BackgroundTargeted therapy approaches have been successfully introduced into the treatment of several cancers. The multikinase inhibitor Sorafenib has antitumor activity in solid tumors and its effects on acute lymphoblastic leukemia (ALL) cells are still unclear.MethodsALL cell lines (SEM, RS4;11 and Jurkat) were treated with Sorafenib alone or in combination with cytarabine, doxorubicin or RAD001. Cell count, apoptosis and necrosis rates, cell cycle distribution, protein phosphorylation and metabolic activity were determined.ResultsSorafenib inhibited the proliferation of ALL cells by cell cycle arrest accompanied by down-regulation of CyclinD3 and CDK4. Furthermore, Sorafenib initiated apoptosis by cleavage of caspases 3, 7 and PARP. Apoptosis and necrosis rates increased significantly with most pronounced effects after 96 h. Antiproliferative effects of Sorafenib were associated with a decreased phosphorylation of Akt (Ser473 and Thr308), FoxO3A (Thr32) and 4EBP-1 (Ser65 and Thr70) as early as 0.5 h after treatment. Synergistic effects were seen when Sorafenib was combined with other cytotoxic drugs or a mTOR inhibitor emphasizing the Sorafenib effect.ConclusionSorafenib displays significant antileukemic activity in vitro by inducing cell cycle arrest and apoptosis. Furthermore, it influences PI3K/Akt/mTOR signaling in ALL cells.

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Several studies have shown that oxidative stress induces apoptosis in many cellular systems including pancreatic acinar cells. However, the exact molecular mechanisms leading to apoptosis remain partially understood. This study aimed to investigate the role of the cytosolic cysteine protease calpain in H2O2-induced apoptosis in pancreatic AR42J cells. Apoptosis was evaluated using flow cytometric analysis of sub-G1 DNA populations, electron-microscopic analysis, caspase-3-specific αII-spectrin breakdown, and measuring the proteolytic activities of the initiator caspase-12 and caspase-8, and the executioner caspase-3. H2O2 induced an increase in the calpain proteolytic activity immediately after starting the experiments that tended to return to a nearly normal level after 8 h and could be attributed to m-calpain. Whereas no caspase-12, caspase-8 and caspase-3 activations could be detected within the first 0.5 h, significantly increased proteolytic activities were observed after 8 h compared with the control. At the same time, the cells showed first ultrastructural hallmarks of apoptosis and a decreased viability. In addition, αII-spectrin fragmentation was identified using immunoblotting that could be attributed to both calpain and caspase-3. Calpain inhibition reduced the activities of caspase-12, caspase-8, and caspase-3 leading to a decrease in the number of apoptotic cells. Immunoblotting analyses of caspase-12 and caspase-8 indicate that calpain may be involved in the activation process of both proteases. The results suggest that H2O2-induced apoptosis of AR42J cells requires activation of m-calpain initiating the endoplasmic reticulum stress-induced caspase-12 pathway and a caspase-8-dependent pathway. The findings also suggest that calpain may be involved in the execution phase of apoptosis.

Background: The phosphatidylinositol 3 Kinase (PI3K)/Akt signalling pathway is involved in the regulation of proliferation, apoptosis and angiogenesis. Dysregulation of PI3K/Akt has been described for several solid and hematological tumors. Its role in acute lymphoblastic leukemogenesis (ALL) is unkown. Here, we investigated whether the inhibition of PI3K/Akt kinases influences apoptosis, necrosis and cell proliferation. Patients and Methods: ALL and high grade NHL cell lines with different cytogenetics and phenotypes were used (SEM, RS4;11, REH, Jurkat, DOGKIT). Phosphorylationstatus of Akt (Ser473, Thr308) and FOXO3A (Thr32) were determined by western blot (WB). Cells were incubated for 96h with LY29004 (5μM, 12.5μM, 25μM; PI3K inhibitor) or sorafenib (0.73μM, 7.3μM). In order to detect synergistic effects with other cytotoxic drugs cells were treated with combinations of sorafenib and doxorubicine, cytosin-arabinoside, and mTOR inhibitor (RAD001), respecitvely. Cell number, apoptosis, necrosis, protein phosphorylation and metabolic activity were determined at 4h, 24h, 48h, 72h, and 96h by microscopy, flow cytometry, WB and WST-1 testing. Whole genome Affymetrix gene expression arrays (U133 Plus 2.0) were performed in order to detect differential gene expression compared to controls treated with DMSO (dimethylsulfoxid) only. Results: In all ALL cell lines phosphorylated Akt (pAKT) was detected. Levels of pAkt and pFOXO3A differed between cell lines significantly, with Jurkat and SEM demonstrating high activation levels. PI3K inhibition by LY29004 led to decreased proliferation in SEM cells with only slight increases in apoptosis and moderate increase in necrosis rates (20%). Sorafenib inhibited the proliferation of SEM, Jurkat and RS4;11 significantly, with most pronounced effects at 96h. Maximal apoptosis and necrosis rates increased and ranged from 11 to 39% and 53 to 84%, respectively. Metabolic activity decreased significantly already after 24h. As early as 0.5h after treatment complete disappearance (SEM, RS4;11) or marked decrease (Jurkat) in levels of pAkt and pFOXO occurred. Combination of sorafenib with conventional cytotoxic drugs failed to demonstrate synergistic effects. Sorafenib treatment induced differential gene expression in several genes e.g. genes involved in apoptosis such as BIMBAM, caspases and CDKs. Conclusions: Inhibition of the PI3K/Akt pathway seems to be a potential therapeutic target in ALL and high grade lymphoma cells. Sorafenib as a multikinase inhibitor approved for clincial application in solid tumors displayes significant antileukemic activity in vitro and might be a potential drug for a targed therapy approach in ALL.

4115 Introduction Inhibition of specific signal transduction pathways in neoplastic cells has been successfully used to regulate aberrantly activated cellular processes such as proliferation. Specific target agents such as tyrosine kinase inhibitors have been successfully introduced into the treatment of several cancers. The novel dual PI3K and mTOR inhibitor, NVP-BEZ235, has shown antitumor activity in vitro and is being investigated in phase I clinical trials in solid tumors. It is currently unclear whether PI3K/Akt and mTOR inhibition might also be usefull in acute leukemia cells, and if so, what would be the best modalities for combination approaches. Here, we examined the antileukemic activity of NVP-BEZ234 in acute lymphoblastic (ALL) and myeloid leukemia (AML) cells. Methods ALL (SEM, RS4;11, Jurkat and MOLT-4) and AML (MV4;11, MONO-MAC-1, HL-60 and NB-4) cell lines with different cytogenetics and PI3K/Akt activity were investigated. Cells were treated for 96h with NVP-BEZ435 (1 nM, 10 nM and 100 nM) alone or in combination with cytarabine, doxorubicin or dexamethasone. Cytotoxic drugs were added simultaneously, 24h before or 24h after treatment with NVP-BEZ235. Cell count, apoptosis rates, necrosis rates, cell cycle distrubution, protein phosphorylation and metabolic activity were determined at 0.5h, 4h, 24h, 48h, 72h, and 96h by microscopy, flow cytometry, western blot and WST-1 testing, respectively. Results NVP-BEZ235 potently inhibited the proliferation of SEM, Jurkat, RS4;11, MV4;11, MONO-MAC-1 and NB-4 significantly. Most pronounced effects were seen after 72h. In HL-60, which has a low PI3K/Akt activation, inhibition of proliferation to a lesser degree was observed. Inhibition of PI3K and mTOR activity and G1/G0 arrest was detected at 10 nM and 100 nM in B- and T-ALL cell lines, respectively. Metabolic activity decreased significantly as early as 48h after exposure. Changes in apoptosis and necrosis rates were not observed in B-ALL and AML cells. However, in Jurkat and MOLT-4 (both T-ALL) highest doses of NVP-BEZ seemed to slightly increase. In a time and concentration dependent manner the phosphorylation status of Akt (Ser473 and Thr308), FoxO3A (Thr32) and p70S6 kinase (Thr389) decreased early (0.5-4h) after treatment. Of interest, subsequently an increase of phosphorylation of the same proteins was observed (24-48h), followed by another dephosphorylation episode. Concomittant treatment with NVP-BEZ235 and cytarabine, doxorubicin or dexamethasone enhanced the cytotoxicity in B-cells compared to single drug treatment. The most significant effects on proliferation inhibition and metabolic activity were observed for the simultaneous exposure to both drugs or pretreatment of cells with NVP-BEZ235 followed by dexamethasone, doxorubicin and cytarabine addition after 24h. No additive effect was observed when cytostatic drugs were added before inhibition of PI3K and mTOR by NVP-BEZ235 was induced. Conclusions NVP-BEZ235 reduces metabolic activity and induces cell cycle arrest in acute lymphoblastic and myeloid leukemia cells. Treatment of ALL cells with NVP-BEZ combined with glucocorticoids or conventional chemotherapy drugs enhances the cytotoxicity of the dual PI3K and mTOR inhibitor when NVP-BEZ235 is given upfront or simultaneously. In contrast, pretreatment with conventional chemotherapy drugs followed by NVP-BEZ235 exposure does not induce additive cytotoxicity. Our data indicate that NVP-BEZ might be a usefull drug in ALL as well AML treatment. Furthermore, simultaneous treatment with chemotherapy seems more advisable compared to NVP-BEZ interruption. Disclosures: No relevant conflicts of interest to declare.