scholarly journals Characterization of a novel glucocorticoid-resistant human B-cell acute lymphoblastic leukemia cell line, with AMPK, mTOR and fatty acid synthesis pathway inhibition

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Yuanyuan Li ◽  
Chuan Zuo ◽  
Ling Gu
Keyword(s):  
Fatty Acid ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Line ◽  
Cell Lines ◽  
Fatty Acid Synthesis ◽  
Lymphoblastic Leukemia ◽  
Acid Synthesis ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Fas Pathway

Abstract Background Acquired glucocorticoid (GC) resistance remains the main obstacle in acute lymphoblastic leukemia (ALL) therapy. The aim of the present study was to establish a novel GC-resistant B-ALL cell line and investigate its biological characteristics. Methods A cell culture technique was used to establish the GC-resistant cell line from the parental cell, NALM-6. Molecular and cellular biological techniques including flow cytometry, MTT assay, western blotting, DNA fingerprinting analysis and whole transcriptome sequencing (WTS) were used to characterize the GC-resistant cell lines. Nude mice were used for xenograft studies. Results The GC-resistant cell line, NALM-6/HDR, was established by culturing NALM-6 cells under hypoxia for 5 weeks with a single dexamethasone (Dex) treatment. We subcloned the NALM-6/HDR cell lines, and got 6 monoclone Dex-resistant cell lines, NALM-6/HDR-C1, C3, C4, C5, C6 and C9 with resistance index (RI) ranging from 20,000–50,000. NALM-6/HDR and its monoclone cell line, NALM-6/HDR-C5, exhibited moderate (RI 5–15) to high resistance (RI > 20) to Ara-c; low or no cross-resistance to L-Asp, VCR, DNR, and MTX (RI < 5). STR analysis confirmed that NALM-6/HDR and NALM-6/H were all derived from NALM-6. All these cells derived from NALM-6 showed similar morphology, growth curves, immunophenotype, chromosomal karyotype and tumorigenicity. WTS analysis revealed that the main metabolic differences between NALM-6 or NALM-6/H (GC-sensitive) and NALM-6/HDR (GC-resistant) were lipid and carbohydrates metabolism. Western blotting analysis showed that NALM-6/HDR cells had a low expression of GR and p-GR. Moreover, AMPK, mTORC1, glycolysis and de novo fatty acid synthesis (FAS) pathway were inhibited in NALM-6/HDR when compared with NALM-6. Conclusions NALM-6/HDR cell line may represent a subtype of B-ALL cells in patients who acquired GC and Ara-c resistance during the treatment. These patients may get little benefit from the available therapy target of AMPK, mTORC1, glycolysis and FAS pathway.

Novel Retinoic Acid Metabolism Blocking Agents (RAMBAs) Induce Differentiation in ATRA Resistant Cell Line: Potential New Theraputics for Acute Promyleocytic Leukemia (APL).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 746-746
Author(s):  
Kavita B. Kalra ◽  
Xiangfei Cheng ◽  
Marion Womak ◽  
Christopher Gocke ◽  
Jyoti B. Patel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Retinoic Acid ◽  
Cell Line ◽  
Cell Lines ◽  
Cancer Cell ◽  
Cancer Cell Lines ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Breast Cancer Cell Lines ◽  
Hematopoietic Stem

Abstract All trans retinoic acid (ATRA) has been used in differentiation therapy for APL and other types of cancers. However, the rapid emergence of ATRA resistance due in part to ATRA-induced acceleration of ATRA metabolism limits its use. A novel strategy to overcome the limitation associated with exogenous ATRA therapy has been developed by inhibiting the cytochrome P450-dependent ATRA-4-hydroxylase enzyme responsible for ATRA metabolism. These inhibitors are referred to as RAMBAs. Novel RAMBAs were developed which demonstrated a superior apoptosis, cell growth inhibition, in vivo anti-tumor effect in addition to the differentiation effect in breast cancer cell lines (Patel JB et al. J. Med. Chem2004,47:6716). We tested 3 RAMBAs, VN/14-1, 50-1, and 66-1 to investigate their activities against APL cell lines. RAMBAs did not confer cytotoxicity or apoptosis induction in vitro at the concentration between 0.5 to 5 μM as opposed to breast or prostate cancer cell lines. However, the differentiation effect was demonstrated by morphological and phenotypic changes using Wright-Giemsa stain and CD11b staining measured by flow cytometric analysis. VN/14-1 and VN/66-1 induced differentiation and apoptosis morphologically and phenotypically in HL60 cells. VN/14-1 and VN/50-1 showed superior differentiation in NB4 cell line compared to ATRA (70%, 69%, and 45%, respectively). Interestingly, HL60 ATRA resistant cell line was induced to undergo differentiation by VN/14-1 (0.5μM) at 55% whereas ATRA (0.5, 1, 5μM) showed less than 5% by flow cytometry analysis. VN/14-1 inhibited cell cycle at S phase whereas ATRA did not attenuate the cell cycle at the same concentration. We also tested the effect of RAMBAs on human CD34+ enriched cell colony formation. RAMBAs were added to the methylcellulose culture plates with CD34+ cells and colonies were determined after 14 days. There was no difference in the CFU-GM or BFU-E colony count between the control and the RAMBAs group. In summary, RAMBAs are promising differentiation agents in the treatment of APL, possibly through an inhibition of Cyp26A leading to increased endogenous ATRA levels. In addition, cell cycle inhibition may be a mechanism of differentiation induction in ATRA resistant cell lines. RAMBAs did not affect normal hematopoietic stem cells. We are currently testing whether RAMBAs can induce acetylation of histones in APL cell lines.

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.

scholarly journals Identifying Mechanisms Associated with Venetoclax Resistance in Multiple Myeloma (MM)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2668-2668
Author(s):  
Yuan Xiao Zhu ◽  
Laura Ann Bruins ◽  
Joseph Ahmann ◽  
Cecilia Bonolo De Campos ◽  
Esteban Braggio ◽  
...  
Keyword(s):  
Cell Line ◽  
Board Of Directors ◽  
Cell Lines ◽  
Acquired Resistance ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Sensitive Cell ◽  
Advisory Committees ◽  
Bcl2 Family ◽  
Family Regulation

Abstract Venetoclax (VTX) is a selective small-molecule inhibitor of BCL-2 that exhibits antitumoral activity against MM cells presenting lymphoid features and those with translocation t(11;14). Despite its impressive clinical activity, VTX therapy for a prolonged duration can lead to drug resistance. Therefore, it is important to understand the underlying mechanisms of resistance in order to develop strategies to prevent or overcome resistance. In the present study, we established four VTX resistant human myeloma cell lines (HMCLs) from four sensitive HMCLs, including three with t(11;14), in culture with a stepwise increase in treatment dose with VTX. To identify the molecular basis of acquired VTX resistance, whole exon sequencing (WES), mRNA-sequencing (mRNAseq), and protein expression assays were performed in the four isogenic VTX-sensitive/resistant HMCLs and three MM patients with samples collected before VTX administration and after clinical resistance to the drug. Compared with sensitive cell lines and patient samples collected before VTX administration, mRNAseq analysis identified downregulation of BIM and upregulation of BCLXL in both resistant cell lines and MM cells from relapse patients. Other transcriptional changes detected included upregulation of AURKA, BIRC3, BIRC5, and IL32. Enrichment analysis of differentially expressed genes suggested involvement of PI3K and MAPK signaling, likely associated with cytokines, growth factors (EGF, FGF and IGF family members), and receptor tyrosine kinase (EGF and FGF). Western blot analysis was performed to compare BCL2 family expression in resistant cell lines versus sensitive cell lines and it showed upregulation of BCL2 survival members (such as MCL-1 and BCLXL), and downregulation of pro-apoptotic BH3 members (such as BIM and PUMA). BIM expression was completely lost in one resistant cell line, and introduction of exogenous BIM into this cell line enhanced VTX sensitivity. Interestingly, BCL2 was upregulated in some resistant cell lines generated after a long-term treatment with VTX, suggesting BCL2 expression level may not be suitable as a marker of VTX sensitivity for acquired resistance. Unlike in CLL, BCL2 mutations were not identified through WES in any resistant cell lines or primary patient sample harvested after relapse. While 8 genes were mutated in two resistant samples , no clear mutational pattern emerged . Based on the above, we further tested some specific inhibitors in in vitro or ex vivo cell models to help understanding resistant mechanism and identify strategies to overcome VTX resistance. We found that inhibition of MCL-1, with the compound S68345, substantially enhanced VTX sensitivity in three resistant HMCLs and in primary cells from one relapsed MM patient. A BCLXL inhibitor (A155463) only significantly enhanced VTX sensitivity in one resistant cell line after co-treatment with VTX. Co-treatment of the other three resistant cell lines with VTX, S68345 and A155463 resulted in the most synergistic anti-myeloma activity, suggesting those cell lines are co-dependent on MCL-1, BCLXL, and BCL2 for survival, although they are more dependent on MCL-1. We also found that inhibition of PI3K signaling, IGF1, RTK (EGF and FGF) and AURKA significantly increased VTX sensitivity, partially through downregulation of MCL-1, and BCLXL, and upregulation of BIM. Conventional anti-MM drugs such as dexamethasone, bortezomib and lenalidomide, were shown to have little activity on augmenting VTX sensitivity in most resistant cell lines. In summary, we find that acquired resistance to VTX in MM is largely associated with BCL2 family regulation, including upregulation of survival members such as MCL-1, BCLXL, BCL2, and downregulation of pro-apoptotic members, especially BIM. Our study also indicates that upstream signaling involved in BCL2 family regulation during acquired resistance is likely related to cytokine, growth factor, and/or RTK-induced cell signaling such as PI3K. Co-inhibition of MCL-1, or BCLXL, as well as the upstream PI3K, RTK (FGF and EGF), IGF-1 mediated signaling were effective in overcoming VTX resistance. Disclosures Fonseca: Mayo Clinic in Arizona: Current Employment; Amgen: Consultancy; BMS: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Bayer: Consultancy; Janssen: Consultancy; Novartis: Consultancy; Pharmacyclics: Consultancy; Sanofi: Consultancy; Merck: Consultancy; Juno: Consultancy; Kite: Consultancy; Aduro: Consultancy; OncoTracker: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Consultancy; AbbVie: Consultancy; Patent: Prognosticaton of myeloma via FISH: Patents & Royalties; Scientific Advisory Board: Adaptive Biotechnologies: Membership on an entity's Board of Directors or advisory committees; Caris Life Sciences: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Arsenic trioxide resistance in acute promyelocytic leukemia: More to it than PML mutations

2020 ◽  
Author(s):  
Nithya Balasundaram ◽  
Saravanan Ganesan ◽  
Ezhilarasi Chendamarai ◽  
Hamenth Kumar Palani ◽  
Arvind Venkatraman ◽  
...  
Keyword(s):  
Cell Line ◽  
Arsenic Trioxide ◽  
Acute Promyelocytic Leukemia ◽  
Cell Lines ◽  
Resistant Cell ◽  
Chemotherapeutic Agents ◽  
Resistant Cell Line ◽  
Promyelocytic Leukemia ◽  
Genetic Mutations ◽  
List Type

AbstractAcquired genetic mutations can confer resistance to arsenic trioxide (ATO) in the treatment of acute promyelocytic leukemia (APL). However, such resistance-conferring mutations are rare and do not explain the majority of disease recurrence seen in the clinic. We have generated a stable ATO resistant promyelocytic cell from a ATO sensitive NB4 cell line. We also noted that another ATRA resistant cell line (UF1) was cross resistant to ATO. We have characterized these resistant cell lines and observed that they significantly differed in their immunophenotype, drug transporter expression, drug resistance mutation profile and were also cross-resistant to other conventional chemotherapeutic agents. The NB4 derived resistant cell line had the classical A216V PML-B2 domain mutation while the UF1 cell line did not. Gene expression profiling revealed prominent dysregulation of the cellular metabolic pathways in the resistant cell lines. Glycolytic inhibition by 2-DG was efficient and comparable to the standard of care (ATO) in targeting the sensitive APL cell lines and was also effective in the in vivo transplantable APL mouse model; however, it did not affect the ATO resistant cell lines. The survival of the resistant cell lines was significantly affected by compounds targeting the mitochondrial respiration irrespective of the existence of ATO resistance-conferring genetic mutations. Our data demonstrate the addition of mitocans can overcome ATO resistance. We further demonstrated that the combination of ATO and mitocans has the potential in the treatment of non-M3 AML and the translation of this approach in the clinic needs to be explored further.Key pointsMetabolic rewiring promotes ATO resistance, which can be overcome by targeting mitochondrial oxidative phosphorylation.Combination of ATO and mitocans can be exploited as a potential therapeutic option for relapsed APL and in non-M3 AML patients.

The Proteome Analysis of an Imatinib-Resistant Cell Line Identifies Changes in Molecular Chaperones Such as Heat Shock Proteins (Hsp): A Possible New Mechanism of Imatinib Resistance.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1981-1981 ◽  
Author(s):  
Jean-Max Pasquet ◽  
Marion Pocaly ◽  
Valérie Lagarde ◽  
Béatrice Turcq ◽  
Josy Reiffers ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Heat Shock ◽  
Cell Line ◽  
Cell Lines ◽  
Molecular Chaperones ◽  
Resistant Cell ◽  
Resistant Cell Line ◽  
Mechanism Of Resistance ◽  
Resistant Phenotype ◽  
Imatinib Resistant

Abstract Targeting the tyrosine kinase activity of Bcr-Abl by imatinib mesylate is an attractive therapeutic strategy in chronic myelogenous leukemia (CML) and in Bcr-Abl positive acute lymphoblastic leukemia. However, resistance to imatinib monotherapy is currently a major issue preventing the successful treatment of CML patients. It may be mainly mediated by mutations within the kinase domain of Bcr-Abl and/or amplification of the BCR-ABL genomic locus. The K562-r imatinib-resistant cell line, derived in our laboratories from the sensitive parental K562-s has neither mechanism of resistance, nor overexpression of Src-kinases such as Lyn and Hck, as described for other cell lines. In the current study we used two-dimensional (2D) difference gel electrophoresis (DIGE) and MALDI-TOF-TOF mass spectrometry to compare the proteome of K562-r and K562-s. With the aid of the Image Master TM 2D platinium software, we detected 31 different proteins in K562-r and K562-s. These proteins were classified in 3 different groups. The first includes proteins involved in the synthesis and stability of RNA (hnRNP K, hnRNP H, CstF , transcription elongation factor A protein 1, PCBP2, TCP1), the second encompasses structural proteins (CAPG, fascin, tubulin, vimentin, laminA, C tubulin beta-1 chain, actin cytoplasmic 1, keratin type I type II), and the third was represented by different enzymes participating in general metabolic pathways (glyceraldehyde 3-phosphate dehydrogenase, malate dehydrogenase, mitochondrial precursor, glutamate dehydrogenase 2, pyruvate kinase, PURH protein). Furthermore, chaperone proteins such as heat-shock protein Hsp60, P60HOP or STI-1, Hsp105 and Hsp70 were differentially expressed in the sensitive and resistant cell lines. Since these proteins complex with Hsp90 and this complex has been reported to interact with the Bcr-Abl protein, we focused on these molecular chaperones. Hsp70 family proteins such as Hsc70 and Hsp74 were found to be more expressed 2.5-fold higher in K562-r than in K562-s, and/or exhibited post translational modifications (phosphorylation and acetylation) confirmed by Western blotting. Hsp70 was recently described as an inhibitor of apoptosis (Ray S et al., JBC 2004) and its overexpression in K562-r could thus contribute to its imatinib-resistant phenotype. Preliminary functional studies showed that whereas K562-s and K562-r were equally sensitive to the apoptotic effect of geldanamycin (an inhibitor of Hsp90), the combination of geldanamycin and a proteasome inhibitor (MG132) was more efficient in K562-r than in K562-s (viability of 16% and 40% respectively after 4 days in culture). Ongoing experiments utilizing siRNA against Hsp70 will help understand the link between the expression profile of Hsp proteins and the imatinib-resistant phenotype of this cell line. In conclusion, the use of a new experimental strategy, i.e. proteomic analysis by DIGE and mass spectrometry, allowed us to identify selected proteins whose patterns of expression and post-translational modification may underlie a new mechanism of resistance to imatinib in Bcr-Abl positive cells.

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.

Identification of Genomic Classifiers That Distinguish Induction Failure in T-Lineage Acute Lymphoblastic Leukemia in COG Study 9404.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1826-1826
Author(s):  
Stuart S. Winter ◽  
Hadya Khawaja ◽  
Zeyu Jiang ◽  
Timothy Griffin ◽  
Barbara Asselin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drug Resistance ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Expression Profiles ◽  
Lymphoblastic Leukemia ◽  
Gene Expression Profiles ◽  
Resistant Cell ◽  
Acquired Drug Resistance ◽  
Induction Failure

Abstract The clinical features of age, white count, and presence of extramedullary disease cannot predict risk for induction failure (IF) in patients who present with T-cell acute lymphoblastic leukemia (T-ALL). On the basis of recent observations that gene expression profiles can distinguish clinicopathologic cohorts of patients with acute leukemia, we hypothesized that microarray analyses performed on diagnostic T-ALL bone marrow samples might identify a genomic classifier for IF patients. Using a case-control study design for children and young adults treated for T-ALL on Children’s Oncology Group Study 9404, we analyzed 50 cryopreserved T-ALL samples using Affymetrix U133A Plus 2 genechips, which have 54,000 genes, ESTs and genomic classifiers. Following RMA normalization, we used Prognostic Multi-array Analysis (PAM) to identify a 116-member genomic classifier that could accurately identify all 6 IF cases from the 44 patients who achieved remission. Within the IF cohort, 37 genes were up-regulated and 79 were down-regulated in comparison to other outcome groups. To further investigate the genetic mechanisms governing IF, we developed four cell lines with acquired drug resistance: Jurkat and Sup T1; each having resistance to daunorubicin (DNR) and asparaginase (ASP). Using a comparative analysis for fold-change in gene expression among 6 IF patients and the T-ALL DNR and ASP-resistant cell lines, we identified seven genes that were up-regulated, and another set of seven genes that were commonly down-regulated. To validate the potential use of our 116-member gene set in predicting IF in T-ALL, we tested our genomic classifier in 42 cases which were treated on COG study 8704 and hybridized to the Affymetrix U133Av.2 chip. Because only 85 probes were shared between U133A Plus 2 and U133Av. 2 chips, we employed shrunken class centroids to constrain our classifier to 25 rank-ordered probes. This smaller classifier correctly identified the single IF case in 8704, as well as another patient who was an early treatment failure, indicating that similar genomic classifiers may identify IF patients in different clinical trials. These results indicate that genetic profiling may be useful in prospectively identifying IF patients in T-ALL. In addition, we identified genes that were commonly upregulated in IF patients and T-ALL cell lines with intrinsic drug resistance.

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