and in REH cells (Fig. 4D), caused downregulation of cell cycle regulatory proteins such as Cyclin B1, Aurora B, Survivin, and Cdc25b, involved in mitotic progression. The expression of the S-phase promoting Cdc25a phosphatase, which plays a major role in G1/S progression, dephosphorylating Cdk2 and activating CDK2-cyclin E activity (26,27), was strongly reduced. Interestingly, both p27^Kip1 and p21^Cip/Waf1, were also downregulated. These two cyclin-dependent kinase inhibitor (CKI) proteins also play a role in the assembly of cyclin-CDK complexes. Altogether, these results indicate that FOXM1 is strongly involved in modulating the expression of cell cycle regulatory proteins at both the G1/S and at the G2/M cell cycle transitions.

To further confirm the role of FOXM1 on B-ALL cell proliferation, we treated cells with the thiazole ring containing antibiotic, thiostrepton, a well-established specific FOXM1 inhibitor (28,29). This drug causes growth inhibition in a small panel of B-ALL cell lines with GI₅₀ in the micromolar and sub-micromolar range (GI₅₀= 0.4–1.4 μM). In this context, we treated both NALM-6 and REH cell lines with two different concentrations of thiostrepton, 0.5 and 1 μM, respectively for 24, 48, and 72 h. Results show a significant reduction in cell viability, revealed by the considerable low numbers of trypan blue-negative cells in both NALM-6 and REH cell lines (Figs. 5A and 6A). Moreover, flow cytometric analysis carried out following different treatment times indicates that thiostrepton induces apoptosis, demonstrated by the appearance of a large percentage of Annexin V-positive cells in both cell lines (Figs. 5B and 6B). Importantly, apoptosis occurs in a concentration- and time-dependent manner. In each assay, FOXM1 downregulation was confirmed by both western blot analysis (Figs. 5C and 6C) and by RT-qPCR (Figs. 5D and 6D). Note that in both cell lines, FOXM1 and its downstream target protein levels were decreased following thiostrepton treatment, in a manner similar to that observed for FOXM1 silencing.

We then analyzed the cell cycle phase distribution in REH and NALM-6 cells following treatment with both 0.5 and 1 μM concentrations of thiostrepton at 24, 48, and 72 h. Results (Fig. 7A and B) show that similarly to FOXM1 knockdown experiments, thiostrepton treatment induces a cellular arrest at the G2/M cell cycle phase as well as a decrease of cells in the S phase.

As FOXM1 down-regulation led to a decrease on B-ALL cell proliferation, we next-tested if thiostrepton could be used in combination with the most commonly used chemotherapeutics in B-ALL treatment. To this end, four different B-ALL cell lines, two glucocorticoid-resistant (REH, SEM) and two glucocorticoid-sensitive (NALM-6, RS4;11), were treated for 48 h with thiostrepton in combination with chemotherapeutic agents (i.e., dexamethasone, asparaginase, daunorubicin, vincristine and Ara-C) normally used to treat pediatric B-ALL patients. More specifically, thiostrepton was combined with different drugs at fixed molar combination ratios, and cell viability analyzed by MTT assay (Figs. 8A and 9A). As described above, thiostrepton has a significant cytotoxicity when used as single agent. Notably, when thiostrepton was used in combination with chemotherapic drugs, we observed a synergistic increase in the cytotoxicity as demonstrated by the values of combination index (CI) according to Chou and Talalay (25,30). As depicted in Table II, reporting CI values calculated at GI₅₀, GI₇₅ and GI₉₀, in almost all cell lines tested, thiostrepton and chemotherapeutic drugs act in a synergistic fashion (CI<1). Our results therefore show that, in general, the pharmacological downregulation of FOXM1 caused by thiostrepton, can significantly increase the cell death induced by the treatment with conventional chemotherapeutic agents.

To prove the above further, we knocked down FOXM1 in REH and NALM-6 cells using siRNA, and then treated them with dexamethasone. After 48 h of treatment we analyzed cell viability by flow cytometry staining with Annexin V and propidium iodide (PI). Results showed a significant decrease in cell viability after 48 h of treatment in FOXM1 silenced cells when compared to its controls, this was particularly noted in glucocorticoid resistant cells, indicating that FOXM1 can be an important therapeutic target for overcoming glucocorticoid resistance in B-ALL (Figs. 8B and 9B).