CCN3, a Novel Growth Inhibitory Factor for Chronic Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4230-4230
Author(s):  
Wanhua Lu ◽  
Lynn McCallum ◽  
Bernard Perbal ◽  
Nourreddine Lazar ◽  
Alexandra Irvine
Keyword(s):  
Cell Cycle ◽  
Growth Inhibition ◽  
Signaling Pathways ◽  
K562 Cells ◽  
Imatinib Treatment ◽  
Down Regulation ◽  
Abl Kinase ◽  
Moderate Growth ◽  
Growth Inhibitory ◽  
Ccn3 Expression

Abstract Chronic Myeloid Leukaemia (CML) is characterized by expression of the constitutively active BCR-ABL tyrosine kinase. Previously, we identified down-regulation of the negative growth regulator, CCN3, as a result of BCR-ABL kinase activity. Reduced CCN3 expression is a prominent feature in both primary human CML cells and cell lines. We now show that CCN3 is growth inhibitory and enhances imatinib induced growth inhibition. To evaluate the biological consequence of CCN3 expression in CML, K562 cells were stably transfected with a construct containing CCN3 (pCMV82-23) and growth characteristics and activation of signaling pathways were compared to cells transfected with empty vector (control). CCN3 expression was undetected by Real-time PCR in control cells whilst pCMV82-23 cells expressed 2.25 × 106 copies per 50ng of cDNA. pCMV82-23 cells showed reduced colony formation capacity (p=0.003) and reduced cell growth over a period of five days (p=0.005). Investigation of cellular signaling showed CCN3 expression resulted in significant down-regulation of three major signaling pathways and demonstrated reduced phosphorylation of ERK (p=0.002), pAKT (p=0.017) and pSTAT5 (p=0.005) compared to control cells. Protein levels for total ERK, AKT and STAT5 were unaffected by CCN3 expression. Flow cytometry showed that sustained CCN3 expression resulted in an accumulation of cells within the subG0 stage of the cell cycle (11.4% ± 3 (p=0.040)). To determine if CCN3 expression could influence sensitivity to the BCR-ABL kinase inhibitor, imatinib, pCMV82-23 cells and control cells were treated with imatinib (5uM) for 48h. Control cells treated with imatinib showed moderate growth inihibition (19.6% ± 2.5) compared to untreated control. pCMV82-23 cells showed a significant increase in the magnitude of imatinib induced growth inhibition (63.3% ± 10.5 (p=0.043)). This was associated with an increased accumulation of cells in the subG0 area of the cell cycle, 34.6% ± 5 for pCMV82-23 cells compared to control cells (21.7% ± 8) in response to imatinib treatment (p=0.006). To then determine if these effects could be reproduced using recombinant CCN3 (rCCN3), K562 cells were treated with imatinib (5uM) alone or in combination with rCCN3 (10nM) for 48h. K562 cells treated with the combination of rCCN3 and imatinib showed enhanced growth inhibition (71.8% ± 7.9) compared to cells treated with imatinib alone (81.1% ± 9.2 (p=0.008)). Loss of CCN3 is consistent with properties associated with the CML phenotype. Sustained expression of CCN3 in K562 cells restores growth control and re-establishes induction of apoptosis. Both increased expression of CCN3 or addition of the recombinant protein provide additional benefit for imatinib induced growth inhibition thus providing a novel avenue for therapeutic intervention.

BCR-ABL Decreases the Expression of CCN3 Leading to Increased Clonogenic Potential and Cell Growth.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1216-1216
Author(s):  
Lynn M.R. McCallum ◽  
Wanhua Lu ◽  
Susan Price ◽  
Nathalie Planque ◽  
Bernard Perbal ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Regulator ◽  
Methyl Cellulose ◽  
K562 Cells ◽  
Fold Increase ◽  
Reciprocal Relationship ◽  
Abl Tyrosine Kinase ◽  
Clonogenic Potential ◽  
Ccn3 Expression ◽  
Negative Growth

Abstract Chronic Myeloid Leukemia (CML) is characterized by expression of the constitutively active BCR-ABL tyrosine kinase. Previously, we have identified downregulation of the negative growth regulator, CCN3, as a result of BCR-ABL kinase activity and detected reduced CCN3 expression in human CML cell lines and primary human CML cells. We now report a reciprocal relationship of BCR-ABL and CCN3 expression and the functional consequence of expressing CCN3 in BCR-ABL+ cells. Real-time PCR was used to examine the relationship between BCR-ABL and CCN3 expression in human K562 cells. Parental K562 cells showed high expression of BCR-ABL (4.68 x104 transcripts in 5 μL of cDNA) whilst CCN3 expression was not detected. Treatment with siRNA directed against BCR-ABL (0.5 μg per106 cells) for 72 hours resulted in a 3.7 fold decrease in BCR-ABL and 6.1 fold increase in CCN3 expression (mean Ct change 1.9 ± 0.2 and 2.6 ± 0.5 for BCR-ABL and CCN3 respectively, n=3, p=0.001). Similarly, K562 cells treated with imatinib (1 micromolar) for 96 hours showed a 5.9 fold decrease in BCR-ABL expression and a 4.2 fold increase in CCN3 expression (mean Ct change 2.5 ± 0.1 and 2.1± 0.2 for BCR-ABL and CCN3 respectively, n=3, p=0.001). To investigate CCN3 function, we expressed CCN3 in BCR-ABL+ cells using Nucleofector technology (Amaxa, GmbH). K562 cells were transfected with either the pCb6+ vector (Invitrogen,UK) or pCb6+ vector containing the CCN3 construct. Cells were analysed 24 hours post-transfection by flow cytometry and also after 7 days in methyl cellulose culture to determine clonogenicity. Cell cycle analysis was performed on 20,000 events using the winMDI software. CCN3 expression in BCR-ABL+ cells resulted in an accumulation of cells in the subG0 phase of the cell cycle (mean for subG0 9.9% ± 4.6 and 21.8% ± 0.7 for the pCb6+ vector alone and pCb6+ vector containing CCN3 construct respectively). CCN3 expression significantly increased the number of cells within the subG0 area of the cell cycle (n=3, p=0.027). In addition, CCN3 expression reduced the clonogenic capacity of BCR-ABL+ cells. K562 cells transfected with the pCb6+ vector containing CCN3 construct formed significantly fewer colonies on methyl cellulose in comparison to cells that had been transfected with the pCb6+ vector alone (n=3, p=0.027). This study demonstrates a reciprocal relationship between CCN3 and BCR-ABL expression. CCN3 is known to be a negative growth regulator and increased expression of CCN3 in BCR-ABL+ cells inhibits proliferation and decreases clonogenic potential. Thus CCN3 down-regulation mediated by BCR-ABL offers growth advantage to hematopoietic cells.

Activity of the Aurora Kinase Inhibitor, MLN8237 (alisertib) Alone or in Combination with Ponatinib Against Imatinib-Resistant BCR-ABL-Positive Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 1333-1333
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Seiichiro Katagiri ◽  
Yuko Tanaka ◽  
Kazuma Ohyashiki
Keyword(s):  
Cell Cycle ◽  
Cell Growth ◽  
Growth Inhibition ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Aurora Kinase ◽  
Dependent Manner ◽  
Cell Growth Inhibition ◽  
Cycle Arrest ◽  
T315i Mutation

Abstract Abstract 1333 Chronic myeloid leukemia (CML) is characterized by cytogenetic aberration (Philadelphia chromosome: Ph) and chimeric tyrosine kinase BCR-ABL. ABL tyrosine kinase inhibitor, imatinib has demonstrated the potency against CML patients. However, resistance to imatinib can develop in CML patients due to BCR-ABL point mutations. One of T315I mutation is resistant to currently available ABL tyrosine kinase inhibitors. Therefore, new approach against T315I mutant may improve the outcome of Ph-positive leukemia patients. Aurora kinases are serine/threonine kinases and upregulated in many malignancies including leukemia, and play an important role in cell cycle control and tumor proliferations. Because Aurora kinases are overexpressed in leukemia cells, Aurora kinases may present attractive targets for leukemia treatment. One of Aurora kinase inhibitor, MLN8237 (alisertib) is an oral and selective Aurora kinase A inhibitor and is currently being investigated in a pivotal phase 3 clinical trial against hematological malignancies. We suggested that alisertib mediated inhibition Aurora kinase activity and in combination with ponatinib, also known as AP24534 may abrogate the proliferation and survival of Ph-positive cells including T315I mutation. In this study, we investigated the combination therapy with a ponatinib and an alisertib by using the BCR-ABL positive cell line, K562, murine Ba/F3 cell line which was transfected with T315I mutant, ponatinib resistant Ba/F3 cells and T315I primary sample. Protein expression of Aurora A and B were increased in Ph-positive leukemia cells. 72 hours treatment of alisertib exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. Alisertib also induced cell cycle arrest. The treatment of ponatinib exhibits cell growth inhibition partially against K562 cells in the presence of feeder cell (HS-5) conditioned media. We found that the treatment of alisertib abrogated the protective effects of HS-5 conditioned media in K562 cells. We investigated the alisertib activity against T315I positive cells. Alisertib potently induced cell growth inhibition of Ba/F3 cells ectopically expressing T315I mutation and induced cell cycle arrest. We investigated the efficacy between ponatinib and alisertib by using these cell lines. Combined treatment of Ba/F3 T315I cells with ponatinib and alisertib caused significantly more cytotoxicity than each drug alone. Ponatinib and alisertib were also effective against T315I primary samples. We examined the intracellular signaling of alisertib. Phosphorylation of Aurora A was inhibited in a time dependent manner. We also found the phosphorylation of histone H3 was also reduced in a dose dependent manner suggested that high concentration of alisertib also inhibits Aurora B activity. We next investigated by using ponatinib resistant Ba/F3 cells. In the ponatinib resistant cell lines, IC50 of ponatinib was up to 200 nM. BCR-ABL triple point mutations (T315I, E255K and Y253H) were detected by direct sequence analysis. The treatment of alisertib exhibits cell growth inhibition against Ba/F3 ponatinib resistant cells in the dose dependent manner. Alisertib induced cell cycle arrest in ponatinib resistant cells. Combined treatment of Ba/F3 ponatinib resistant cells with ponatinib and alisertib caused significantly more cytotoxicity. To assess the activity of alisertib and ponatinib, we performed to test on CML tumor formation in mice. We injected nude mice subcutaneously with 1×107 Ba/F3 T315I cells. A dose of 30 mg/kg/day p.o of ponatinib and 30 mg/kg/day p.o of alisertib inhibited tumor growth and reduced tumor volume compared with control mice. The treatments were well tolerated with no animal health concerns observed indicating the feasibility of alisertib combination strategies in the clinic. Data from this study suggested that administration of the ponatinib and Aurora inhibitor, alisertib may be a powerful strategy against BCR-ABL mutant cells including T315I. Disclosures: No relevant conflicts of interest to declare.

Bcr-Abl kinase down-regulates cyclin-dependent kinase inhibitor p27 in human and murine cell lines

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1933-1939 ◽  
Author(s):  
Tarja Jonuleit ◽  
Heiko van der Kuip ◽  
Cornelius Miething ◽  
Heike Michels ◽  
Michael Hallek ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Cell Cycle Progression ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Down Regulation ◽  
Cycle Progression ◽  
Cyclin Dependent Kinases ◽  
Abl Kinase

Abstract Chronic myeloid leukemia (CML) is a malignant stem cell disease characterized by an expansion of myeloid progenitor cells expressing the constitutively activated Bcr-Abl kinase. This oncogenic event causes a deregulation of apoptosis and cell cycle progression. Although the molecular mechanisms protecting from apoptosis in CML cells are well characterized, the cell cycle regulatory event is poorly understood. An inhibitor of the cyclin-dependent kinases, p27, plays a central role in the regulation of growth factor dependent proliferation of hematopoietic cells. Therefore, we have analyzed the influence of Bcr-Abl in the regulation of p27 expression in various hematopoietic cell systems. An active Bcr-Abl kinase causes down-regulation of p27 expression in murine Ba/F3 cells and human M07 cells. Bcr-Abl blocks up-regulation of p27 after growth factor withdrawal and serum reduction. In addition, p27 induction by transforming growth factor-beta (TGF-β) is completely blocked in Bcr-Abl positive M07/p210 cells. This deregulation is directly mediated by the activity of the Bcr-Abl kinase. A Bcr-Abl kinase inhibitor completely abolishes p27 down-regulation by Bcr-Abl in both Ba/F3 cells transfected either with a constitutively active Bcr-Abl or with a temperature sensitive mutant. The down-regulation of p27 by Bcr-Abl depends on proteasomal degradation and can be blocked by lactacystin. Overexpression of wild-type p27 partially antagonizes Bcr-Abl–induced proliferation in Ba/F3 cells. We conclude that Bcr-Abl promotes cell cycle progression and activation of cyclin-dependent kinases by interfering with the regulation of the cell cycle inhibitory protein p27.

A Potent Src/Abl Kinase Inhibitor (Ap23464) Potently Inhibits the Growth of Myeloid Leukemic Cells Characterized by Flt3-Itd Expression, GM-CSF Dependency, or G-CSF Responsiveness Via an Abl-Independent Mechanism.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4479-4479
Author(s):  
Jukka Kanerva ◽  
Ogonna Nwawka ◽  
Kevin Hwang ◽  
William C. Shakespeare ◽  
Chester A. Metcalf ◽  
...  
Keyword(s):  
Growth Inhibition ◽  
Cell Lines ◽  
Western Blotting ◽  
Tyrosine Kinases ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Positive Control ◽  
Gm Csf ◽  
Abl Kinase ◽  
Imatinib Resistant

Abstract AP23464 is a potent Src and Abl inhibitor (in vitro IC50 < 1 nM), which has been shown to effectively inhibit growth of imatinib-resistant Bcr-Abl+ cells, and thus, is a promising compound for treating patients with imatinib-resistant leukemia. We conducted a study to evaluate growth inhibition and inhibition of Src versus Abl protein tyrosine kinases in human myeloid cell lines: MV4-11 expressing an internal tandem duplication of Flt3 (Flt3-ITD), the murine pro-B cell line Ba/F3 that expresses the Flt3-ITD, the GM-CSF dependent Mo7e, and the G-CSF-responsive BaF3-GR (Ba/F3 cells expressing the human G-CSF receptor). We compared AP23464 with the PP1, a previously described Src kinase inhibitor (IC50 < 1 uM). We sought to correlate growth inhibition with Src or Abl inhibition. Methods: Growth inhibition was assessed by Trypan blue exclusion and MTT assay using drug concentrations 0.1 uM – 10 uM. Drugs were added daily to the cell suspension during the 3-day experiment. After a 60 min incubation at concentrations 0.1 nM – 1 uM, Src or Abl kinase inhibition was analyzed by blotting with a polyclonal phospho-Src (Tyr416) antibody or a polyclonal phospho-Abl (Tyr245) antibody. Results: In MV4-11 cells AP23464 was more potent than PP1 in causing growth inhibition with IC50 at <1 uM vs 2.5 to 5 uM. By western blotting, inhibition of phospho-Src 416 occurred at the lowest dose of AP23464 and PP1 studied (0.1 nM and 1 nM, respectively). Abl was not detected in MV4-11 cells. In Ba/F3-ITD cells, the IC50 for AP23464 was 1 uM (grown in IL-3) and 0.01–0.1 uM (grown without IL-3). In Ba/F3-ITD cells, IC50 for phosphoSrc was 0.1 uM for both AP23464 and PP1. Abl was present in Ba/F3-ITD cells, but no phospho-Abl was detected. In Mo7e cells grown in the presence of GM-CSF, the IC50 was 1 uM for AP23464 v. 10 uM for PP1. In Mo7e cells treated with Lyn siRNA, there was >50% growth inhibition with 70% knock-down of Lyn. The IC50 for phosphoSrc was 1 nM for both AP23464 and PP1. Abl was present in Mo7e cells, but no phosphoAbl was demonstrated (K562 cells served as positive control). In BaF3-GR cells grown in G-CSF, the IC50 was 1 uM for AP23464 vs. 10 uM for PP1. In western blotting, the IC50 for phospho-Src 416 was detected at 10 nM AP23464. Abl was present in Ba/F3GR cells, but no phosphoAbl was demonstrated (K562 cells served as positive control). Conclusions: AP23464 is more potent than PP1 in causing growth inhibition and Src kinase inhibition in these cell lines that serve as models for acute myeloid leukemia. It is unlikely that Abl is the drug target, because MV4-11 cells do not express detectable Abl, and phospho-Abl was not detected in Mo7e or Ba/F3 cells. These results suggest that inhibition of Src tyrosine kinases contributes predominantly to growth inhibition caused by the Src/Abl kinase inhibitor AP23464 in these cell types.

Identification and Functional Significance of Genes Regulated by Structurally Different ABL Kinase Inhibitors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2860-2860
Author(s):  
Kousuke Nunoda ◽  
Tetsuzo Tauchi ◽  
Tomoiku Takaku ◽  
Masahiko Sumi ◽  
Seiichi Okabe ◽  
...  
Keyword(s):  
Gene Expression ◽  
Growth Inhibition ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Specific Inhibitor ◽  
K562 Cells ◽  
Myelogenous Leukemia ◽  
Altered Expression ◽  
Abl Kinase ◽  
Abl Kinase Inhibitors

Abstract Imatinib is an ABL-specific inhibitor that binds with high affinity to the inactive conformation of the BCR-ABL tyrosine kinase and has been shown to be effective in the treatment of chronic myelogenous leukemia. Dasatinib is an ATP-competitive, dual-spesific SRC and ABL kinase inhibitor that can bind BCR-ABL in both the active and inactive conformations. From a clinical stand point, dasatinib is particular attractive because it has been shown to induce hematologic and cytogenetic responses in imatinib-resistant CML patients. In the view of the fact that the combination of imatinib and dasatinib shows the additive/synergistic growth inhibition on a wild type p210 BCR-ABL expressing cells, we reasoned that these ABL kinase inhibitors might induce the different molecular pathways. To address this question, we used DNA microarrays to identify genes whose transcription was altered by imatinib and dasatinib. K562 cells were cultured with imatinib or dasatinib for 16 hrs, and gene expression data was obtained from three independent microarray hybridizations. Almost all of the imatinib- and dasatinib- responsive genes appeared to be similarly increased or decreased in K562 cells; however, small subsets of genes were identified as selectively altered expression by either imatinib or dasatinib. The genes whose expression was affected by imatinib and dasatinib were categorized into different functional groups based on their biological function, and genes in the cell proliferation and apoptosis categories were examined in detail. Imatinib and dasatinib affected the expression of several cyclin-dependent kinases (CDK2, CDK4, CDK6, CDK8, and CDK9), cell division cycle genes (CDC6, CDC7, CDC25C, and CDC34), and cyclones (cyclin A2, C, D2, D3, E1, E2, F, G1, G2, and H). Imatinib and dasatinib also modulated the expression of apoptosis-related genes including APAF1, BAK1, BCL2, BCL10, MCL1, CASP3, and CASP6). One of the distinct genes which are selectively modulated by dasatinib are CDK2 and CDK8, which had a maximal fold reduction of <8-fold in microarray screen. Immunoblotting confirmed that gene expression changes induced only by dasatinib correlated with changes in protein expression. To assess the functional importance of dasatinib regulated genes, we used RNA interference to determine whether reduction of CDK2 and CDK8 affected the growth inhibition. The siRNA to CDK2 or CDK8 specifically reduced cdk2 or cdk8 in K562 cells. K562 cells pretreated with CDK2 or CDK8 siRNA showed the additive growth inhibition with imatinib but not with dasatinib. These finding demonstrate that the additive/synergistic growth inhibition by imatinib and dasatinib may be mediated by CDK2 and CDK8.

Effective Targeting of MEK/ERK Signalling at Nanomolar Concentrations by the Novel Small Molecule Inhibitor PD0325901 in Hematopoietic and Solid Tumors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3370-3370 ◽  
Author(s):  
Maria R. Ricciardi ◽  
Maria C. Scerpa ◽  
Ludovica Ciuffreda ◽  
Sabina Chiaretti ◽  
Simona Tavolaro ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Inhibition ◽  
Cell Lines ◽  
Breast Cancer Cell Lines ◽  
Cell Cycle Distribution ◽  
Erk Activation ◽  
Erk Phosphorylation ◽  
Growth Inhibitory ◽  
Molecule Inhibitor ◽  
Dose Dependent

Abstract In the MAPK module, MEK lies upstream of ERK which is found constitutively activated in a host of human tumors. We already demonstrated the growth inhibitory activity of MEK inhibitors in myeloid cells (JCI 2001, Leukemia 2005). PD0325901 is the latest small-molecule inhibitor of MEK with promising effects at lower concentrations. We tested its activity (0.1–1000 nM) in a broad spectrum of leukemia, melanoma and breast cancer cell lines evaluating changes on cell cycle distribution, apoptosis, protein and gene expression profiles, aiming at defining the molecular signature induced by this MEK inhibitor. Among hematopoietic cell lines, PD0325901 induced a marked growth inhibition in myeloid cells with constitutive ERK activation (IC50=11 and 12 nM for OCI-AML3 and OCI-AML2, respectively). Conversely, relative resistance to PD0325901-mediated growth inhibition (IC50>1μM) was observed in myeloid cell lines without constitutive ERK activation (U937, KG-1) and in lymphoid cell lines (Raji, Jurkat). Among the solid tumor cell lines, the M14 melanoma was markedly sensitive to PD0325901-induced growth inhibition (IC50=24 nM), even with forced Bcl-2 expression (IC50 ranging from 64 to 200 nM in Bcl-2-overexpressing clones). Conversely, the breast cancer cell lines tested (SKBr3, BT474, MDA-MB-231 and ZR75-1) proved relatively resistant (IC50≥1 μM), regardless of the ERK phosphorylation status. In responsive cells (OCI-AML3, OCI-AML2 and M14), PD0325901 inhibited ERK phosphorylation in a dose-dependent manner, the effect was already evident at 15 min. Cell cycle distribution analysis demonstrated a dramatic dose-dependent decrease in the proliferative compartment in OCI-AML3. Subsequently (48–72 h), PD0325901 induced apoptosis in a dose- and time-dependent fashion, as demonstrated by the increase in the percentage of AnnV+ cells from 6.3%±1.1 (DMSO control) to 15.5%±3.9, 31.5%±2.8 and 45.3%±0.14 (10, 100, and 1000 nM PD0325901, respectively). Gene expression profiling was conducted using AffymetrixTM HG-U133A 2.0 GeneChip® in OCI-AML3 cells exposed to DMSO (control) or PD0325901 at 10nM. Using a p<0.05 and a fold changes ≥ 2.0 cut-off, 16 genes were found to be differentially expressed (3 upregulated and 13 downregulated) after 6h of PD0325901 treatment. These effects were even more pronounced after 24h of treatment and, in addition, at this time other genes turned out to be modulated by PD0325901 treatment (a total of 37 were upregulated, 59 downregulated); PD0325901 induced transcriptional changes mostly in genes responsible for cell growth/proliferation, DNA replication and cell signalling. In conclusion, we found that PD0325901, at nanomolar concentrations, displays a promising growth-inhibitory and pro-apoptotic activity in cells with constitutive ERK activation (particularly myeloid leukemias and melanoma). We demonstrated that the expression gene profile of OCI-AML3 is profoundly altered by PD0325901 treatment, particularly reflecting changes in genes involved in the MEK-dependent regulation of cell cycle, as well as new genes potentially useful candidates for further investigation.

The Efficacy of Dual PI3K and mTOR Inhibitor, NVP-BEZ235 against BCR-ABL Positive Leukemia Cells Include ABL Kinase Domain Mutation in Combination with Nilotinib.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3784-3784
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Shinya Kimura ◽  
Taira Maekawa ◽  
Kazuma Ohyashiki
Keyword(s):  
Cell Growth ◽  
Growth Inhibition ◽  
K562 Cells ◽  
Kinase Domain ◽  
Dependent Manner ◽  
Cell Growth Inhibition ◽  
Dual Inhibitor ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Primary Sample

Abstract Abstract 3784 Poster Board III-720 Imatinib has shown clinical efficacy against chronic myeloid leukemia (CML) and it is now the standard care fore initial therapy of CML. However, a substantial number of patients are either primary refractory or acquire resistance to imatinib. Mutation in the ABL kinase domain is the principal mechanism of imatinib resistance in patients with BCR-ABL-positive leukemia and this becomes a problematic in clinically. Phosphoinositide 3 kinase (PI3K)/Akt /mammalian target of rapamycin (mTOR) pathway regulates various processes including cell proliferation, survival and anti-apoptosis activity. Moreover, PI3K/Akt/mTOR pathway is desregulated in hematological malignancies and activated by upstream proteins such as BCR-ABL. NVP-BEZ235 is a dual inhibitor of PI3K and mTOR. It has been reported NVP-BEZ235 showed high target specificity and demonstrated antiproliferative activity against tumor. However, the molecular and functional consequences of NVP-BEZ235 against BCR-ABL expressing cells with BCR-ABL kinase domain mutation have not fully known. In this study, we investigated the NVP-BEZ235 efficacy by using the BCR-ABL positive cell line, K562 and murine Ba/F3 cell line which was transfected wild type (Wt) p210 BCR-ABL or imatinib resistant BCR-ABL mutants such as E255K, T315I and primary sample with T315I mutation. 48 hours treatment of NVP-BEZ235 exhibits cell growth inhibition and induced apoptosis against K562 cells in a dose dependent manner. We also found that NVP-BEZ235 potently induced cell growth inhibition of murine Ba/F3 cells ectopically expressing wild type (Wt) p210 and imatinib resistant BCR-ABL mutants such as E255K and T315I mutation. We next examined the intracellular signaling by using these cell lines. We found that phosphorylation of Akt, eukaryotic initiation factor 4-binding protein 1 (4E-BP1) and p70 S6 kinase were decreased after NVP-BEZ235 treatment in a dose dependent manner. However, Mitogen-activated Protein Kinase (MAPK) activation was not reduced after NVP-BEZ235 treatment. Because Abl kinase inhibitor, nilotinib has shown efficacy against imatinib resistant BCR-ABL mutant, we investigated the efficacy between NVP-BEZ235 and nilotinib by using these cell lines. We found that combination of NVP-BEZ235 and nilotinib more potentially cell growth inhibition of K562, Ba/F3 Wt BCR-ABL, BCR-ABL mutations (E255K, T315I) Ba/F3 cells. We found that combination of NVP-BEZ235 and nilotinib more potentially induced apoptosis in 48 hours treatment. Phosphorylation of Akt, 4E-BP-1, S6 kinase was reduced after NVP-BEZ235 and nilotinib treatment in K562 cells. Cyclin D1 and BCL-XL were also decreased. PARP activation was synergistically increased after NVP-BEZ235 and nilotinib treatment. We next examined the affectivity of NVP-BEZ235 and nilotinib by using T315I positive primary sample. We found that NVP-BEZ235 and nilotinib potently induced cell growth inhibition and also increased apoptosis of primary T315I cells in 48 hours treatment. Data from this study suggested that administration of the a dual inhibitor of PI3K and mTOR, NVP-BEZ235 may be a powerful strategy against BCR-ABL mutant cells and enhance cytotoxic effects of nilotinib in those imatinib resistant BCR-ABL mutant cells. Disclosures: No relevant conflicts of interest to declare.

Potentiation of the Antileukemic Effects of Imatinib through the Modulation of BCRP/ABCG2 Activity.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3400-3400
Author(s):  
Eliza Glodkowska-Mrowka ◽  
Magda Winiarska ◽  
Piotr Mrowka ◽  
Grzegorz Basak ◽  
Joanna Niesiobedzka-Krezel ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Lines ◽  
Abc Transporters ◽  
Kinase Inhibitors ◽  
K562 Cells ◽  
Intracellular Concentration ◽  
Drug Efflux ◽  
Antileukemic Activity ◽  
Abl Kinase ◽  
Cd34 Cells

Abstract Abstract 3400 Resistance to tyrosine kinase inhibitors (TKI) constitutes a growing problem in chronic myeloid leukemia (CML) therapy. Variations of intracellular concentration of TKIs, dependable on the activity of drug transporters, are among major mechanisms responsible for the development of resistance. Intracellular imatinib concentration depends on the balance of active transport of the drug into the cell, mediated mainly by OCT-1 transporter, and drug efflux out of the cell, mediated by transmembrane pumps belonging to ABC transporters family. While there are reports that imatinib is a substrate for ABCG2 efflux pump, other studies show that it rather acts as an inhibitor of this drug transporter. According to the latest reports both views may be true – in low concentration imatinib seems to be a substrate of ABCG2 pump while in high concentrations it can inhibit activity of the pump. Drugs able to modulate membrane-bound proteins conformation, including drug transporters, may influence intracellular concentration of imatinib. Statins, inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase, widely used in the treatment of hypercholesterolemia, can modulate activity of ABC transporters. Therefore, we evaluated the potential influence of statins on the antileukemic activity of imatinib in cell lines transformed with BCR/ABL oncoprotein and primary human CML CD34+ cells. Lovastatin, a member of statin family, not only exerted antileukemic activity alone, but also significantly enhanced cytotoxicity of imatinib in BCR/ABL-positive murine 32Dcl3 cells and in human K562 cells as compared to 32Dcl3 wild-type counterparts and HL-60 cells respectively, which was measured in cytotoxic assays (i.e. trypan blue, XTT) and clonogenic tests. Similar effects were observed for other statins (incl. atorvastatin, simvastatin and mevastatin). Potentiation of antileukemic activity of imatinib by lovastatin was also observed for primary CML CD34+ cells from patients in different stages of the disease. In contrast, this antileukemic effect was not observed in peripheral blood leukocytes from healthy blood donors. Moreover, in comparison to monotherapy the co-treatment induced cell cycle arrest and increased percentage of apoptotic leukemic cells measured using flow cytometric analysis after propidium iodide staining. Lovastatin also increased imatinib-induced reduction of the phosphorylated form of the adaptor protein CrkL, which serves as a marker for efficient BCR/ABL kinase inhibition in both stable cell lines and primary CML CD34+ cells. Additionally, co-treatment with statin and imatinib has modified cell cycle/apoptosis controlling proteins profile. Cytometric analysis of intracellular concentration of ABCG2 substrate (fluorescent dye BODIPY-prazosin), revealed that lovastatin significantly decreased ABCG2 mediated efflux capacity in BCR/ABL-positive cells. The effect was completely reversed by the addition of cholesterol to the sample previously incubated with lovastatin. These results suggest that statin-mediated depletion of cholesterol may influence conformational status of the efflux pumps and hence can modulate their activity. Such phenomenon was observed both for stable cell lines and primary CD34+ CML cells including cells from patients clinically resistant to imatinib with no detectable ABL kinase domain mutations. To confirm the results of indirect experiments with BODIPY-prazosin we examined whether statins modulate intracellular level of imatinib itself, employing radiolabeled 14C-imatinib. Pretreatment of K562 cells with lovastatin resulted in significant increase in intracellular labeled drug concentration (at least 2-fold). This effect was due to diminished efflux of the drug, since we did not observe changes in initial influx of imatinib between statin-treated and control cells. This suggests that statins may exert significant effect on activity of ABCG2, but not Oct1 transporter. Our results indicate that statins can positively influence the therapeutic potency of imatinib and modification of drug efflux may be a major mechanism responsible for such effect. We conclude that addition of statins to the treatment regimen may become an effective treatment modality capable of potentiating the antileukemic effectiveness of tyrosine kinase inhibitors. Disclosures: No relevant conflicts of interest to declare.

The Heat Shock Protein 32 (Hsp32)/HO-1-Targeting Drug SMA-ZnPP and the Triterpenoid CDDO-Me Exert Synergistic Growth-Inhibitory Effects on TKI-Resistant Leukemic Cells in Ph+ CML

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4414-4414
Author(s):  
Karoline V. Gleixner ◽  
Harald Herrmann ◽  
Katharina Blatt ◽  
Winfried F Pickl ◽  
Marina Konopleva ◽  
...  
Keyword(s):  
Heat Shock ◽  
Growth Inhibition ◽  
Synergistic Effects ◽  
K562 Cells ◽  
Leukemic Cells ◽  
Drug Resistant ◽  
Inhibitory Effects ◽  
Growth Inhibitory ◽  
Imatinib Resistant ◽  
Ic50 Values

Abstract Abstract 4414 Resistance against one or more tyrosine kinase inhibitors (TKI) prevents eradication of Ph+ chronic myeloid leukemia (CML). In many patients BCR/ABL1 mutations are detectable. We have recently identified two targeted drugs that exert major growth-inhibitory effects on drug-resistant CML cells, the triterpenoid CDDO-Me (Bardoxolone-methyl, REATA Pharma) that blocks several signalling molecules including mTOR, Akt, and STAT3, and upregulates expression of heat shock protein 32 (Hsp32 = heme oxygenase 1, HO-1), and styrene-maleic acid-copolymer micelle-encapsulated ZnPP (SMA-ZnPP), a water-soluble inhibitor of Hsp32/HO-1. In the current project, we asked whether CDDO-Me exerts inhibitory effects on growth of TKI-resistant CML cells and whether the combination of CDDO-Me and SMA-ZnPP would produce synergistic effects in drug-resistant CML cells. As determined by 3H-thymidine incorporation, CDDO-Me was found to inhibit the proliferation of imatinib-responsive and imatinib-resistant K562, imatinib-resistant KCL-22, KU812, and Ba/F3 cells transfected with various TKI-resistant mutants of BCR/ABL1 (T315I, E255K, Y253F, H396P). In each case, IC50 values <1 μM were obtained without major differences between imatinib-responsive and imatinib-resistant cells. Growth-inhibition was accompanied by apoptosis as assessed by combined AnnexinV/PI staining as well as by an increase in expression of HO-1 in KU812 and KCL-22 cells. CDDO-Me was also found to inhibit proliferation of leukemic cells in all patients with TKI-resistant CML (n=4), with IC50 values ranging between <0.1 and 0.5 μM. No differences in IC50 values were observed between treatment-naïve and TKI-resistant cells. Next, we applied the combination CDDO-Me+SMA-ZnPP and found that this combination acts highly synergistically on imatinib-responsive and imatinib-resistant K562 cells as well as primary CML cells isolated from imatinib-naïve CML patients (n=2) or from patients with imatinib-resistant CML (n=2), including one patient in whom BCR/ABL1 T315I was detected. We also examined whether CDDO-Me would exert synergistic effects on CML cells when combined with BCR/ABL1 TKI. In these experiments, we applied the combinations CDDO-Me+dasatinib and CDDO-Me+nilotinib on K562 cells. Both combinations were found to synergistically induce growth inhibition. In conclusion, CDDO-Me inhibits the proliferation of imatinib-resistant BCR/ABL1+ cells, including primary CML cells isolated from untreated patients and cells derived from patients with TKI-resistant CML carrying the BCR/ABL1 mutant T315I. Our data also show that CDDO-Me + SMA-ZnPP and CDDO-Me + BCR/ABL1 TKI synergize in producing growth inhibition in CML cells. Whether these drug combinations also produce synergistic effects in vivo in patients with TKI-resistant CML remains to be evaluated. Disclosures: Valent: Novartis: Consultancy, Honoraria, Research Funding.

Bcr-Abl kinase down-regulates cyclin-dependent kinase inhibitor p27 in human and murine cell lines

Blood ◽  
2000 ◽  
Vol 96 (5) ◽  
pp. 1933-1939 ◽  
Author(s):  
Tarja Jonuleit ◽  
Heiko van der Kuip ◽  
Cornelius Miething ◽  
Heike Michels ◽  
Michael Hallek ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Growth Factor ◽  
Cell Cycle Progression ◽  
Molecular Mechanisms ◽  
Transforming Growth Factor ◽  
Kinase Inhibitor ◽  
Down Regulation ◽  
Cycle Progression ◽  
Cyclin Dependent Kinases ◽  
Abl Kinase

Chronic myeloid leukemia (CML) is a malignant stem cell disease characterized by an expansion of myeloid progenitor cells expressing the constitutively activated Bcr-Abl kinase. This oncogenic event causes a deregulation of apoptosis and cell cycle progression. Although the molecular mechanisms protecting from apoptosis in CML cells are well characterized, the cell cycle regulatory event is poorly understood. An inhibitor of the cyclin-dependent kinases, p27, plays a central role in the regulation of growth factor dependent proliferation of hematopoietic cells. Therefore, we have analyzed the influence of Bcr-Abl in the regulation of p27 expression in various hematopoietic cell systems. An active Bcr-Abl kinase causes down-regulation of p27 expression in murine Ba/F3 cells and human M07 cells. Bcr-Abl blocks up-regulation of p27 after growth factor withdrawal and serum reduction. In addition, p27 induction by transforming growth factor-beta (TGF-β) is completely blocked in Bcr-Abl positive M07/p210 cells. This deregulation is directly mediated by the activity of the Bcr-Abl kinase. A Bcr-Abl kinase inhibitor completely abolishes p27 down-regulation by Bcr-Abl in both Ba/F3 cells transfected either with a constitutively active Bcr-Abl or with a temperature sensitive mutant. The down-regulation of p27 by Bcr-Abl depends on proteasomal degradation and can be blocked by lactacystin. Overexpression of wild-type p27 partially antagonizes Bcr-Abl–induced proliferation in Ba/F3 cells. We conclude that Bcr-Abl promotes cell cycle progression and activation of cyclin-dependent kinases by interfering with the regulation of the cell cycle inhibitory protein p27.

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