A COMBINATION OF BORTEZOMIB WITH CX-4945, A CASEIN KINASE 2 (CK2) INHIBITOR, HAS SYNERGISTIC CYTOTOXIC EFFECTS IN ACUTE LYMPHOBLASTIC LEUKEMIA (ALL) CELL LINES

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

Therapeutic targeting of Polo-like kinase-1 and Aurora kinases in T-cell acute lymphoblastic leukemia

Polo-like kinases (PLKs) and Aurora kinases (AKs) act as key cell cycle regulators in healthy human cells. In cancer, these protein kinases are often overexpressed and dysregulated, thus contributing to uncontrolled cell proliferation and growth. T-cell acute lymphoblastic leukemia (T-ALL) is a heterogeneous malignancy arising in the thymus from T-cell progenitors. Primary chemoresistant and relapsed T-ALL patients have yet a poor outcome, therefore novel therapies, targeting signaling pathways important for leukemic cell proliferation, are required. Here, we demonstrate the potential therapeutic effects of BI6727, MK-5108, and GSK1070916, three selective inhibitors of PLK1, AK-A, and AK-B/C, respectively, in a panel of T-ALL cell lines and primary cells from T-ALL patients. The drugs were both cytostatic and cytotoxic to T-ALL cells by inducing G2/M-phase arrest and apoptosis. The drugs retained part of their pro-apoptotic activity in the presence of MS-5 bone marrow stromal cells. Moreover, we document for the first time that BI6727 perturbed both the PI3K/Akt/mTORC2 and the MEK/ERK/mTORC1 signaling pathways, and that a combination of BI6727 with specific inhibitors of the aforementioned pathways (MK-2206, CCI-779) displayed significantly synergistic cytotoxic effects. Taken together, our findings indicate that PLK1 and AK inhibitors display the potential for being employed in innovative therapeutic strategies for improving T-ALL patient outcome. © 2014 Landes Bioscience

ASSESSMENT OF THE EFFECT OF SPHINGOSINE KINASE INHIBITORS ON APOPTOSIS, UNFOLDED PROTEIN RESPONSE AND AUTOPHAGY OF T-ACUTE LYMPHOBLASTIC LEUKEMIA CELLS: INDICATIONS FOR NOVEL THERAPEUTICS

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

Long-term health-related quality of life and psychological adjustment in children after haemolytic-uraemic syndrome

BACKGROUND: In children after haemolytic-uraemic syndrome (HUS), little is known about long-term health-related quality of life (HRQoL) and psychological adjustment as defined by behavioural problems, depressive symptoms and post-traumatic stress symptoms. METHODS: Sixty-two paediatric patients with a history of HUS were included in this study. Medical data of the acute HUS episode were retrieved retrospectively from hospital records. Data on the clinical course at study investigation were assessed by clinical examination and laboratory evaluation. HRQoL and psychological adjustment data were measured by standardised, parent- and self-reported questionnaires. RESULTS: Haemolytic-uraemic syndrome was diagnosed at a mean of 6.5 years before the initiation of the study (standard deviation 2.9, range 0.1-15.7) years. Among the preschool children, parents reported that their child was less lively and energetic (HRQoL emotional dimension), while no increased behavioural problems were reported. In the school-age children, self- and proxy-reported HRQoL was well within or even above the norms, while increased total behavioural problems were found. The school-age children reported no increased depression scores. Also none of the children met the criteria for full or partial HUS-associated posttraumatic stress disorder. CONCLUSIONS: Healthcare providers should be particularly alert to behavioural problems in school-age children with a history of HUS and to lower HRQoL in preschool children

Hemolytic-uremic syndrome in Switzerland: a nationwide surveillance 1997-2003

Hemolytic-uremic syndrome (HUS) is a leading cause of acute renal failure in childhood. In its typical presentation, it is preceded by an episode of diarrhea mostly due to Shiga-toxin-producing Escherichia coli. There is important geographical variation of many aspects of this syndrome. Nationwide data on childhood HUS in Switzerland have not been available so far. In a prospective national study through the Swiss Pediatric Surveillance Unit 114 cases (median age 21 months, 50% boys) were reported between April 1997 and March 2003 by 38 pediatric units (annual incidence 1.42 per 10(5) children < or =16 years). Shiga-toxin-producing E. coli were isolated in 32 (60%) of tested stool samples, serotype O157:H7 in eight. Sixteen children presented with only minimal renal involvement, including three with underlying urinary tract infection. Six patients presented with atypical hemolytic-uremic syndrome, and six with HUS due to invasive Streptococcus pneumoniae infection. Mortality was 5.3%, including two out of six children with S. pneumoniae infection. The severity of thrombocytopenia and the presence of central nervous system involvement significantly correlated with mortality. In conclusion, childhood HUS is not rare in Switzerland. Contrasting other countries, E. coli O157:H7 play only a minor role in the etiology. Incomplete manifestation is not uncommon