261 research outputs found
Abcg2 overexpression represents a novel mechanism for acquired resistance to the multi-kinase inhibitor Danusertib in BCR-ABL-positive cells in vitro.
No full textThe success of Imatinib (IM) therapy in chronic myeloid leukemia (CML) is compromised by the development of IM resistance and by a limited IM effect on hematopoietic stem cells. Danusertib (formerly PHA-739358) is a potent pan-aurora and ABL kinase inhibitor with activity against known BCR-ABL mutations, including T315I. Here, the individual contribution of both signaling pathways to the therapeutic effect of Danusertib as well as mechanisms underlying the development of resistance and, as a consequence, strategies to overcome resistance to Danusertib were investigated. Starting at low concentrations, a dose-dependent inhibition of BCR-ABL activity was observed, whereas inhibition of aurora kinase activity required higher concentrations, pointing to a therapeutic window between the two effects. Interestingly, the emergence of resistant clones during Danusertib exposure in vitro occurred considerably less frequently than with comparable concentrations of IM. In addition, Danusertib-resistant clones had no mutations in BCR-ABL or aurora kinase domains and remained IM-sensitive. Overexpression of Abcg2 efflux transporter was identified and functionally validated as the predominant mechanism of acquired Danusertib resistance <i>in vitro</i>. Finally, the combined treatment with IM and Danusertib significantly reduced the emergence of drug resistance <i>in vitro</i>, raising hope that this drug combination may also achieve more durable disease control <i>in vivo</i>
STAP-2 interacts with and modulates BCR-ABL-mediated tumorigenesis
No full textIn chronic myeloid leukemia (CML), the BCR-ABL fusion oncoprotein activates multiple pathways involved in cell survival, growth promotion and disease progression. In this report, we show that the signal transducing adaptor protein-2 (STAP-2) is involved in BCR-ABL activity. We demonstrate that STAP-2 bound to BCR-ABL, and BCR and ABL proteins, depending on the STAP-2 Src homology 2-like domain. BCR-ABL phosphorylates STAP-2 Tyr250 and the phosphorylated STAP-2 in turn up-regulated BCR-ABL phosphorylation, leading to enhanced activation of downstream signaling molecules including ERK, STAT5, BCL-xL and BCL-2. In addition, STAP-2 interacts with BCR-ABL to alter chemokine receptor expression leading to downregulation of CXCR4 and upregulation of CCR7. The interaction between STAP-2 and BCR-ABL plays a crucial role in conferring a growth advantage and resistance to imatinib, a BCR-ABL inhibitor, as well as tumor progression. Notably, mice injected with BCR-ABL/STAP-2-expressing Ba/F3 cells developed lymph node enlargement and hepatosplenomegaly. Moreover, suppression of STAP-2 in K562 CML cells resulted in no tumor formation in mice. Our results demonstrate a critical contribution of STAP-2 in BCR-ABL activity, and suggest that STAP-2 might be an important candidate for drug development for patients with CML. Further, the expression of STAP-2 provides useful information for estimating the characteristics of individual CML clones
Elevated Bcr-Abl expression levels are sufficient for a haematopoietic cell line to acquire a drug-resistant phenotype
No full textA characteristic feature of chronic myeloid leukaemia (CML) is the inevitable advancement from a treatable chronic phase to a fatal, drug-resistant stage referred to as blast crisis. The molecular mechanisms responsible for this disease transition remain unknown. As increased expression of Bcr-Abl has been associated with blast crisis CML, we have established transfectants in 32D cells that express low and high levels of Bcr-Abl, and assessed their drug sensitivity. Cells with high Bcr-Abl expression levels are resistant to conventional cytotoxic drugs, and also require higher levels of STI571 (an inhibitor of Bcr-Abl), to induce cell death. Co-treatment with cytotoxic drugs and STI571 increased the sensitivity of the drug-resistant cells. Despite the drug-resistant phenotype, high Bcr-Abl levels concomitantly increased the expression of p53, p21, Bax and down-regulated Bcl-2. These cells maintain a survival advantage irrespective of a reduced proportion of cycling cells and the pro-apoptotic shift in gene expression. In addition, the level of Bcr-Abl expression (high or low) does not alter the growth factor independence and elevated Bcl-xL expression observed. Our study indicates that drug resistance can be primarily attained by increased Bcr-Abl expression, and highlights the potential of therapy which combines STI571 with conventional cytotoxic drugs.LR: 20061115; PUBM: Print; JID: 8704895; 0 (Antineoplastic Agents); 0 (Enzyme Inhibitors); 0 (Fusion Proteins, bcr-abl); 0 (Interleukin-3); 0 (Piperazines); 0 (Pyrimidines); 152459-95-5 (imatinib); EC 2.7.1.112 (Protein-Tyrosine Kinases); 2001/05/28 [received]; 2001/08/09 [accepted]; ppublishSource type: Electronic(1
CCR7 is involved in BCR-ABL/STAP-2-mediated cell growth in hematopoietic Ba/F3 cells
No full textChronic myeloid leukemia is a clonal disease characterized by the presence of the Philadelphia chromosome and its oncogenic product, BCR-ABL, which activates multiple pathways involved in cell survival, growth promotion, and disease progression. We previously reported that in murine hematopoietic Ba/F3 cells, signal transducing adaptor protein-2 (STAP-2) binds to BCR-ABL and up-regulates BCR-ABL phosphorylation, leading to enhanced activation of its downstream signaling molecules. The binding of STAP2 to BCR-ABL also influenced the expression levels of chemokine receptors, such as CXCR4 and CCR7. For the induction of CCR7 expression, signals mediated by the MAPK/ERK pathway were critical in Ba/F3 cells expressing BCR-ABL and STAP-2. In addition, STAP-2 cooperated with BCR-ABL to induce the production of CCR7 ligands, CCL19 and CCL21. Our results demonstrate a contribution of CCR7 to STAP-2-dependent enhancement of BCR-ABL-mediated cell growth in Ba/F3 cells. (C) 2015 Elsevier Inc. All rights reserved
Bcr-Abl upregulates cytosolic p21WAF-1/CIP-1by a phosphoinositide-3-kinase (PI3K)-independent pathway
No full textChronic myeloid leukaemia invariably progresses from a drug-sensitive to a drug-resistant, aggressive acute leukaemia. The mechanisms responsible for this are unknown, although loss of p53 has been reported in approximately 25% of cases. Elevated expression of Bcr-Abl is also associated with disease progression. We have shown that cells expressing high levels of Bcr-Abl also express elevated levels of p53 and the cell cycle inhibitor, p21WAF-1. Despite this, cells continue to cycle and are drug resistant. As p21WAF-1 inhibitory activity is associated with nuclear localization, we investigated its localization in Bcr-Abl-expressing cells, and found that it is predominantly cytoplasmic. We have also shown that it associates physically with the serine/threonine kinase AKT, but this association and the cytosolic location of p21WAF-1 are phosphinositide-3-kinase (PI3K) independent. Cytosolic p21WAF-1 has been reported to have a prosurvival role in other transformed cells. In Bcr-Abl-expressing cells, p21WAF-1 rapidly diminishes as the cells are sensitized to apoptosis, using the inhibitor STI571. It is possible therefore that p21WAF-1 could also have a positive, prosurvival role in these cells. This study suggests that, by retaining p21WAF-1 in a cytosolic location, Bcr-Abl can evade the cell cycle arrest normally induced by nuclear p21WAF-1 and therefore also enable the cells to negate an important feature of a tumour suppressor response.LR: 20061115; PUBM: Print; JID: 0372544; 0 (Biological Markers); 0 (CDKN1A protein, human); 0 (Cyclin-Dependent Kinase Inhibitor p21); 0 (Cyclins); 0 (Fusion Proteins, bcr-abl); 0 (Proto-Oncogene Proteins); 0 (Pyrimidines); 0 (ST 1571); 136601-57-5 (Cyclin D1); EC 2.7.1.137 (1-Phosphatidylinositol 3-Kinase); EC 2.7.1.37 (AKT1 protein, human); EC 2.7.1.37 (Protein-Serine-Threonine Kinases); EC 2.7.1.37 (Proto-Oncogene Proteins c-akt); ppublishSource type: Electronic(1
Positive interactions between STAP-1 and BCR-ABL influence chronic myeloid leukemia cell proliferation and survival
No full textChronic myeloid leukemia (CML) is a clonal disease characterized by the presence of the Philadelphia chromosome and its oncogenic product, BCR-ABL, which activates multiple pathways involved in cell survival, growth promotion, and disease progression. We recently reported that signal-transducing adaptor protein 1 (STAP-1) is upregulated in CML stem cells (LSCs) and functions to reduce the apoptosis of CML LSCs by upregulating the STAT5-downstream anti-apoptotic genes. In this study, we demonstrate the detailed molecular interactions among BCR-ABL, STAP-1, and signal transducer and activator of transcription 5 (STAT5). Studies with deletion mutants have revealed that STAP-1 interacts with BCR-ABL and STAT5a through its SH2 and PH domains, respectively, suggesting the possible role of STAP-1 as a scaffold protein. Furthermore, the binding of STAP-1 to BCR-ABL stabilizes the BCR-ABL protein in CML cells. Since STAP-1 is highly expressed in CML cells, we also analyzed the STAP-1 promoter activity using a luciferase reporter construct and found that NFATc1 is involved in activating the STAP-1 promoter and inducing STAP-1 mRNA expression. Our results demonstrate that STAP-1 contributes to the BCR-ABL/STAT5 and BCR-ABL/Ca2+/NFAT signals to induce proliferation and STAP-1 mRNA expression in CML cells, respectively
Apoptosis and mitotic slippage following drug intervention in leukaemia cells
No full textPhDThe response of leukaemia cells to therapeutic agents includes cell cycle arrest and apoptosis. The former response is useful in retarding disease progression, but induction of the latter is essential for disease eradication. Cell death is often related to toxicity so reducing drug-concentration or sensitising target cells to apoptosis is desirable. The relationship between the cell cycle and cell death has been at the centre of recent investigation focusing on mechanisms of cell death that are not driven directly by apoptotic responses. These mechanisms included mitotic catastrophe and mitotic slippage.
The K562 myeloid leukaemia cell line exhibits a combination of p53 negativity and carries the Bcr-Abl t (9:22) Philadelphia chromosome. Bcr-Abl is a powerful anti-apoptotic translocation and is the hallmark of chronic myeloid leukaemia (CML). The absence of p53-mediated apoptosis and the anti-apoptotic effects of Bcr-abl delays drug-induced cell death, leaving a window of opportunity to investigate the effects of different agents on leukaemia cells.
My investigations show that when DNA-targeting agents are used against myeloid leukaemia cells, G2 cell cycle arrest and apoptosis do not occur together i.e. cell cycle arrest precludes cell death; cells may escape G2 arrest as a result of mitotic slippage. In contrast, when anti-mitotic agents are used, it is necessary to induce mitotic arrest to subsequently induce apoptosis; thus lower concentrations are more effective in inducing apoptosis than higher drug concentrations. Evidence is provided suggesting reduced concentrations of both genotoxic agents and anti-mitotic agents may share a common pathway in inducing cell death that is related to events at mitosis and I suggest that this pathway has potential for exploitation by new agents currently in clinical trials, such as UCN-01, Purvanol, Roscovitine and agents that target the passenger proteins, in reducing the concentration of more conventional agents required to kill the Bcr-Abl positive leukaemias
The activation of IgM- or isotype-switched IgG- and IgE-BCR exhibits distinct mechanical force sensitivity and threshold
No full textB lymphocytes use B cell receptors (BCRs) to sense the physical features of the antigens. However, the sensitivity and threshold for the activation of BCRs resulting from the stimulation by mechanical forces are unknown. Here, we addressed this question using a double-stranded DNA-based tension gauge tether system serving as a predefined mechanical force gauge ranging from 12 to 56 pN. We observed that IgM-BCR activation is dependent on mechanical forces and exhibits a multi-threshold effect. In contrast, the activation of isotype-switched IgG- or IgE-BCR only requires a low threshold of less than 12 pN, providing an explanation for their rapid activation in response to antigen stimulation. Mechanistically, we found that the cytoplasmic tail of the IgG-BCR heavy chain is both required and sufficient to account for the low mechanical force threshold. These results defined the mechanical force sensitivity and threshold that are required to activate different isotyped BCRs
STAP‐2 negatively regulates BCR‐mediated B cell activation by recruiting tyrosine‐protein kinase CSK to LYN
No full textAlthough signal-transducing adaptor protein-2 (STAP-2) acts in certain immune responses, its role in B cell receptor (BCR)-mediated signals remains unknown. In this study, we have revealed that BCR-mediated signals, cytokine production and antibody production were increased in STAP-2 knockout (KO) mice compared with wild-type (WT) mice. Phosphorylation of tyrosine-protein kinase LYN Y508 was reduced in STAP-2 KO B cells after BCR stimulation. Mechanistic analysis revealed that STAP-2 directly binds to LYN, dependently of STAP-2 Y250 phosphorylation by LYN. Furthermore, phosphorylation of STAP-2 enhanced interactions between LYN and tyrosine-protein kinase CSK, resulting in enhanced CSK-mediated LYN Y508 phosphorylation. These results suggest that STAP-2 is crucial for controlling BCR-mediated signals and antibody production by enhanced CSK-mediated feedback regulation of LYN
A Novel FRET-Based Biosensor for the Measurement of BCR-ABL Activity and Its Response to Drugs in Living Cells
No full textPurpose: To develop a novel diagnostic method for the assessment of drug efficacy in CML patients individually, we generated a biosensor that enables the evaluation of BCR-ABL kinase activity in living cells using the principle of fluorescence resonance energy transfer (FRET). Experimental Design: To develop FRET-based biosensors, we utilized CrkL, the most characteristic substrate of BCR-ABL, and designed a protein in which CrkL is sandwiched between Venus, a variant of YFP, and ECFP, so that CrkL intra-molecular binding of the SH2 domain to phosphorylated tyrosine (Y207) increases FRET efficiency. After evaluation of the properties of this biosensor by comparison to established methods including western blotting and flow cytometry, BCR-ABL activity and its response to drugs were examined in CML patient cells. Results: After optimization, we obtained a biosensor that possesses higher sensitivity than that of established techniques with respect to measuring BCR-ABL activity and its suppression by imatinib. Thanks to its high sensitivity, this biosensor accurately gauges BCR-ABL activity in relatively small cell numbers and can also detect less than 1% minor drug-resistant populations within heterogeneous ones. We also noticed that this method enabled us to predict future onset of drug resistance as well as to monitor the disease status during imatinib therapy, using patient cells. Conclusion: In consideration of its quick and practical nature, this method is potentially a promising tool for the prediction of both current and future therapeutic responses in individual CML patients, which will be surely beneficial for both patients and clinicians
- …
