364 The SRC-kinase inhibitor AZD0530 efficiently counteracts the transformation potential of BCR/ABL by targeting its kinase activity

Imatinib mesylate (imatinib) is highly effective in the treatment of chronic myeloid leukemia (CML) but is less effective in eliminating CML stem cells. We investigated whether SKI-606, a potent Bcr-Abl and Src kinase inhibitor without anti-PDGF or c-Kit activity, could effectively target primitive CML progenitors. CML and normal progenitors were cultured with SKI-606 or imatinib. SKI-606 effectively inhibited Bcr-Abl kinase activity in CML CD34+ cells and inhibited Src phosphorylation more potently than imatinib. However, SKI-606 and imatinib resulted in similar suppression of CML primitive and committed progenitor proliferation and growth in CFC and LTC-IC assays. Exposure to either agent alone or in combination resulted in only modest increase in apoptosis. Evaluation of downstream signaling pathways indicated that Akt and STAT5 activity was not changed, but a delayed increase in MAPK activity was seen at high concentrations of SKI-606. SKI-606 inhibited normal progenitor proliferation to a lesser extent than imatinib. SKI-606 effectively inhibits Bcr-Abl and Src kinase activity and inhibits CML progenitor growth with relatively little effect on normal progenitors. However, SKI-606 does not demonstrate increased ability to eliminate primitive CML progenitors by apoptosis compared with imatinib, emphasizing the need for additional strategies besides Bcr-Abl kinase inhibition for curative therapy of CML.

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Targeting of the N-terminal coiled coil oligomerization interface of BCR interferes with the transformation potential of BCR-ABL and increases sensitivity to STI571

Blood ◽

10.1182/blood-2003-03-0811 ◽

2003 ◽

Vol 102 (8) ◽

pp. 2985-2993 ◽

Cited By ~ 24

Author(s):

Tim Beissert ◽

Elena Puccetti ◽

Andrea Bianchini ◽

Saskia Güller ◽

Simone Boehrer ◽

...

Keyword(s):

Kinase Activity ◽

Kinase Inhibitor ◽

Coiled Coil ◽

Chromosome 9 ◽

Abl Kinase ◽

Kinase C ◽

Coiled Coil Region ◽

Increased Sensitivity ◽

Transformation Potential

Abstract Translocations involving the abl locus on chromosome 9 fuses the tyrosine kinase c-ABL to proteins harboring oligomerization interfaces such as BCR or TEL, enabling these ABL-fusion proteins (X-ABL) to transform cells and to induce leukemia. The ABL kinase activity is blocked by the ABL kinase inhibitor STI571 which abrogates transformation by X-ABL. To investigate the role of oligomerization for the transformation potential of X-ABL and for the sensitivity to STI571, we constructed ABL chimeras with oligomerization interfaces of proteins involved in leukemia-associated translocations such as BCR, TEL, PML, and PLZF. We assessed the capacity of these chimeras to form high molecular weight (HMW) complexes as compared with p185(BCR-ABL). There was a direct relationship between the size of HMW complexes formed by these chimeras and their capacity to induce factor independence in Ba/F3 cells, whereas there was an inverse relationship between the size of the HMW complexes and the sensitivity to STI571. The targeting of the oligomerization interface of p185(BCR-ABL) by a peptide representing the coiled coil region of BCR reduced its potential to transform fibroblasts and increased sensitivity to STI571. Our results indicate that targeting of the oligomerization interfaces of the X-ABL enhances the effects of STI571 in the treatment of leukemia caused by X-ABL.

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Endothelial contraction and monolayer hyperpermeability are regulated by Src kinase

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00862.2002 ◽

2003 ◽

Vol 284 (3) ◽

pp. H994-H1002 ◽

Cited By ~ 25

Author(s):

David R. Mucha ◽

Carter L. Myers ◽

Richard C. Schaeffer

Keyword(s):

Tyrosine Phosphorylation ◽

Myosin Light Chain ◽

Kinase Activity ◽

Kinase Inhibitor ◽

Src Kinase ◽

Endothelial Monolayer ◽

Herbimycin A ◽

Barrier Formation ◽

Monolayer Barrier Function

Endothelial monolayer hyperpermeability is regulated by a myosin light chain phosphorylation (MLCP)-dependent contractile mechanism. In this study, we tested the role of Src-dependent tyrosine phosphorylation to modulate endothelial contraction and monolayer barrier function with the use of the myosin phosphatase inhibitor calyculin A (CalA) to directly elevate MLCP with the Src family tyrosine kinase inhibitor herbimycin A (HA) in bovine pulmonary artery endothelial cells (EC). CalA stimulated an increase in MLCP, Src kinase activity, an increase in the tyrosine phosphorylation of paxillin and focal adhesion (FA) kinase (p125FAK), and monolayer hyperpermeability. Microscopic examination of CalA-treated EC revealed a contractile morphology characterized by peripheral contractile bands of actomyosin filaments and stress fibers linked to phosphotyrosine-containing FAs. These CalA-dependent events were HA sensitive. HA alone stimulated an improvement in monolayer barrier formation by reducing the levels of MLCP and phosphotyrosine-containing proteins and the number of large paracellular holes. These data show that Src kinase plays an important role in regulating monolayer hyperpermeability through adjustments in tyrosine phosphorylation, MLCP, and EC contraction.

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Activation of Src kinase Lyn by the Kaposi sarcoma–associated herpesvirus K1 protein: implications for lymphomagenesis

Blood ◽

10.1182/blood-2004-07-2781 ◽

2005 ◽

Vol 105 (10) ◽

pp. 3987-3994 ◽

Cited By ~ 59

Author(s):

Om Prakash ◽

O. Rama Swamy ◽

Xiochang Peng ◽

Zhen-Ya Tang ◽

Li Li ◽

...

Keyword(s):

Kinase Activity ◽

Kinase Inhibitor ◽

Src Kinase ◽

Kaposi Sarcoma ◽

B Cell Lymphoma ◽

Proliferation Index ◽

Kinase Activation ◽

Transmembrane Glycoprotein ◽

Lyn Kinase ◽

A Cell

AbstractThe K1 gene of Kaposi sarcoma–associated herpesvirus (KSHV) encodes a transmembrane glycoprotein bearing a functional immunoreceptor tyrosine-based activation motif (ITAM). Previously, we reported that the K1 protein induced plasmablastic lymphomas in K1 transgenic mice, and that these lymphomas showed enhanced Lyn kinase activity. Here, we report that systemic administration of the nuclear factor kappa B (NF-κB) inhibitor Bay 11-7085 or an anti–vascular endothelial growth factor (VEGF) antibody significantly reduced K1 lymphoma growth in nude mice. Furthermore, in KVL-1 cells, a cell line derived from a K1 lymphoma, inhibition of Lyn kinase activity by the Src kinase inhibitor PP2 decreased VEGF induction, NF-κB activity, and the cell proliferation index by 50% to 75%. In contrast, human B-cell lymphoma BJAB cells expressing K1, but not the ITAM sequence–deleted mutant K1, showed a marked increase in Lyn kinase activity with concomitant VEGF induction and NF-κB activation, indicating that ITAM sequences were required for the Lyn kinase–mediated activation of these factors. Our results suggested that K1-mediated constitutive Lyn kinase activation in K1 lymphoma cells is crucial for the production of VEGF and NF-κB activation, both strongly implicated in the development of KSHV-induced lymphoproliferative disorders.

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H2O2-mediated permeability II: importance of tyrosine phosphatase and kinase activity

AJP Cell Physiology ◽

10.1152/ajpcell.2001.281.6.c1940 ◽

2001 ◽

Vol 281 (6) ◽

pp. C1940-C1947 ◽

Cited By ~ 58

Author(s):

Christopher G. Kevil ◽

Naotsuka Okayama ◽

J. Steven Alexander

Keyword(s):

Phosphatase Activity ◽

Protein Tyrosine Phosphatase ◽

Kinase Activity ◽

Kinase Inhibitor ◽

Biological Effects ◽

Tyrosine Phosphatase ◽

Src Kinase ◽

Cell Junctions ◽

Endothelial Monolayer ◽

Solute Permeability

We previously reported that exposure of endothelial cells to H2O2results in a loss of cell-cell apposition and increased endothelial solute permeability. The purpose of this study was to determine how tyrosine phosphorylation and tyrosine phosphatases contribute to oxidant-mediated disorganization of endothelial cell junctions. We found that H2O2caused a rapid decrease in total cellular phosphatase activity that facilitates a compensatory increase in cellular phosphotyrosine residues. H2O2exposure also results in increased endothelial monolayer permeability, which was attenuated by pp60, an inhibitor of src kinase. Inhibition of protein tyrosine phosphatase activity by phenylarsine oxide (PAO) demonstrated a similar permeability profile compared with H2O2, suggesting that tyrosine phosphatase activity is important in maintaining a normal endothelial solute barrier. Immunofluorescence shows that H2O2exposure caused a loss of pan-reactive cadherin and β-catenin from cell junctions that was not blocked by the src kinase inhibitor PP1. H2O2also caused β-catenin to dissociate from the endothelial cytoskeleton, which was not prevented by PP1. Finally, we determined that PP1 did not prevent cadherin internalization. These data suggest that oxidants like H2O2produce biological effects through protein phosphotyrosine modifications by decreasing total cellular phosphatase activity combined with increased src kinase activity, resulting in increased endothelial solute permeability.

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Activation of RBL-2H3 Mast Cells Is Dependent on Tyrosine Phosphorylation of Phospholipase D2 by Fyn and Fgr

Molecular and Cellular Biology ◽

10.1128/mcb.24.16.6980-6992.2004 ◽

2004 ◽

Vol 24 (16) ◽

pp. 6980-6992 ◽

Cited By ~ 33

Author(s):

Wahn Soo Choi ◽

Takaaki Hiragun ◽

Jun Ho Lee ◽

Young Mi Kim ◽

Hyoung-Pyo Kim ◽

...

Keyword(s):

Kinase Activity ◽

Kinase Inhibitor ◽

Immunoglobulin E ◽

Tyrosine Phosphatase ◽

Src Kinase ◽

Activation Mechanism ◽

Src Kinases ◽

Small Interfering Rnas ◽

Phospholipase D2 ◽

Phospholipase D1

ABSTRACT Both phospholipase D1 (PLD1) and PLD2 regulate degranulation when RBL-2H3 cells are stimulated via the immunoglobulin E receptor, FcεRI. However, the activation mechanism for PLD2 is unclear. As reported here, PLD2 but not PLD1 is phosphorylated through the Src kinases, Fyn and Fgr, and this phosphorylation appears to regulate PLD2 activation and degranulation. For example, only hemagglutinin-tagged PLD2 was tyrosine phosphorylated in antigen-stimulated cells that had been made to express HA-PLD1 and HA-PLD2. This phosphorylation was blocked by a Src kinase inhibitor or by small interfering RNAs directed against Fyn and Fgr and was enhanced by overexpression of Fyn and Fgr but not by other Src kinases. The phosphorylation and activity of PLD2 were further enhanced by the tyrosine phosphatase inhibitor, Na3VO4. Mutation of PLD2 at tyrosines 11, 14, 165, or 470 partially impaired, and mutation of all tyrosines blocked, PLD2 phosphorylation and activation, although two of these mutations were detrimental to PLD2 function. PLD2 phosphorylation preceded degranulation, both events were equally sensitive to inhibition of Src kinase activity, and both were enhanced by coexpression of PLD2 and the Src kinases. The findings provide the first description of a mechanism for activation of PLD2 in a physiological setting and of a role for Fgr in FcεRI-mediated signaling.

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Effectiveness of the dual specific Src/Abl kinase inhibitor AZD0530 in combination with tamoxifen in preventing acquired anti-estrogen resistance in breast cancer cells

Journal of Clinical Oncology ◽

10.1200/jco.2007.25.18_suppl.14054 ◽

2007 ◽

Vol 25 (18_suppl) ◽

pp. 14054-14054 ◽

Cited By ~ 1

Author(s):

S. Hiscox ◽

T. P. Green ◽

C. Smith ◽

N. Jordan ◽

M. James ◽

...

Keyword(s):

Breast Cancer ◽

Cancer Cells ◽

Breast Cancer Cells ◽

Kinase Activity ◽

Kinase Inhibitor ◽

Acquired Resistance ◽

Src Kinase ◽

Abl Kinase ◽

Invasive Capacity

14054 Background: AZD0530 is a novel, orally potent, once-daily, highly selective and dual-specific Src/Abl kinase inhibitor with potential for activity in a wide range of tumors. In the context of breast cancer, where tamoxifen resistance presents a major problem, Src inhibition may be a particularly valuable therapeutic strategy since we have previously observed that elevated Src kinase activity accompanies anti-estrogen resistance in vitro, promoting an aggressive cell phenotype. Here, we have explored the potential therapeutic effects of Src inhibition with AZD0530, alone and in combination with tamoxifen, on the acquisition of endocrine resistance in breast cancer cells. Methods: MCF7 and T47D breast cancer cells were exposed to tamoxifen (10–7 M), AZD0530 (1 μM), or both agents in combination for a minimum of 10 months with passaging as necessary, or until total cell death occurred. Cells were assayed at monthly intervals for intracellular signaling pathway activity (Western Blotting) and in vitro invasive capacity (Matrigel invasion assays). Apoptosis and proliferation were assessed by ELISA and Ki67 staining, respectively. Changes in c-Myc and cyclin-D1 were measured with RT-PCR. Results: Treatment of cells with tamoxifen alone ultimately resulted in acquired resistance, elevated Src kinase activity, and a Src- dependent increase in invasive capacity. Chronic exposure to AZD0530 alone resulted in outgrowth of AZD0530 resistant cells, in which Src kinase activity remained suppressed as did their in vitro invasiveness. Treatment of MCF7 and T47D cells with AZD0530 and tamoxifen combined resulted in a reduction of Src, FAK, and Akt activity, inhibition of c-Myc gene expression, and complete abrogation of their in vitro invasive behavior. Furthermore, combination treatment completely prevented cell proliferation and the subsequent emergence of a resistant phenotype, with a total loss of cells by 12 weeks. Conclusions: Inhibition of Src kinase with AZD0530, when used in conjunction with anti-estrogen therapies, effectively prevents acquired resistance in breast cancer cells in vitro suggesting a potential novel therapeutic benefit of Src kinase inhibitors clinically. No significant financial relationships to disclose.

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Faculty Opinions recommendation of 2-aminothiazole as a novel kinase inhibitor template. Structure-activity relationship studies toward the discovery of N-(2-chloro-6-methylphenyl)-2-[[6-[4-(2-hydroxyethyl)-1- piperazinyl)]-2-methyl-4-pyrimidinyl]amino)]-1,3-thiazole-5-carboxamide (dasatinib, BMS-354825) as a potent pan-Src kinase inhibitor.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.1085984.538933 ◽

2007 ◽

Author(s):

Andrew Combs

Keyword(s):

Kinase Inhibitor ◽

Src Kinase ◽

Structure Activity Relationship ◽

Activity Relationship ◽

Structure Activity ◽

Template Structure

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ADAM12 localizes with c-Src to actin-rich structures at the cell periphery and regulates Src kinase activity

Experimental Cell Research ◽

10.1016/j.yexcr.2009.09.017 ◽

2010 ◽

Vol 316 (1) ◽

pp. 55-67 ◽

Cited By ~ 32

Author(s):

Dorte Stautz ◽

Archana Sanjay ◽

Matilde Thye Hansen ◽

Reidar Albrechtsen ◽

Ulla M. Wewer ◽

...

Keyword(s):

Kinase Activity ◽

Src Kinase ◽

Cell Periphery

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Regulation of protein kinase by vasopressin in renal medulla in situ

AJP Renal Physiology ◽

10.1152/ajprenal.1977.232.1.f50 ◽

1977 ◽

Vol 232 (1) ◽

pp. F50-F57

Author(s):

T. P. Dousa ◽

L. D. Barnes

Keyword(s):

Protein Kinase ◽

Cyclic Amp ◽

Kinase Activity ◽

Kinase Inhibitor ◽

Renal Medulla ◽

Protein Kinase Activity ◽

Kinase Activation ◽

Heat Stable ◽

Protein Kinase Activation ◽

Heat Stable Protein

Results of this study demonstrate that vasopressin activates protein kinase in intact renal medullary cells as detected by measurement of the (-cyclic AMP/+cyclic AMP) protein kinase activity ratios in freshly prepared tissue extracts (40,000 X g supernates) from bovine renal medullary slices. The activation of protein kinase was specific for vasopressin since parathyroid hormone, histamine, angiotensin II, or the inactive analog of vasopressin did not activate protein kinase. There was a direct correlation between the extent of protein kinase activation and the elevation in tissue levels of cyclic AMP elicited by increasing doses of vasopressin or with an increase in incubation time. The elevation of tissue cyclic AMP level and maximum activation of protein kinase reached maximum level at a vasopressin concentration of about 2 X 10(-9) M. Incubation of slices with vasopressin caused a dose-dependent decrease in the cyclic AMP-dependent protein kinase activity in the 40,000 X g supernate of homogenate from the renal medullary slices. This effect of vasopressin was specific for protein kinase since activity of lactate dehydrogenase or a specific [3H]colchicine-binding activity was not affected, and the decrease in the protein kinase was not due to the accumulation of a heat-stable protein kinase inhibitor. There was an increase in protein kinase was not due to the accumulation of a heat-stable protein kinase inhibitor. There was an increase in protein kinase activity extracted from 40,000 X g pellets of homogenate prepared from slices exposed to vasopressin. Results thus provide evidence that cyclic AMP-mediated protein kinase activation in the intact cells is an integral part of cellular response of the mammalian renal medulla to vasopressin.

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