Bcl-2 and mTOR as Effective Targets for Molecular Therapy of Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3228-3228
Author(s):  
Stefano Iacovelli ◽  
Maria Rosaria Ricciardi ◽  
Andrea Miele ◽  
Paola Bergamo ◽  
Roberto Licchetta ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Growth Inhibition ◽  
Cell Line ◽  
Cell Lines ◽  
Synergistic Effects ◽  
Lymphoblastic Leukemia ◽  
Mtor Inhibitors ◽  
Primary Cells ◽  
Apoptosis Induction ◽  
Combined Use

Abstract Abstract 3228 Although they frequently achieve complete remission (CR), adult patients with acute lymphoblastic leukemia (ALL) subsequently experience leukemia relapse, which represents an unresolved therapeutic problem. Based on the observation that ALL cells are frequently characterized by the deregulation of the apoptotic machinery, we and others have evaluated pre-clinically the activity of ABT-737 (kindly provided by Abbott Laboratories), a BH3-mimetic Bcl-2/Bcl-XL inhibitor, displaying a potent growth-inhibitory activity in ALL cell lines and primary cells. However ABT-737 binds to the anti-apoptotic protein Mcl-1 with low affinity and Mcl-1 expression may mediate resistance to ABT-737. Since ALL is also characterized by the aberrant activation of the mTOR and related signalling pathways, in the present study we further evaluated the combined Bcl-2/Bcl-XL (by ABT-737) and mTOR (by CCI-779) inhibition focusing, in particular, on the activity of combined molecularly targeted therapies on resistant cells. In MOLT-4 cells, ABT-737 induced dose and time-dependent growth inhibition (IC-50= 198nM) followed, at higher concentrations (250-500nM), by apoptosis induction. In contrast, the CEM-S, CEM-R, JURKAT, DAUDI and RAJI cells proved resistant (IC-50 >5 μM). When we explored the effects of CCI-779 on the aforementioned cell lines, only minor cytostatic effects were observed (IC-50 0.5–28.2μM). MOLT-4 cells, for example, showed a flat dose-response curve (35-55% growth inhibition) at concentrations ranging between 1 and 5000 nM (IC50=9,87μM) and apoptosis induction was not seen until 5000 nM. We next investigated the effects of the combined use of ABT-737 and CCI-779 (each at 1000nM) in the ABT-resistant JURKAT cells. A significant (p= 0.04) induction of apoptosis was observed with the combination, as compared with single agents, after 24 h (47.7% ±5.9 of cells with sub-G1 DNA content with ABT-737 + CCI-779, compared to 17.4% ±1.5 and 4.2% ±1.5 with ABT-737 and CCI-779 as single agents, respectively). Similarly, when we exposed CEM-R cells to the drug combination (ABT-737 1000nM and CCI-779 5000nM) for 24 h, a strikingly stronger apoptosis induction (sub-G1 peak= 75.3% ±16.8) was observed, compared to single agents (15.8% ±7.2 and 4.2% ±1.9 with ABT-737 and CCI-779 alone, respectively) (p=0.0003). These effects were confirmed by measuring Annexin V binding. WB analysis showed decreased Mcl-1 levels, following exposure to CCI-779 and further downregulation in response to combined ABT-737+CCI-779 in the CEM-R cell line. These effects, however, were not seen in the parental CEM-S cell line. Primary cells, obtained from 10 ALL patients, showed an increase of the sub-G1 peak in 7/10 and in 4/10 samples, after exposure to ABT-737 (50nM) and CCI-779 (5000 nM), while synergistic effects on apoptosis induction were observed in 4/10 samples after exposure to the combination. In summary, we observed that the combined use of Bcl-2/Bcl-XL and mTOR inhibitors may exert synergistic cytotoxic effects in some resistant ALL models and this effect is associated with CCI-779-induced Mcl-1 down-regulation. Synergistic effects between these inhibitors were also found in a proportion of primary ALL samples, thus supporting further studies of combined Bcl-2/Bcl-XL and mTOR inhibitors, to overcome ALL resistance. Disclosures: Petrucci: Celgene: Honoraria; Janssen Cilag: Honoraria. Tafuri:Sigma-Tau: Research Funding.

Can Bortezomib Treatment Overcome Glucocorticoid Resistance of Childhood Acute Lymphoblastic Leukemia Cells?.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2801-2801
Author(s):  
Stefanie V. Junk ◽  
Melchior Lauten ◽  
Gunnar Cario ◽  
Nicole Wittner ◽  
Martin Schrappe ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
Proteasome Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Childhood Acute Lymphoblastic Leukemia ◽  
Strong Increase ◽  
Rna Expression ◽  
Bortezomib Treatment ◽  
Multiagent Chemotherapy

Abstract The response to initial glucocorticoid (gc) therapy in childhood acute lymphoblastic leukemia (ALL) reliably predicts the response to multiagent chemotherapy. In a recent study, we identified the valosin-containing protein (VCP) as a part of the ubiquitin proteasome degradation pathway (UPDP) as one of the proteins overexpressed in prednisone poor responder (PPR) patients. Therefore, we investigated whether treatment of ALL cell lines with the proteasome inhibitor bortezomib acted synergistically with glucocorticoid treatment. Human B-cell precursor leukemic cell lines MHH cALL 2 (PPR) and MHH cALL 3 (PGR) were treated with prednisone(6.3μM) as baseline and also with different concentrations of the proteasome inhibitor bortezomib for 96hours (h). To study drug effects, cells were sampled every 24h for immunofluorescence (IF) staining, protein and RNA extraction, viability (Trypan blue, WST-1) and apoptosis assays. Western blot analyses using an anti-p97 antibody were performed on whole cell lysates (wcl) and fractions and separated by differential detergent fractionation. VCP RNA expression was analyzed by real-time PCR. Single bortezomib treatment with 3nM or higher concentrations led to a significant decline in vitality of both cell lines. Within 24h, the PPR cell line lost about half and the PGR about one-fourth of their vitality. In combination with prednisone, 1.5nM bortezomib reduced the vitality by about 50% within 96h for both cell lines. Combining both drugs decreased the vitality rate by about 10% in the PPR cell line, whereas the PGR cells showed no decrease compared to single gc treatment. In FACS analyses, stages of different quantities of apoptosis were detected in PPR and PGR cells. PPR cells treated with both drugs showed a strong increase of necrotic cells at 24h. PGR cells started with an accession of apoptotic cells and initially had no necrotic cells, but started to rise from 48h on. We hence propose that the PPR cells react more quickly to the combined therapy. Under single gc treatment, VCP RNA expression increased in the PPR cells to a maximum of about 1.8- and in PGR cells to 1.5-fold. In PGR cells treated only with 1.5nM or 3nM bortezomib, VCP RNA rose to 1.4- and 2-fold respectively. Drug combination led to a 1.4-fold increase of VCP RNA in PPR compared to untreated cells, whereas RNA was reduced compared to single gc-treated cells. Protein levels of VCP in PPR cells remained high during drug treatment. VCP increased to a maximum of 1.6-fold in the cytosol of PGR cells, using bortezomib only. In the combination experiments, the amount doubled within 48h and thence decreased to initial levels. Single gc treatment caused a VCP increase to 1.5-fold within 24h. In the wcl, we found the VCP levels for the PGR cells converted to the cytosolic patterns. The results of IF staining supported the different VCP concentrations and exposed formation of aggresome-like complexes in the PPR cell line. The results of this study suggest that the multiagent chemotherapy resistance is indicated by differentially expressed VCP and related to the deregulation of the UPDP. Using inhibitors appears to chemisensitize the PPR for gc treatment. Therefore, drug targeting the proteasome, as in other hematological cancer therapies, might improve the overall therapy outcome.

Dual Inhibition of Phosphatidylinositol 3-Kinase and Mammalian Target of Rapamycin with NVP-BEZ235 as a New Therapeutic Option for T-Cell Acute Lymphoblastic Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2025-2025
Author(s):  
Francesca Chiarini ◽  
Cecilia Grimaldi ◽  
Francesca Ricci ◽  
Pierluigi Tazzari ◽  
Camilla Evangelisti ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Mtor Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Mammalian Target Of Rapamycin ◽  
Mtor Inhibitors ◽  
Jurkat Cells ◽  
Phosphatidylinositol 3 Kinase ◽  
Pathway Activation ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 2025 Poster Board II-2 Introduction: Recent findings have highlighted that constitutively active phosphatidylinositol 3-kinase (PI3K)/Akt/mammalian Target of Rapamycin (mTOR) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL) where it strongly influences cell proliferation and survival. Pathway activation could be due to several reasons which include Notch1 activation leading to HES1-mediated transcriptional suppression of PTEN gene, PTEN phosphorylation or oxidation, and inactivation of SHIP1 phosphatase. These findings lend compelling weight for the application of PI3K/Akt/mTOR inhibitors in T-ALL. Rapamycin and its analogues have shown some promising effects in pre-clinical models of T-ALL. However, mTOR inhibitors are mainly cytostatic and could hyperactivate Akt due to the existence of feedback loops between mTOR, p70 S6 kinase, PI3K, and Akt. Recently, dual PI3K/mTOR inhibitors have been synthesized. Here, we have analyzed the therapeutic potential of the novel, dual PI3K/mTOR inhibitor, NVP-BEZ235, an orally bioavailable imidazoquinoline derivative, which has entered clinical trials for solid tumors, on both T-ALL cell lines and patient samples. Methods and Patients: We employed a panel of cell lines with up-regulated PI3K/Akt/mTOR signaling, including CEM-R cells [which overexpress high levels of the membrane transporter, 170-kDa P-glycoprotein (P-gp)], MOLT-4 and CEM-S cells (which lack PTEN expression), Jurkat cells (which do not express both PTEN and SHIP1), and RPMI-8402 and BE-13 cells. MOLT-4, CEM, and Jurkat cells have a non-functional p53 pathway. Moreover, both Jurkat and MOLT-4 cells have aberrant Notch1 signaling. Patients samples displayed pathway activation as documented by increased levels of p-Akt, p-4E-BP1, and p-S6 ribosomal protein, as well as low/absent PTEN expression. Results: NVP-BEZ235 was cytotoxic to the panel of cell lines as documented by MTT assays. NVP-BEZ235 IC50 ranged from 80 to 280 nM at 24 h. A comparison between NVP-BEZ235 and the dual PI3K/mTOR inhibitor PI-103, a small synthetic molecule of the pyridofuropyrimidine class with the same targets, demonstrated that NVP-BEZ235 was more effective than PI-103 when employed at equimolar concentrations. NVP-BEZ235 did not significantly affect the proliferation of peripheral blood T-lymphocytes from healthy donors stimulated with phytohemagglutinin and interleukin-2, whereas it blocked leukemic cells in the G1 phase of the cell cycle, and this was accompanied by decreased levels of phosphorylated Retinoblastoma protein. NVP-BEZ235 treatment also resulted in apoptotic cell death (about 20-30% at 6 h of exposure, when employed at 200 nM), as documented by Annexin V/propidium iodide staining and cytofluorimetric analysis. Moreover, NVP-BEZ235 activated caspase-8 and caspase-3, as demonstrated by western blot. Western blot documented a dose- and time-dependent dephosphorylation of Akt and its downstream target, GSK-3β, in response to NVP-BEZ235. mTOR downstream targets were also efficiently dephosphorylated, including p70S6 kinase, S6 ribosomal protein, and 4E-BP1. Remarkably, NVP-BEZ235 targeted the side population (SP, identified by Hoechst 33342 staining and ABCG2 expression) of T-ALL cell lines, which might correspond to leukemia initiating cells, and synergized with several chemotherapeutic agents (dexamethasone, vincristine, cyclophosphamide, Ara-C) currently employed for treating T-ALL patients. NVP-BEZ235 reduced chemoresistance to vincristine induced in Jurkat cells by co-culturing with MS-5 stromal cells which mimic the bone marrow microenvironment. NVP-BEZ235 was cytotoxic (IC50: 10-15 nM at 96 h) to primary lymphoblasts from patients with T-ALL, where the drug dephosphorylated 4E-BP1, at variance with rapamycin. Of note, NVP-BEZ235 targeted the SP also in T-ALL patient samples. Conclusions: NVP-BEZ235 was cytotoxic to T-ALL cell lines and patient lymphoblasts (including SP cells) at concentrations that have been previously reported to be achievable in vivo. Taken together, our findings indicate that longitudinal inhibition at two nodes of the PI3K/Akt/mTOR network with NVP-BEZ235, either alone or in combination with other drugs, may serve as an efficient treatment towards T-ALL cells (including those overexpressing P-gp and independently from p53 status) which require upregulation of this signaling pathway for their survival and growth. Disclosures: No relevant conflicts of interest to declare.

Vincristine and Prednisolone Combination Reduces MDR1 and Microenvironment-Mediated Treatment Resistance In Acute Lymphoblastic Leukemia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2529-2529
Author(s):  
Nan Jiang ◽  
Zhenhua Li ◽  
Grace Shimin Koh ◽  
Yi Lu ◽  
Shirley K.Y. Kham ◽  
...  
Keyword(s):  
Treatment Outcome ◽  
Acute Lymphoblastic Leukemia ◽  
Treatment Response ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Treatment Resistance ◽  
Intrathecal Methotrexate ◽  
The Cost

Abstract Acute lymphoblastic leukemia (ALL) is the most common form of childhood cancer with excellent treatment outcome where >80% are cured. However, relapse and therapy-related toxicities limit further improvements and greatly increase the cost of therapy. Vincristine (VCR) is cheap, well tolerated, and highly effective. Using VCR optimally will help improve the cost-benefit ratio favorably by allowing us to reduce toxicities like infections from myelosuppression and yet improving cure. The highly successful BFM-ALL treatment backbone starts with a single intrathecal methotrexate on Day 1 followed by 7 days of oral prednisolone (PRED). The persistence of absolute blasts count >1,000/µL at Day 8 (D8), known as PRED poor response, confers a significantly poorer treatment outcome. To avoid seeding the CNS with leukemia from traumatic taps, the new Ma-Spore ALL 2010 treatment protocol, omitted intrathecal methotrexate at Day 1 and replaced with VCR at Day 0. By June 2013, a total of 133 patients have been enrolled. We found that the number of poor PRED responders was halved from the historical 9.5% in the previous Ma-Spore ALL 2003 study (Yeoh et al. J Clin Oncol 2013) to only 4.7% of patients in the ALL 2010 study. In addition, the percentage of MRD standard risk patients (Day 33 blast count ≤1x10-4) increased from 38.9% in the Ma-Spore ALL 2003 to 51.8% in the Ma-Spore ALL 2010 study (P<0.001). The 2-year event-free survival (EFS) for good and poor D8 response patients under the Ma-Spore ALL 2010 trial remained similar to the ALL 2003 study despite only half the number of PRED poor responders (Fig. 1). These data taken together suggests that VCR and PRED combination is highly synergistic and can improve early treatment response. We investigated VCR and PRED combination in PRED and VCR-resistant (VCR-R) cell lines. Specifically, REH cell line is intrinsically resistant to PRED in vitro because of a mutation in its glucocorticoid receptor. We exposed the REH cell line to increasing concentrations of VCR over 6 months and generated a VCR resistant REH cell line (Fig. 2). This VCR-R REH cell line is resistant to both PRED or VCR when exposed individually in vitro. However when exposed to both PRED and VCR in combination, only 30% of the resistant cells survived (P<0.01). We found that the drug efflux transporter multi-drug resistance protein 1 (MDR1) was preferentially highly expressed in our VCR-R cell line models. To determine if the highly expressed MDR1 is responsible for treatment resistance, we exposed the VCR-R cell lines to VCR, verapamil (an MDR1 inhibitor) and combination of both VCR and verapamil. The combination of VCR and verapamil increased the G2 cell cycle arrest by 3- folds compared to when VCR was used alone (Fig. 3), supporting the role of MDR1 in treatment resistance. Interestingly we also found that the combination of VCR and PRED led to a decrease in levels of MDR1 expression by western blot, suggesting that depletion of MDR1 may be a mechanism through which VCR and PRED combination therapy enhances leukemic cell killing. We also investigated microenvironment-mediated resistance to VCR and PRED using mesenchymal stromal cells (MSC) co-culture systems. It was found that after co-culture with MSC or in conditional medium containing soluble factors secreted by MSC, leukemic cells were resistant to VCR and PRED mono-treatment, but the resistance was abrogated after combinatorial therapy. In conclusion, VCR in combination with PRED improves D8 peripheral blood treatment response during early induction in our Ma-Spore 2010 trial. This synergistic combination results from its ability to reverse resistance from intrinsic overexpression of MDR1 in resistant leukemia cells and decrease microenvironment-contributed resistance by mesenchymal cells. Disclosures: No relevant conflicts of interest to declare.

AMN107, Novel Aminopyrimidine Inhibitor of Bcr-Abl, Is Significantly More Potent Than Imatinib Mesylate Against Philadelphia Chromosome Positive Acute Lymphoblastic Leukemia Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2737-2737
Author(s):  
Mirna Golemovic ◽  
Miloslav Beran ◽  
Francis Giles ◽  
Taghi Manshouri ◽  
Deborah Thomas ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Line ◽  
Imatinib Mesylate ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Single Agent ◽  
Philadelphia Chromosome ◽  
Imatinib Treatment ◽  
Significant Activity ◽  
Mts Assay

Abstract Imatinib mesylate is effective against Philadelphia chromosome (Ph)-positive acute lymphoblastic leukemia (ALL) but, when used as a single agent, responses are transient and most patients relapse within 4–6 months. AMN107 is a novel oral aminopyrimidine ATP-competitive inhibitor of the protein tyrosine kinase activity of Bcr-Abl. Following oral administration to animals, AMN107 is well absorbed, has a good pharmacokinetic profile, and is well tolerated. The activity of AMN107, relative to imatinib, in both Ph-positive (Z-119 and Z-181) and Ph-negative (Z-138) ALL cell lines was studied. Z-119 and Z-181 cells were derived from Ph-positive ALL patients and retained typical B-cell characteristics and phenotypes of the original leukemia, including cytogenetic abnormality t(9;22) and p190 Bcr/Abl kinase. Z-138, a Ph-negative cell line, was derived from a patient with chronic lymphocytic leukemia and supervening ALL. Treatment with AMN107 or imatinib for 3 days (MTS assay) inhibited proliferation of Z-119 cells with the IC50 values of 19.3 nM and 620.0 nM, respectively, revealing AMN107 to be 32 fold more potent than imatinib. Treatment of Z-181 cell line lasted for 4 days (MTS assay) because of lower growth rate of these cells: IC50 for AMN107 and imatinib were 1.6 nM and 63.9 nM, respectively, showing AMN107 to be 40 fold more potent than imatinib. Neither drug showed activity against Ph-negative Z-138 cells. We also compared the activity of AMN107 in Ph-positive ALL cell lines expressing p190 Bcr/Abl protein to that in Ph-positive chronic myeloid leukemia cell lines KBM5 and KBM7 expressing p210 Bcr/Abl protein. The activity was similar with IC50 in KBM5 cells of 11.3 nM and in KBM7 cells of 4.3 nM. In experiments focused on cell cycle analysis we found that at equipotent doses (as determined by MTS assay) both drugs induced cell accumulation in G0/G1 phase in Z-119 but not in Z-181. We demonstrated that increasing equipotent concentrations of AMN107 and imatinib induced activation of caspase-3 that resulted in apoptosis, as assessed by propidium iodide staining, in Z-119 cells, while Z-181 cells showed lack of apoptotic response. Following treatment with a broad range of AMN107 and imatinib doses for 3 hrs, Bcr/Abl expression and phosphorylation were determined in Z-119 cells by immunoprecipitation and Western blotting: Bcr/Abl phosphorylation was inhibited completely with AMN107 at 125.0 nM, and with imatinib at 2500 nM, confirming again the higher potency of AMN107. Finally, similar differential effect of AMN107 and imatinib on Bcr/Abl protein expression and phosphorylation was observed in leukemic cells obtained from blood of Ph-positive ALL patients. We conclude that AMN107 has significant activity against Ph-positive ALL cells and warrants investigation in patients with Ph-positive ALL.

The Methylation Status of p21 Is Not Relevant in Adult Acute Lymphoblastic Leukemia Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4552-4552
Author(s):  
Chiara Gregorj ◽  
Fabiana De Cave ◽  
Maria R. Ricciardi ◽  
Maria C. Scerpa ◽  
Cristina M. Precupanu ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Protein Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Induction Therapy ◽  
Lymphoblastic Leukemia ◽  
Raji Cell ◽  
Methylation Status ◽  
Philadelphia Chromosome ◽  
Normal Peripheral Blood

Abstract Methylation of CpG islands in the 5′ gene region is associated with transcriptional silencing of gene expression. The hypermethylation of tumor suppressor genes has been described in various tumor tissues, as in gastric and pancreatic cancer, as well as in acute myeloid leukemia, suggesting its potential role in tumorigenesis. Among the three members of the Kip/Cip family of cyclin dependent kinase inhibitors (CKI) p21, p27 and p57, little is known of their methylation status in hematological malignancies and contrasting studies have been reported on the role of p21 hypermethylation in the pathogenesis of acute lymphoblastic leukemia (ALL). The aim of our study was to analyze in primary blasts from adult ALL enrolled in the GIMEMA protocols 0496 and LAL2000 the methylation status of p21, defining in addition its protein expression by Western blot using the monoclonal antibody p21-WAF1 (Santa Cruz, CA). Primary samples from 81 untreated ALL patients were processed using a widely accepted method based on bisulfite modification of DNA, followed by the use of methylation-specific PCR assay (MSP). The human lymphoblastic cell lines (Jurkat, RPMI8866 and CEM), the myeloid cell line OCI-AML3 and normal peripheral blood lymphocytes (PBL) from 10 healthy donors were characterized by a consistent p21 promoter unmethylation (negative controls). In contrast, it was weakly methylated in the Raji cell line and strongly methylated in the Rael (Burkitt’s lymphoma) cell line (positive controls). This assay was further validate in vitro by SsI methylase. In the present study we analyzed 54 B-lineage ALLs, 25 T-ALLs and 2 biphenothypic leukemias; the mean WBC value at diagnosis was 125.6x109/L and 20 samples were Philadelphia chromosome positive. 71/81 of patients studied for p21 methylation were evaluated for response: 53 (74.6%) achieved complete remission (CR) after induction therapy, 8 (11.3%) patients were resistance and 10 (14.1%) died during induction therapy. DNA methylation was not observed in any of the adult ALL patients. p21 protein expression was found in OCI-AML3, Raji and RPMI8866 cell lines, while resulted negative in the Jurkat cell line and in normal PBL. Preliminary results obtained in the ALL samples showed that this protein was expressed in 8/29 (27.6%) cases. In summary, we demonstrated in a large number of primary ALL cases studied at presentation that the p21 gene is not methylated in this population and therefore that the status of p21 methylation does not play a role in the pathogenesis of adult ALL.

To Study the Proliferative Inhibition of Anticancer Drug in Two Kinds of Ph (+) Leukemia Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4110-4110
Author(s):  
Yuping Gong ◽  
Xi Yang ◽  
Ting Niu
Keyword(s):  
Gene Expression ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
K562 Cell ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Myelogenous Leukemia ◽  
Leukemia Cell Line ◽  
Leukemia Cell Lines

Abstract Abstract 4110 Objective To study the proliferative inhibition of imatinib, daunorubicin and bortezomib in two kinds of Ph(+) leukemia cell lines: chronic myelogenous leukemia cell line K562 expressing P210 protein and acute lymphoblastic leukemia cell line SUP-B15 expressing P190 protein. Methods (1) Cell proliferation with imatinib, daunorubicin and bortezomib for 72 hours was analyzed by the MTT assay and displayed by growth curve and IC50 value. (2) The change of bcr-abl gene mRNA levels after the 48 hours' intervention of imatinib (final concentration at 0μM, 0.35μM, 1 μM) was detected by reverse transcription polymerase chain reaction (RT-PCR). Results (1) The IC50 values of K562 and SUP-B15 cells inhibited by imatinib, daunorubicin and bortezomib for 72 hours was respectively 0.286±0.06 (μmol/L), 0.303±0.009 (μmol/L), 22.127±3.592 (nmol/L) and 1.387±0.180(μmol/L), 0.117±0.017 (μmol/L), 12.350±0.740 (nmol/L), which indicated that the K562 cell line was the more sensitive to imatinib than SUP-B15 cell line, whereas the SUP-B15 cell line had the more sensitivity to daunorubicin and bortezomib. (2) There was no change of bcr-abl gene expression after the 48 hours' intervention of imatinib in both cell lines. Conclusion (1) Imatinib, daunorubicin and bortezomib had good anti-cancer effect to Ph+ leukemia cells in vitro. What's more, the K562 cell was the more sensitive to imatinib and only imatinib will have good effect on chronic myelogenous leukemia. Whereas the SUP-B15 cell had the more sensitivity to daunorubicin and bortezomib and combining imatinib with daunorubicin or bortezomib, the effect will be better on Ph(+) acute lymphoblastic leukemia. (2) The short time intervention of imatinib had no effect on the bcr-abl gene expression and imatinib could need long time to show curative effect for the Ph+ leukemia. Disclosures: No relevant conflicts of interest to declare.

New LMO2 Translocations in T-Cell Acute Lymphoblastic Leukemia, Involving STAG2 at Xq25 and MBNL1 at 3q25: A Positive Change?.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2641-2641
Author(s):  
Suning Chen ◽  
Stefan Nagel ◽  
Bjoern Schneider ◽  
Maren Kaufmann ◽  
Ursula R. Kees ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
T Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
B Cell Lymphoma ◽  
Small Sample ◽  
Upstream Region ◽  
Cell Acute Lymphoblastic Leukemia

Abstract Abstract 2641 Poster Board II-617 Background: In T-cell acute lymphoblastic leukemia (T-ALL) the LMO2 transcription factor locus is juxtaposed with T-cell receptor (TCR) genes by a recurrent chromosome translocation, t(11;14)(p13;q11). Recent molecular cytogenetic data indicate that unlike classical TCR rearrangements, t(11;14) operates synonymously with submicroscopic del(11)(p13p13) by removing a negative upstream LMO2 regulator (Dik et al., Blood 2007;110:388). The combined incidence of both LMO2 rearrangements is ∼10-15% (Van Vlierberghe and Huret, Atlas Genet Cytogenet Oncol Haematol, November 2007). However, aberrant LMO2 expression occurs in nearly half of all T-ALL cases, a discrepancy which may indicate a significant contribution by cryptic chromosome alterations. We attempted the extended characterization of the LMO2 genomic region in T-ALL cell lines to look for such rearrangements. Cells and Methods: We investigated a panel of 26 well characterized and authenticated T-ALL cell lines using parallel fluorescence in situ hybridization (FISH) with a tilepath BAC/fosmid contig and both conventional and quantitative reverse transcriptase (Rq)-PCR. Global gene expression was additionally measured in some cell lines by Affymetrix array profiling. Results: LMO2 rearrangements were detected in 5/26 (19.2%) cell lines including both established rearrangements, t(11;14) and del(11)(p13p13) in one cell line apiece (3.8%). Interestingly, we found two novel LMO2 translocations: t(X;11)(q25;p13) in 2/26 (7.7%), and t(3;11)(q25;p13) in 1/26 (3.8%) cell lines, respectively. Comparing transcription levels in cell lines with and without genomic rearrangements showed that LMO2 expression was significantly higher in T-ALL cell lines carrying LMO2 rearrangements (P<0.001). Rq-PCR revealed that 5 of the top 10 (50%) LMO2 expressing cell lines carry cytogenetic rearrangements at this locus, compared to 0/16 remaining examples. Loss of a recently defined LMO2 negative regulatory element was identified in the del(11)(p13p13) cell line but no other deletions were detected. Two genes STAG2 at Xq25 and MBNL1 at 3q25 were identified as novice LMO2 partners in t(X;11) and t(3;11), respectively. In both genes breakpoints lay at intron 1 close to deeply conserved noncoding regulatory regions. Both t(X;11) cell lines displayed conspicuous silencing of the ubiquitously expressed STAG2 gene highlighting the transcriptional significance of the region displaced. Unlike t(11;14)/del(11)(p13p13) both new rearrangements carry LMO2 breakpoints in the far upstream region (at minus 80–150 Kbp), and appear to result in upregulation of LMO2 by juxtaposition rather than via covert deletion. STAG2 is a component of the chromosomal cohesin complex which acts as a transcriptional coactivator, and which has been recently identified as a potential driver of oncogene transcription in acute myeloid leukemia (Walter et al., Proc Natl Acad Sci U S A. 2009;106:1295). MBNL1 controls RNA splicing and is a rare BCL6 partner gene in B-cell lymphoma, but this is the first report of its involvement in T-ALL. Conclusion: Given their frequency and variety in a small sample, we propose that cryptic chromosome rearrangements targeting LMO2 upregulation may be significantly more frequent than hitherto appreciated in T-ALL. Unlike canonical LMO2 rearrangements, both t(X;11) and t(3;11) would appear to function positively by upregulation of LMO2 via juxtaposition with noncoding driver elements within these novel partner genes. Perspectives: Future work will address the regulatory potential of candidate enhancer sequences embedded within conserved noncoding intronic sequences of MBNL1 and STAG2. Cytogenetically inconspicuous cell lines displaying LMO2 upregulation will be subjected to more detailed scrutiny using high density genomic SNP arrays. Disclosures: No relevant conflicts of interest to declare.

Association of Asparagine Synthetase Expression and Sensitivity to L-Asparaginase in Cell Lines and Primary Pediatric Acute Lymphoblastic Leukemia Samples.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2738-2738
Author(s):  
Ivana Hermanova ◽  
Jan Trka ◽  
Julia Starkova
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Amino Acid ◽  
Mrna Expression ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Asparagine Synthetase ◽  
Leukemic Cells ◽  
Mrna Levels ◽  
Basal Expression

Abstract Abstract 2738 Poster Board II-714 L-Asparaginase (L-Asp) is an important component in the combined chemotherapy for childhood acute lymphoblastic leukemia (ALL). Administration of L-Asp leads to depletion of plasmatic asparagine and consequently causes loss of intracellular asparagine. As a non-essential amino acid, asparagine is synthesized from aspartate and glutamine by asparagine synthetase (ASNS). Primary ALL cells are believed to have low ASNS expression and therefore to be sensitive to asparagine depletion. Although increased ASNS level was shown to be connected with L-Asp resistance the exact relationship between ASNS expression and L-Asp sensitivity is not clear. We and others have previously shown TEL/AML1[+] ALL blasts express more ASNS mRNA than TEL/AML[-] do although primary TEL/AML[+] cell are in vitro more sensitive to treatment with L-Asp. Hutson et al (1997) showed that amino acid deprivation led to increased expression of ASNS on mRNA and protein level as well as to increased biological activity. On the other hand, Nan Su et al described negative correlation between L-Asp sensitivity and ASNS protein rather than mRNA levels. Therefore, in our studies we concentrated on protein expression of ASNS in patients' samples. So far, there has been no reproducible published data on ASNS protein detection by Western blot in primary patients' samples. Despite using 3 different antibodies and precise optimization we were not able to detect ASNS protein in patients' samples in contrast to cell lines. Transcripts' levels confirmed significantly lower (2 log) expression of ASNS in patients' leukemic cells compared to leukemic cell lines. Therefore, for further studies on gene and protein relation we had to rely on cell lines as a model. We detected ASNS gene expression and ASNS protein content in four ALL cell lines: REH (TEL/AML1[+]), UOCB6 (TEL/AML1[+]), NALM6 (TEL/PDGFRB[+]) and RS4;11 (MLL/AF4[+]). ASNS mRNA levels were in accord with sensitivity to L-Asp. UOCB6 as the most resistant cell line (IC50=0.04U/ml) had the highest expression of ASNS (normalized ASNS, nASNS=4.946), then NALM6 (IC50=0.01U/ml; nASNS=1.8), REH (IC50=0.6.10−4; nASNS=1.176) and RS4;11 (IC50<0.3.10−4; nASNS=0.024). ASNS protein levels significantly differed through passages in REH cells, likely due to rapid turnover. For the remaining three cell lines L-Asp sensitivity correlated also with protein content. We have previously shown that different basal expression levels do not affect short-term dynamics of ASNS expression after L-Asp administration. Here we were interested to see the changes of sensitivity to L-Asp using gradient silencing of ASNS by RNAi in two cell lines with different basal expression: REH cell line with intermediate ASNS mRNA expression and RS4;11 cell line with very low mRNA expression. Gradient silencing revealed that L-Asp sensitivity correlated with ASNS expression till 50% decrease; further silencing did not potentiate the effect. The same response was seen in both cell lines despite different basal ASNS expression and sensitivity to L-Asp. The ASNS is glutamine dependent enzyme therefore we also studied expression of glutamate dehydrogenase (GDH), an enzyme necessary for glutamine synthesis. We found significantly lower GDH mRNA expression in primary TEL/AML1[+] blasts in comparison with TEL/AML[-] blasts (p=0.019), which might lead to deficiency of glutamine in these cells and consequently higher sensitivity to L-Asp. Accordingly, silencing of ASNS in REH tended to increase GDH expression levels. Our data confirm that generally, both ASNS mRNA and protein expression inversely correlate with the sensitivity to L-Asp in the cell lines. However, it may be misleading to draw conclusions for the patients' cells directly from the results obtained in cell line models. The expression patterns of ASNS in primary leukemic cells differ even from those of genotypically identical cell lines. The control of basal levels of ASNS in leukemic cells remains to be elucidated. Our results implicate an important role of GDH and glutamine metabolic pathway in the regulation of ASNS activity. This work was supported by MSM0021620813 and GAUK 7835. Disclosures: No relevant conflicts of interest to declare.

Effect of Protein Kinase C β Specific Inhibition On Acute Lymphoblastic Leukemia Cell Lines.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4817-4817
Author(s):  
Nakhle S Saba ◽  
Hana F Safah ◽  
Laura S Levy
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Growth Inhibition ◽  
Cell Viability ◽  
B Cell ◽  
Cell Lines ◽  
Chromosomal Abnormality ◽  
Lymphoblastic Leukemia ◽  
Specific Inhibitor ◽  
B Cell Lymphoma ◽  
Specific Inhibition

Abstract Abstract 4817 Acute lymphoblastic leukemia (ALL) is the most common leukemia in children and accounts for 20% of acute leukemia in adults. The intensive induction–consolidation–maintenance therapeutic regimens used currently have improved the 5-year disease free survival to around 80% in children and to 25%-40% in adults. The poorer response in adults is basically due to the inability to tolerate the intensive chemotherapy, and to the biology of adult disease which is associated with poor-risk prognostic factors. In the present era of target-specific therapy, PKCβ targeting arose as a new, promising, and well tolerated treatment strategy in a variety of neoplasms, especially in B-cell malignancies. It showed encouraging results in preclinical and clinical studies involving chronic lymphocytic leukemia, diffuse large B-cell lymphoma and multiple myeloma. PKCβ plays a major role in B-cell receptor signaling, but studies describing the role of PKCβ in B-cell ALL are lacking. In the present study, we measured the sensitivity of a variety of B-cell ALL cell lines to PKCβ specific inhibition. Three cell lines were studied: RS4;11 (characterized by the t(4;11) chromosomal abnormality), TOM-1 (characterized by the t(9;22) chromosomal abnormality), and REH (characterized by the t(12;21) chromosomal abnormality). Cells were tested for PKCβ1 and PKCβ2 expression by immunoblot. Cell viability was measured when PKCβ-specific inhibitor at concentrations of 1, 2.5, 5, 10, 20 and 30 μM was added for 48 hours in the presence of 10% fetal bovine serum (FBS). MTS assay was performed to quantify cell viability. Results showed that all three cell lines express PKCβ1 and PKCβ2. Treatment with PKCβ-specific inhibitor resulted in a dose-dependent inhibition of cell proliferation; Sensitivity was evident at 1 μM for RS4;11 cell line, and at 2.5 μM for TOM-1 and REH cell lines, with 10% cell growth inhibition; Growth inhibition increased to 90% for all cell lines at an inhibitor concentration of 30 μM. These results indicate that PKCβ plays an important role in the malignant process in B-cell ALL, and suggest that PKCβ targeting should be considered as a potential treatment, whether in combination with the current regimens used or as a single agent monotherapy. Ongoing studies in our lab will detail the mechanism of PKCβ and adverse cytogenetics like t(4;11) and t(9;22). Disclosures: No relevant conflicts of interest to declare.

Simultaneous Targeting of Bcl-2/Bcl-Xl and MEK/ERK Results in Highly Synergistic Induction of Apoptosis in Human AML, Both in Vitro and In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 2757-2757
Author(s):  
Marina Konopleva ◽  
Michele Milella ◽  
Julie C Watts ◽  
Maria Rosaria Ricciardi ◽  
Borys Korchin ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Cell Line ◽  
Cell Lines ◽  
Single Agent ◽  
Mek Inhibitor ◽  
Human Leukemia ◽  
Apoptosis Induction ◽  
Mek Inhibitors ◽  
Synergistic Induction ◽  
Induction Of Apoptosis

Abstract Abstract 2757 Poster Board II-733 Previous experience from our group suggests that simultaneous inhibition of MEK-to-ERK signaling and interference with the anti-apoptotic activity of Bcl-2/Bcl-xL, using a variety of different agents, may result in synergistic anti-leukemic activity (Milella M, Blood 2002; Ricciardi MR, ASH 2004; Konopleva M, Cancer Cell 2006; Milella M, ASH 2008). Here, we screened AML cell lines (OCI-AML3, HL-60, MOLM-13, and U937) for the growth inhibitory/pro-apoptotic effects of the combination of the BH3 mimetic ABT-737 and the selective MEK inhibitor PD0325901. With the notable exception of U937 cells all the cell lines tested displayed highly synergistic growth inhibition/induction of apoptosis in response to the combination (CIs ranging from 0.01 to 0.43); the 25:1 and 1:2 ABT-737/PD0325901 ratios appeared to have optimal synergistic effects in OCI-AML3 and MOLM-13 cells, respectively. Cell growth inhibition was primarily due to the highly synergistic induction of apoptosis in sensitive cell line models. From a mechanistic standpoint, ABT-737 induced ERK activation and Mcl-1 protein expression, two putative resistance mechanisms, both of which were efficiently abrogated by co-treatment with PD0325901. In addition to the modulation of Mcl-1, both single agent PD0325901 and combined ABT-737/PD0325901 treatment rapidly (1-6 hrs) induced BimEL dephosphorylation and Bak expression, thereby contributing to Mcl-1 inactivation. To further assess the role of pro-apoptotic Bcl-2 family members in the observed proapoptotic synergism betweeen ABT-737 and PD0325901, we analyzed the effects of the combination in Bim-, Bak-, and Bax-KO MEFs, as well as in double (Bak/Bax) KO; while these experiments demonstrated that all three proapoptotic proteins play a role in apoptosis induction by combined ABT-737/PD0325901, siRNA-mediated silencing of either Bim or Bak clearly indicated Bim as the most important player in the AML cell line OCI-AML3. In addition to cell line models of leukemia, striking apoptosis induction (20-75% net apoptosis induction) was also observed with the combination of Bcl-2 and MEK inhibitors in ex vivo-cultured primary AML samples (n=8); most interestingly, the ABT-737/PD0325901 combination appeared to selectively kill leukemic stem cells, with < 20% of CD34+/CD38- cells surviving after exposure to relatively low doses of the combination (50 nM for each agent). Finally, we tested the combination of ABT-737 and the MEK inhibitor CI-1040 in nude mice injected with GFP/luciferase bearing MOLM-13 human leukemia cells. Two weeks after leukemia transplantation, mice were randomized and treated with liposomal ABT-737 (i.v. 20 mg/kg, qod for three weeks), CI1040 (i.p. 50 mg/kg qod for three weeks), ABT-737 in combination with CI1040 (ABT-737 + CI1040), or with empty liposomes (i.v.; control). Engraftment of MOLM-13 cells was shown by immunohistochemical detection of GFP-positive cells in the spleen of control mice five weeks after transplantation. Notably, while control and CI1040 treated mice demonstrated progressive increases in leukemia-derived bioluminescence, ABT-737 treated mice, and to a greater extent ABT-737 + CI1040 treated mice, appeared to resist tumor burden progression. In addition, quantitation of leukemia-derived bioluminescence demonstrated that ABT-737 + CI1040 treated mice had significantly (p<0.00001) lower leukemia burden than control mice or ABT-737 treated mice at all time points (7, 14 and 21 days of treatment). Overall our data demonstrate that an anti-apoptotic crosstalk between the Bcl-2 and the MEK/ERK pathway is operative in AML cells and could be exploited therapeutically by targeting both pathways simultaneously. The combination of BH3 mimetics (such as ABT-737) and MEK inhibitors warrants clinical testing as a novel therapeutic strategy for patients with AML. Disclosures: No relevant conflicts of interest to declare.

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