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Materials and methods. (DOCX 22Â kb)

Introduction. The proteasome inhibitor bortezomib is a new treatment option for patients with refractory or relapsed ALL, particularly when used in combination with conventional chemotherapy or other targeted agents. Indeed, a limited efficacy of bortezomib alone in ALL patients has been reported. A terminal pro-apoptotic endoplasmic reticulum (ER) stress/unfolded protein response (UPR) is one of the several mechanisms of bortezomib-induced apoptosis. CX-4945, a potent CK2 inhibitor, has been found to induce apoptotic cell death in T-ALL preclinical models, via perturbation of ER/UPR pathway. Here, we have explored the cytotoxic effects of a bortezomib/CX-4945 combination in a panel of B- and T-ALL cell lines. Methods. B- (KOPN-8, NALM-6, RS4;11) and T- (MOLT-4, Jurkat, CEM-R) ALL cell lines were pretreated with bortezomib (Selleck Chemicals, Houston TX, USA), for six hours and then with CX-4945 (Selleck Chemicals, Houston TX, USA), for 16/24 hours. MTT assays were performed to analyze cell viability. Apoptosis induction was evaluated by Annexin V/PI staining and flow cytometric analysis. Protein expression was studied by western blot. Results. Cells were cultured in the presence of bortezomib or CX-4945, either alone or in combination at a fixed ratio. The combined treatment was more effective in reducing cell viability in all B-ALL cell lines and in MOLT-4 cells. Annexin V/PI staining was performed; interestingly, no synergism was detected if the two drugs were administered together since the beginning of treatment. In response to treatment with 2.5 nM bortezomib followed by 5 μM CX-4945, we detected an increase in apoptosis in B-ALL cell lines and in MOLT-4 cells after 24 h. Western blot analysis for the cleaved forms of caspase-3 and PARP, confirmed a higher apoptosis induction by the combined treatment. A reduction in anti-apoptotic Bcl2 concomitant with an increase in proapoptotic Bax, suggested that bortezomib/CX-4945 treatment caused a mitochondrial apoptosis. IRE1a and CHOP (established markers of ER stress/UPR-mediated apoptosis) levels increased in response to the combined treatment, in contrast the expression of GRP78/BIP (a marker of UPR activation) decreased, suggesting that a potential mechanism by which the drug combination induced cell death, involved ER stress induction by bortezomib and the inability to respond by adequate activation of the UPR signaling which was blocked by CX-4945. Conclusions. Here, we demonstrated that the proteasome inhibitor bortezomib and the CK2 inhibitor CX-4945 interact in a synergistic manner to induce apoptosis both in B- and in T-ALL cell lines. Drug cytotoxicity was associated with modulation of the ER stress/UPR signaling pathway. Importantly, the synergism was observed only when bortezomib treatment preceded CX-4945 administration. Therefore, our findings support clinical application of bortezomib in combination with CX- 4945 in B- and T-ALL treatment.

Expression of dCK and dGK in T-ALL cell lines. Western blotting analyses for the expression of dCK and dGK proteins in T-ALL cell lines. Fifty micrograms of protein was blotted to each lane. Antibody to β-actin served as a loading control. Molecular weights are indicated on the right. One representative of two different experiments is shown.

Specific effects of nelarabine on PI3K/AKT and MEK/ERK1/2 pathways. Western blotting analyses for the expression of p-AKT and p-ERK in resistant T-ALL cell lines treated with the specific inhibitors LY294002 (PI3K inhibitor), CCI-779 (mTOR allosteric inhibitor) or trametinib (MEK1/2 inhibitor) alone or in combination with nelarabine. Thirty micrograms of protein was blotted to each lane. Antibody to β-actin served as a loading control. Molecular weights are indicated on the right. CTRL: untreated cells; Nela and N: nelarabine at 10 μM; LY: LY294002 at 10 μM; CCI: CCI-779 at 100 nM; Tram: trametinib at 1 μM. Cells were treated for 48 h.

The combination of nelarabine and ZSTK-474 is synergistic in CEM-R cells, which overexpress P-gp. Cell viability assay of CEM-R cell line treated for 48Â h with increasing concentrations of nelarabine alone or combined with the pan PI3K p110 inhibitor ZSTK-474. One representative of two different experiments is shown.

Bcl2 expression in T-ALL cell lines. Western blotting analyses for the basal expression of Bcl2 in untreated T-ALL cell lines. Twenty micrograms of protein was blotted to each lane. Antibody to β-actin served as a loading control. Molecular weights are indicated on the right. Densitometric analysis was performed using a Chemidoc 810 Imager with the appropriate software (UVP, Upland, CA, USA), and for each cell line, Bcl2 protein expression is indicated relatively to β-actin expression.

DNA damage and ROS production. A. Flow cytometric analysis of γH2AX in MOLT-4 and Jurkat sensitive to nelarabine cells (upper panel). Percentages of γH2AX are shown in the table (lower panel). B. Reactive oxygen species (ROS) production was assessed in MOLT-4 and Jurkat sensitive T-ALL cell lines after 1 and 24 h treatment with nelarabine. Fluorescence values (DCF) were reported as the percentage of intracellular ROS in respect to controls. ROS increased significantly in both 1- and 24-h treatment compared to controls in MOLT-4 and Jurkat cells. Statistical analyses were performed with the Dunnett’s multiple comparison test.

HS-5 stromal cells protect MOLT-4 cells from nelarabine cytotoxicity at least in part through CXCL12/CXCR4 interactions. A. MTT assays of MOLT-4 cells growing alone or in co-culture system with HS-5 cells and treated with nelarabine (2 μM for 48 h) in a Transwell@ system. Three hundred seventy-five thousand MOLT-4 cells were seeded in the upper chamber of the Transwell@ system. Results are the mean of three different experiments ± SD. B. Western blot analysis for p-ERK, p-CXCR4, and their total forms in MOLT-4 cells grown either in the absence or the presence of HS-5 stromal cells in a Transwell@ system. Nelarabine treatment (2 μM) was for 24 h. Fifty micrograms of protein was blotted to each lane. Antibody to β-actin served as a loading control. Molecular weights are indicated on the right. One representative of two different experiments is shown.

Class I phosphatidylinositol 3-kinases (PI3Ks) are heterodimeric lipid kinases consisting of a regulatory subunit and one of four catalytic subunits (p110α, p110β, p110γ or p110δ). p110γ/p110δ PI3Ks are highly enriched in leukocytes. In general, PI3Ks regulate a variety of cellular processes including cell proliferation, survival and metabolism, by generating the second messenger phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3). Their activity is tightly regulated by the phosphatase and tensin homolog (PTEN) lipid phosphatase. PI3Ks are widely implicated in human cancers, and in particular are upregulated in T-cell acute lymphoblastic leukemia (T-ALL), mainly due to loss of PTEN function. These observations lend compelling weight to the application of PI3K inhibitors in the therapy of T-ALL. At present different compounds which target single or multiple PI3K isoforms have entered clinical trials. In the present research, it has been analyzed the therapeutic potential of the pan-PI3K inhibitor BKM120, an orally bioavailable 2,6-dimorpholino pyrimidine derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. BKM120 treatment resulted in cell cycle arrest and apoptosis, being cytotoxic to a panel of T-ALL cell lines and patient T-lymphoblasts. Remarkably, BKM120 synergized with chemotherapeutic agents currently used for treating T-ALL patients. BKM120 efficacy was confirmed in in vivo studies to a subcutaneous xenotransplant model of human T-ALL. Because it is still unclear which agents among isoform-specific or pan inhibitors can achieve the greater efficacy, further analyses have been conducted to investigate the effects of PI3K inhibition, in order to elucidate the mechanisms responsible for the proliferative impairment of T-ALL. Overall, these results indicated that BKM120 may be an efficient treatment for T-ALLs that have aberrant up-regulation of the PI3K signaling pathway and strongly support clinical application of pan-class I PI3K rather than single-isoform inhibitors in T-ALL treatment.

Enrichment analysis report: enrichment by process networks. (PDF 35 kb)

Basal expression of Chk1 and Chk2 (mRNA level) in the cell lines treated, the results are expressed as 2exp(-ΔΔCt) (A). Schematic representation of the statistically significance of the ANOVA multiple comparison test in which the basal expression of each cell lines is compared to the value of the basal expression of all the other cell lines (B). Figure S2. Oncomine expression analysis of Chk1 (A) and Chk2 (B) levels between normal monunuclear cells (MNCs) from bone marrow and different subtypes of leukemia (AML, B-ALL, T-ALL). Figure S3. Apoptosis following treatment with PF-00477736 at 24 and 48 hours in B-ALL and T-ALL cells(A). Cell cycle analysis of RPMI-8402, BV-173, SUP-B15 and NALM-6 cell lines treated for 24 hours with increasing concentrations of PF-00477736 (B). Figure S4. Western Blot analysis in all leukemia cell lines after exposure to PF-00477736 (+) at the concentration closest the IC50 or DMSO 0.1 %(-) (A). Western Blot analysis of BV-173 and NALM-6 cell lines treated with or without PF-00477736 (IC50) for 3, 6 and 9 hours (B). Figure S5. Cell cycle_ESR1 regulation of G1/S transition: the top scored map (map with the lowest p-value) based on the enrichment distribution sorted by 'Statistically significant Maps' set. Figure S6. Apoptosis and survival_Granzyme A signaling: the second scored map (map with the second lowest p-value) based on the enrichment distribution sorted by 'Statistically significant Maps' set. Figure S7. DNA damage_ATM/ATR regulation of G1/S checkpoint: the third scored map (map with the third lowest p-value) based on the enrichment distribution sorted by 'Statistically significant Maps' set. Figure S8. Protein expression level of c-Jun in B-/T-ALL cell lines (A). Western Blot analysis of normal MNCs and ALL MNCs after exposure to PF-0047773(B). mRNA expression of CDK4, Chk2, GADD45a and PLK3 in B-/T-ALL cell lines treated with or without PF-00477736(IC50 value) for 24 hours. The results are expressed as 2exp(-ΔΔCt)(C). (PPTX 2905 kb)

Table S1. List of differentially expressed genes between treated cells and their untreated counterpart with a false discovery rate less than 0.05. Table S2. Main clinical and molecular characteristics of ALL patients whose primary ALL samples have been tested in vitro. Table S3. TP53 primer sequences. Table S4. Antibody reactivity and sources as well as the antigen retrieval protocols, dilutions, and revelation systems used in immunohistochemistry. (DOCX 28 kb)

Treatment resulted in a differential expression of 941 genes ( pâ

Enrichment analysis report: enrichment by GO processes. (PDF 41 kb)

Introduction. Sphingosine 1-phosphate (S1P) is involved in many processes such as cell survival, growth, migration, and cancer. S1P is formed by the phosphorylation of sphingosine by sphingosine kinase 1 (SK1) or sphingosine kinase 2 (SK2). While the role of SK1 in cancer is well established, the role of SK2 in regulating apoptosis is still a matter of debate, even though emerging evidence has highlighted the importance of SK2 in cancer. Therefore, SKs represent a promising target for cancer therapy. Recently, the importance of S1P in hematological malignancies has been described. Here, we analyze the therapeutic effects of 2-(p-Hydroxyanilino)-4-(p-chlorophenyl)thiazole (SKi), an SK1/2 inhibitor, and (R)-FTY720 methyl ether (ROMe), a SK2-selective inhibitor, in T-ALL cell lines. Methods. T- ALL cell lines (Molt-4, Jurkat, CEM-R) were treated with SKi and ROMe for 40 h, then MTT assays were performed to analyze cell viability. Apoptosis induction was evaluated by Annexin V/PI staining. Protein expression was studied by western blot (WB). Results. SKi and ROMe induced a decrease in cell viability, as demonstrated by MTT assays. Annexin-V/PI staining and flow cytometric analysis of cells treated with a SKi concentration equivalent to the IC50 documented the occurrence of apoptotic cell death. This was confirmed by WB analysis for caspase and PARP cleavage in all the cell lines. However, we also observed a SKi-induced autophagy by means of WB and transmission electron microscopy analysis in Jurkat and CEM-R cells. We then analyzed the expression of ER stress/UPR hallmarks. SKi activated the ER stress/UPR pathway and this occurred following apoptosis and correlated with autophagy. The combination of SKi and chloroquine (an autophagy inhibitor) induced a decrease in cell viability, indicating that the UPR/autophagic response is likely to be a protective mechanism. In Molt-4 cells, we detected an increase in SK1 expression after 40 h of treatment with SKi, that could represent an attempt of cells to escape SKi-induced apoptosis, as high SK1 expression is known to enhance cell growth and survival. Notably, SKi synergized with vincristine at concentrations of SKi that were much below its respective IC50, suggesting that vincristine sensitized T-ALL cells to SKi and that combining chemotherapeutic agents with SK inhibitors could be feasible in the treatment of T-ALL. Finally, ROMe treatment induced an autophagic type of cell death. Conclusions. We report herein that SKi and ROMe affect T-ALL cell viability, but they exert their effects through different mechanisms. While ROMe induces an autophagic cell death, SKi induces apoptosis. Moreover, for the first time, we demonstrated that SKi activates an ER stress/UPR pathway in T-ALL cells and this is linked with a protective autophagic response. Thus, our findings indicate that SK1 or SK2 may represent potential targets for treating T-ALL, thereby enabling better management of this cancer.