Loss of SYK and LYN Tyrosine Kinase Expression Impair Ponatinib-Induced Apoptosis in K562 Cells

2015 ◽  
Vol 2 ◽  
pp. 15-19
Author(s):  
Jean-Max Pasquet ◽  
Valérie Lagarde ◽  
François-Xavier Mahon
2001 ◽  
Vol 156 (6) ◽  
pp. 751-760 ◽  
Author(s):  
Soo-Jin Jeong ◽  
Young-Hee Jin ◽  
Chang-Woo Moon ◽  
Hae-Rahn Bae ◽  
Young-Hyun Yoo ◽  
...  

1997 ◽  
Vol 83 (4) ◽  
pp. 756-761 ◽  
Author(s):  
Arunkumar B. Deora ◽  
Michelle B. Miranda ◽  
S.G. Anand Rao

Aims and background K562 cells are growth factor independent and neither function as stem cells nor differentiate into functional end cells. They are blast cells. There is evidence that the constitutively expressed bcr-abl tyrosine kinase might be responsible for the maintenance of the blast state of CML cells. We have studied the effect of two tyrosine kinase inhibitors, quercetin and genistein, on K562 cells. Methods K562 cells were treated with quercetin/genistein for a period of 72 hrs and then subjected to staining for apoptosis and erythroid differentiation and Western blotting with c-abl and phosphotyrosine monoclonal antibodies. Results The IC50 value was found to be 9.2 μg/ml for quercetin and 11.8 μg/ml for genistein. Quercetin-treated cells did not show any differentiation but showed 68% apoptosis as compared to 7% in control. Genistein-treated cells showed 16% apoptosis and 15% erythroid differentiation. Quercetin reduced the level of p210 by 74% and its phosphotyrosine content by 67.6%. Genistein reduced p210 by 77.8% and its phosphotyrosine content by 16%. Conclusion Both quercetin and genistein are able to down-modulate the tyrosine kinase activity of p210 as well as bring about a decrease in the content of the protein with different effects: quercetin induced apoptosis while genistein brought about both differentiation and apoptosis.


Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3342-3342
Author(s):  
Ajoy K. Samanta ◽  
Sandip Chakravorty ◽  
Yan Wang ◽  
Jorge Cortes ◽  
Hagop Kantarjian ◽  
...  

Abstract Chronic myelogenous leukemia (CML) patients treated with imatinib mesylate (IM) results in drug resistance in accelerated and blast crisis stages predominantly involving either mutation of the kinase domain of Bcr-Abl or increased expression of the Lyn tyrosine kinase, whose regulation by Bcr-Abl is not yet understood. Treatment of CML patients with IM can at times cause a decline in levels of wild-type Bcr-Abl protein followed in some cases by activation of Lyn/Hck tyrosine kinases. Lyn is reported to be involved in Bcr-Abl signaling pathway and the Bcr-Abl oncoprotein activates Lyn. Although increased expression of Lyn with the progression of the disease is known and Lyn is involved in the survival of CML cells, its exact position in the Bcr-Abl driven signal transduction pathway, its down-stream targets, its regulation by upstream regulators and the mechanism of continuous activation of Lyn in accelerated and blast crisis stages of CML are still unclear. Our previous reports suggested that Bcr-Abl activates the Jak2 tyrosine kinase, by phosphorylation of the Tyr 1007 which involves the enhanced expression of c-Myc. Recently, we have shown that Jak2 and the Bcr-Abl tyrosine kinase are part of a large Network of signaling proteins (Samanta et al Cancer Research, 2006). Here we report that inhibition of Jak2, a target of Bcr-Abl, by either Jak2-specific siRNA or by the Jak2 inhibitor II (1,2,3,4,5,6-hexabromocyclohexan, Sandberg. et al J.Med. Chem. 2005) or AG490 reduced the level of activated Lyn, pGab2 (YxxM), pAkt, pGSK3 and c-Myc. Inhibition of Lyn by knocking down by Lyn specific siRNA or by Lyn inhibitor resulted the similar results further supporting that Lyn is a downstream target of Jak2. We further explored the possible regulatory mechanism of Lyn by Jak2. Jak2 inhibition decreased the level of the SET protein, which strongly inhibits activity of PP2A. Activation of PP2A combined with Jak2 inhibition enhanced the reduction of activated Lyn compared to Jak2 inhibition alone in BCR-ABL+ cells. In contrast suppression of both PP2A and the tyrosine phosphatase Shp1 interfered with the loss of activated Lyn kinase caused by Jak2 inhibition, indicating the involvement of PP2A and Shp1 in the inactivation of the Lyn kinase by Jak2 inhibition. Inhibition of either Jak2 or Lyn kinase induced apoptosis in BCR-ABL+ cell lines, including imatinib-resistant (IM) T315I Bcr-Abl+ cells. Jak2 inhibition induced apoptosis in CML patient cells accelerated and blast crisis stages but not normal cells including cells resistant to IM. In conclusion, our findings indicate that Lyn is downstream of Jak2 and is regulated by Jak2 in Bcr-Abl+ cells. We have shown that activated Jak2 prevents the dephosphorylation of Tyr 396 of Lyn (the active form of Lyn) in Bcr-Abl+ cells through a SET-dependent inactivation of the PP2A tumor suppressor/Shp1 pathway. Our studies in Bcr-Abl driven CML cells indicate that therapeutic strategies to inhibit Jak2 and its downstream target Lyn may be a powerful approaches for the management of IM-resistant CML specifically T315I where the dual kinase inhibitor -dasatanib fails to kill CML cells.


Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4218-4218
Author(s):  
Francis Belloc ◽  
Kelly Airiau ◽  
Vanessa Augis ◽  
Marie Jeanneteau ◽  
Melissa Garcia ◽  
...  

Abstract Imatinib is an effective first line therapy for early chronic phase chronic myelogenous leukaemia (CML) that acts by targeting the tyrosine kinase activity of BCR-ABL. However, mutations occurred that procure imatinib resistance and, to overcome this, second generation inhibitors of BCR-ABL have been developed. Among these, nilotinib is significantly more potent against BCR-ABL than imatinib but is used in the same order of concentration as imatinib in vivo. Moreover, nilotinib is more specific towards BCR-ABL than imatinib which, at efficient concentration, also inhibits c-KIT. In the current study an in vitro flow cytometry assay to analyze imatinib- and nilotinib-induced apoptosis in CML cells has been developed. Both drugs induced significant apoptosis in CD34 positive cells from 36 CML bone marrow samples collected at diagnosis while CD34 positive cells from 12 other myeloproliferative disorders samples were unaffected, confirming the high specificity of both inhibitors on BCR-ABL positive cells (p<10−9). Nilotinib was confirmed to be 100 times more potent than imatinib to induce apoptosis of CD34 positive cells. When the experiments were performed in the presence of a cocktail of cytokines, inhibitions of 26% for spontaneous apoptosis, 22% for imatinib-induced apoptosis (n=20, p<0.005) and 71% for nilotinib-induced apoptosis (p<10−6) were observed. The inhibition essentially occurred in CD117+/CD34+ cells. This differential inhibition by cytokines was confirmed on K562 cells (34% for imatinib and 71% for nilotinib). A blocking anti-CD117 antibody alleviated the anti-apoptotic effect of cytokines against nilotinib. Moreover, using shRNA against BCR-ABL, we demonstrated that K562 cells were not dependant of BCR-ABL expression as long as the SCF receptor pathway was activated. We conclude that the c-KIT pathway may substitute for BCR-ABL tyrosine kinase to activate survival signals and that c-KIT must be inhibited beside BCR-ABL to allow apoptosis of CML cells.


Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2578-2578
Author(s):  
Zhanxiang Wang ◽  
Seiji Fukuda ◽  
Louis M. Pelus

Abstract The Bcr-abl oncogene is found in cells of >95% of patients with CML and encodes a cytoplasmic protein with constitutive tyrosine kinase activity. Bcr-abl induces hematopoietic cell transformation and protects cells from apoptosis induced by numerous stimuli. Bcr-abl activates Myc, Ras, raf, Pl3K, and c-jun kinases that are critical for transforming activity, however, the signaling pathways between Bcr-abl and the apoptosis machinery are just beginning to be determined. It is known that Bcr-abl can exert an antiapoptotic effect by blocking mitochondrial release of cytochrome C. Although increased knowledge of Bcr-abl pathways resulted in the design of selective tyrosine kinase inhibitors such as STI571 (Gleevec), the development of drug resistance limits efficacy. Survivin is a member of the highly conserved inhibitor-of-apoptosis (IAP) family of endogenous caspase inhibitors. Similar to other IAPs, Survivin blocks apoptosis by inhibiting caspases 3, 7 and 9. However in contrast to other IAPs, Survivin is not expressed in most adult tissues but aberrantly overexpressed in all cancers and hematopoietic malignancies. Targeting of Survivin by antisense (AS) or dominant-negative (DN) strategies in transformed cell models induces apoptosis. Survivin is expressed in Bcr-abl+ CML cells in blast crisis, but not in cells from patients with Bcr-ablneg CML. Furthermore, high expression of Survivin is found in Adriamycin resistant Bcr-abl+ K562 cells. These findings led us to investigate whether Survivin is involved in the antiapoptotic effects of Bcr-abl and if Survivin disruption can facilitate apoptosis in Bcr-abl+ cells. Transient transfection of human Mo7e and mouse BaF3 hematopoietic cells with the Bcr-abl oncongene results in significantly elevated expression of Survivin mRNA and protein. The mRNA of 2 other Survivin splice variants, Survivin-2B and Survivin-ΔEx3 were also upregulated. In transfected Mo7e cells, Survivin promoter activity was upregulated 2–4 fold compared to parental Mo7e cells, determined using a luciferase reporter construct. In addition to Survivin, the IAP family member ILP was also upregulated by Bcr-abl. Disruption of Survivin expression/function in K562 cells that contain endogenous Bcr-abl and in Bcr-abl transfected Mo7e cells by ectopic expression of AS or DN T34A or C84A mutant Survivin constructs, significantly promoted apoptosis induced by the Bcr-abl tyrosine kinase inhibitor, Gleevec by ≥2-fold, in a time and dose dependent manner. Enhanced apoptosis induced by AS or DN Survivin was accompanied by caspase dependent cleavage of Bcr-abl oncoprotein (>50% decrease in Bcr-abl protein) that was blocked by the caspase inhibitor Z-VAD-fmk; disruption of mitochondria membrane potential (>35% increase in TMRMnegexpression); and enhanced cytochrome C release, quantified by westerns. Similarly, disruption of Survivin mRNA in K562 cells by a 20-mer antisense oligonucleotide resulted in >40% increase in mitochondrial disruption. In contrast, forced expression of wild-type Survivin in K562 cells protected cells from Gleevec-induced apopotosis. In summary, our results demonstrate that the Bcr-abl oncogene regulates Survivin transcription and production, which represents a new signaling pathway downstream of Bcr-abl that may be helpful in understanding the pathophysiology of CML. Targeted Survivin disruption may sensitize Bcr-abl+ CML cells to Gleevec-induced apoptosis and have therapeutic potential, particularly in the development of drug resistance.


2008 ◽  
Vol 31 (4) ◽  
pp. 22
Author(s):  
Jonathan So ◽  
Kelly Elder ◽  
Anna Dai ◽  
Claus Jorgensen ◽  
Rune Linding ◽  
...  

Networks of kinases play a role in the transmission and integration of signals from the membrane to the nucleus. We aim to elucidate kinase phosphorylation and interaction partners in these networks through the immuno-precipitation and mass spectrometric analysis of a representative set of 100 Flag-tagged kinases stably expressed in human colorectal cancer cells. The goal is to generate a comprehensive set of interactions and dynamic phosphorylation sites which correlate with cell phenotypes such as apoptosis and proliferation. The techniques of mass-spectrometry have allowed for the identification of proteins and their phosphorylation sites in complex samples. Various labeling methods such as iTRAQ has enabled the relative quantification of these sites as afunction of time (White et al. PNAS, 2007). However, kinases usually work in the context of particular signaling stimuli. We aim to characterize the role of these over-expressed kinases in the context of Trail-induced apoptosis. This isparticularly relevant to tumorigenesis in that many cancers are resistant to apoptosis and recombinant Trail therapies are currently undergoing clinical trials. We present assays to correlate the proliferative ability and sensitivity to apoptosis of various stable cell lines with kinase expression levels through flow cytometry. We also present efforts to trace downstream signaling through the monitoring of MAP kinase phosphorylation using a high-throughput bead array.


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