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.

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.

Anti-Leukemic Property of Sulphoraphane In Precursor B Cell Acute Lymphoblastic Leukemia

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3974-3974
Author(s):  
Koramit Suppipat ◽  
Xiao Zhu ◽  
Chun Shik Park ◽  
H. Daniel Lacorazza
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Annexin V ◽  
Dependent Manner ◽  
Carcinogenic Activity ◽  
Childhood All

Abstract Abstract 3974 Acute lymphoblastic leukemia (ALL) is the most common form of hematologic malignancy in children. In spite of significant advances achieved in the treatment of childhood ALL, one fifth of these patients still relapse after the standard treatment. Moreover, relapse ALL is the second most common cause of cancer-related deaths in children. The development of novel therapies is prevented by a limited understanding of the exact pathobiology. There are emerging evidences that the transcription factor KLF4 has a tumor suppressor property in ALL. Recently, a gene expression classifier study in pediatric precursor B-cell ALL (pre-B ALL) showed that KLF4 expression was significantly reduced in high risk ALL patients with positive MRD after induction. This finding suggests a possible role of this cell cycle inhibitor on the development, expansion and drug-resistant of leukemic cells. Several studies demonstrate that overexpression of KLF4 in normal B cells and BCR transformed B cells show increased apoptosis and reduced proliferation. Furthermore, we recently described that KLF4 inhibits proliferation of naïve lymphocytes by activating p21 (Yamada, et al, 2009). Sulphoraphane (SF; 4-methylsulfonylbutyl isothiocyanate) is a dietary compound derived from Cruciferae vegetables with anti-carcinogenic activity in colon cancer by upregulating KLF4 and p21 among other genes. Thus, we hypothesized that SF could also exhibit anti-leukemic activity in human-derived acute lymphoblastic leukemia cells via the activation of KLF4. The pre-B ALL cell lines (Nalm6, REH, RS-4, SUP-B15) and an EBV transformed B cell line were treated with different concentrations of SF (0-40 μM) for 24–48 hours. Then, cell number was estimated using an ATP-based viability method. Flow cytometric analysis of ANNEXIN-V/7-AAD binding as well as CFSE dilution was used to measure apoptosis and proliferation respectively. We found that SF induced cytotoxicity in Nalm-6, REH and RS-4 cell lines in a dose and time dependent manner. This cytotoxic effect was less pronounced in EBV-transformed B cell line. SF treatment led to increased ANNEXIN-V and 7-AAD positive cells (82% apoptotic cells in SF-treated versus 9% in DMSO control). Further, SF-treated cells displayed significantly less proliferation in comparison to DMSO controls thus suggesting that SF inhibits cellular proliferation. Preliminary data also suggest that SF-mediated apoptosis is caused by upregulation of KLF4. In conclusion, Sulphoraphane exhibits an anti-leukemic property by inducing apoptosis and abrogating proliferation in pre-B ALL cell lines. Thus, sulphoraphane could potentially be used as an adjunctive therapy in a subgroup of pre-B ALL patients who have decreased expression of KLF4. Disclosures: No relevant conflicts of interest to declare.

Quantitative Proteomic Analysis Reveals Maturation As a Mechanism Underlying Glucocorticoid Resistance in Childhood Acute Lymphoblastic Leukemia and PAX5 As a Re-Sensitising Therapeutic Target

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1411-1411
Author(s):  
Lindsay Nicholson ◽  
Caroline Evans ◽  
Elizabeth C Matheson ◽  
Lynne Minto ◽  
Christopher Keilty ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Cell Viability ◽  
B Cell ◽  
Cell Line ◽  
Cell Lines ◽  
Therapeutic Target ◽  
Proteomic Analysis ◽  
Lymphoblastic Leukemia ◽  
Childhood All ◽  
Resistant Phenotype

Abstract Abstract 1411 Glucocorticoids (GC), such as prednisolone and dexamethasone, are an integral component of the multi-agent treatment of childhood acute lymphoblastic leukemia (ALL). GC-resistance is a significant prognostic indicator of a poor treatment outcome and remains a clinical problem, with the underlying mechanisms still unclear. Mutation or loss of the primary mediator of GC-action, the glucocorticoid receptor (GR), underlies the GC-resistant phenotype in several commonly used leukemic cell lines. However, these events are rare in primary leukemic cells, with relatively few examples in vivo. This suggests that it may be possible to reverse the GC-resistant phenotype pharmacologically. We have used an iTRAQ proteomics approach for hypothesis generation of potential mechanisms for GC-resistance in childhood ALL. To achieve this, we compared a well-characterized GC-sensitive cell line, PreB 697, and a GC-resistant sub-clone (R3F9), both bearing wildtype GR, in a comparative proteomic experiment using 4-channel isobaric tagging for relative and absolute quantification (iTRAQ). A comparison of protein profiles before and after dexamethasone exposure of the two cell lines identified two transcription factors involved in B-cell differentiation, PAX5 and IRF4, to be differentially upregulated in the PreB 697 compared to the R3F9 cell line in response to GC. Experimentally, there was approximately 50% reduction in PAX5 basal protein expression in R3F9 compared to its GC-sensitive parent, a finding which was also evident in four other resistant sub-lines. This was accompanied by a decreased expression of CD19 and CD10, indicative of an increased B-cell maturation state. The reduced PAX5 level in the GC-resistant cell lines was not due to mono-allelic loss or mutation and mRNA levels were not significantly altered, suggestive of a post-transcriptional mechanism for PAX5 protein reduction. Paradoxically, knockdown of PAX5 reversed the GC-resistant phenotype of the R3F9 cell line such that the apoptotic response to dexamethasone was similar to that of the GC-sensitive parent line as measured by Annexin V staining (R3F9: mean 52.22%, SD 12.54%, n=3; PreB 697: mean 67.23%, SD 9.96%, n=3) and cell viability assays. This chemosensitization after PAX5 knockdown was specific to GC, with no difference in cell viability observed in either cell line after exposure to daunorubicin, vincristine or L-asparaginase when compared to negative siRNA or mock controls. This increase in GC-sensitivity was coupled with a significant upregulation of GR and its transcriptional target, GILZ. We also showed an enhanced GC response after PAX5 knockdown in two out of eight primary, diagnostic pre-B lineage ALL patient samples. Thus, in this ALL cell line model, quantitative proteomic analysis revealed increased maturation as a recurrent mechanism underlying GC-resistance and identifies PAX5 as a possible therapeutic target to fully re-sensitise GC-response in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

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