Lyn Kinase Alters Gab2 Phosphorylation and c-Cbl Protein Levels To Regulate Imatinib Sensitivity and Survival of Chronic Myelogenous Leukemia Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2132-2132
Author(s):  
Ji Wu ◽  
Feng Meng ◽  
Moshe Talpaz ◽  
Nicholas J. Donato
Keyword(s):  
Tyrosine Phosphorylation ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Imatinib Resistance ◽  
Kinase Inhibition ◽  
Protein Levels ◽  
Abl Kinase ◽  
Lyn Kinase ◽  
Imatinib Resistant ◽  
Cbl Protein

Abstract The tyrosine kinase inhibitor imatinib mesylate (Gleevec) is effective in controlling BCR-ABL expressing leukemias but resistance occurs in some early phase patients while it is more common in advanced disease. Resistance has been generally associated with mutations in the BCR-ABL kinase that effect drug affinity. However patients are also increasingly reported to fail imatinib therapy while retaining wild-type BCR-ABL expression. Our previous studies suggested a role for Lyn, a Src-related kinase, in imatinib resistance. K562 cells selected for imatinib resistance (K562R) overexpress Lyn kinase and its targeted silencing overcomes imatinib resistance and engages apoptosis. Overexpression of Lyn in K562 cells reduces imatinib sensitivity (3-fold) and patients that fail imatinib therapy in the absence of BCR-ABL mutations express a highly activated Lyn kinase that is not suppressed by imatinib. Silencing Lyn expression in patient specimens induces changes in cell survival that are proportional to the level of Lyn protein reduction. To understand the role of Lyn kinase in imatinib resistance and apoptosis we examined proteins associated with this kinase in imatinib resistant cell lines, leukemic cells overexpressing Lyn and specimens derived from imatinib resistant patients. Lyn overexpression blocked complete suppression of BCR-ABL tyrosine phosphorylation by imatinib and affected BCR-ABL signaling adaptors. Although BCR-ABL forms a stable complex with the leukemogenic-critical adaptor protein Gab2 in imatinib sensitive cells, Lyn overexpression resulted in the formation of Lyn:Gab2 complexed in resistant cells. BCR-ABL kinase inhibition failed to reduce tyrosine phosphorylation of Gab2 in these cells while Lyn silencing or kinase inhibition (with dasatinib) completely suppressed Gab2 tyrosine phosphorylation and correlated with the induction of apoptosis. Lyn silencing in K562R cells also lead to a reciprocal increase in the tyrosine phosphorylation and association with a protein of ~120kDa, identified as the E3 ligase, c-Cbl. Lyn overexpression in K562 cells reduced their imatinib sensitivity and reduced c-Cbl protein levels. Kinase inhibitor and co-transfection studies demonstrated that tyrosine phosphorylation of c-Cbl at a critical signaling site (Y774) is primarily controlled by BCR-ABL and deletion or mutation of the c-Cbl RING domain altered its BCR-ABL phosphorylation. These results suggest that c-Cbl complexes are regulated at both the protein and phosphorylation level by Lyn and BCR-ABL kinase activities, respectively. Overexpression and/or activation of Lyn may disrupt the balance and regulation of critical regulators of leukemogenic signaling (Gab2) or protein trafficking and stability (c-Cbl), resulting in increased cell survival and reduced responsiveness to BCR-ABL kinase inhibition. We conclude that Lyn alters the level and function of critical signaling adaptor proteins in CML cells.

scholarly journals Lyn regulates BCR-ABL and Gab2 tyrosine phosphorylation and c-Cbl protein stability in imatinib-resistant chronic myelogenous leukemia cells

Blood ◽  
2008 ◽  
Vol 111 (7) ◽  
pp. 3821-3829 ◽  
Author(s):  
Ji Wu ◽  
Feng Meng ◽  
Henry Lu ◽  
Ling Kong ◽  
William Bornmann ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Chronic Myelogenous Leukemia ◽  
Myelogenous Leukemia ◽  
Imatinib Resistance ◽  
Kinase Inhibition ◽  
Signaling Complex ◽  
Abl Kinase ◽  
Lyn Kinase ◽  
Imatinib Resistant ◽  
Cbl Protein

Abstract Lyn kinase functions as a regulator of imatinib sensitivity in chronic myelogenous leukemia (CML) cells through an unknown mechanism. In patients who fail imatinib therapy but have no detectable BCR-ABL kinase mutation, we detected persistently activated Lyn kinase. In imatinib-resistant CML cells and patients, Lyn activation is BCR-ABL independent, it is complexed with the Gab2 and c-Cbl adapter/scaffold proteins, and it mediates persistent Gab2 and BCR-ABL tyrosine phosphorylation in the presence or absence of imatinib. Lyn silencing or inhibition is necessary to suppress Gab2 and BCR-ABL phosphorylation and to recover imatinib activity. Lyn also negatively regulates c-Cbl stability, whereas c-Cbl tyrosine phosphorylation is mediated by BCR-ABL. These results suggest that Lyn exists as a component of the BCR-ABL signaling complex and, in cells with high Lyn expression or activation, BCR-ABL kinase inhibition alone (imatinib) is not sufficient to fully disengage BCR-ABL–mediated signaling and suggests that BCR-ABL and Lyn kinase inhibition are needed to prevent or treat this form of imatinib resistance.

The SRC/ABL Inhibitor BMS-354825 Overcomes Resistance to Imatinib Mesylate in Chronic Myelogenous Leukemia Cells through Multiple Mechanisms.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1989-1989 ◽  
Author(s):  
Nicholas J. Donato ◽  
Ji Wu ◽  
Ling Y. Kong ◽  
Francis Lee ◽  
Moshe Talpaz
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitor ◽  
Kinase Inhibitor ◽  
Myelogenous Leukemia ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Lyn Kinase ◽  
Abl Kinases ◽  
Imatinib Resistant ◽  
Dual Inhibition

Abstract BCR-ABL is an oncogenic tyrosine kinase expressed in chronic myelogenous leukemia (CML) cells and is the main target of the tyrosine kinase inhibitor imatinib mesylate. Imatinib-based CML therapy induces hematological and cytogenetic remission in early phase CML patients whereas more advanced patients frequently develop resistance to imatinib by multiple mechanisms, including mutations in the BCR-ABL kinase domain and over-expression of tyrosine kinases that are not inhibited by imatinib. These observations suggest that dual inhibition of src and abl kinases may circumvent imatinib resistance and provide more effective therapy for CML. BMS-354825 is a novel tyrosine kinase inhibitor that inhibits both abl and src kinases at low nM concentrations and is currently being clinically evaluated in imatinib resistant or intolerant CML patients. Our earlier studies demonstrated that increased expression of the src-related kinase Lyn in BCR-ABL expressing K562 cells was associated with imatinib resistance in this cell model and some CML patients. To determine whether inhibition of SRC/ABL kinases differentially affects imatinib sensitive K562 (BCR-ABL +, Lyn −) and resistant K562R (BCR-ABL +, Lyn +) cells were treated with imatinib or BMS-354825 before analysis of cell growth, survival and signaling. BMS-354825 induced apoptosis in both K562 and K562R cells which correlated with inhibition of both Lyn activation and BCR-ABL signaling (CrkL). BMS-354825 effectively reduced both K562 and K562R tumor growth in nude mice whereas imatinib had minimal effects on K562R tumors. Clinical specimens from imatinib resistant CML patients (with and without BCR-ABL kinase mutations) were treated with imatinib or BMS-354825 and analyzed for changes in Lyn and Hck activation. While imatinib had minimal inhibitory effects on Lyn/Hck activation, BMS-354825 completely suppressed Lyn/Hck phosphorylation which correlated with its greater anti-tumor activity in CML samples. BCR-ABL tyrosine phosphorylation was not inhibited by imatinib in Cos cells co-expressing BCR-ABL and Lyn kinase and loss of imatinib sensitivity was totally dependent on Lyn kinase activity. BMS-354825 reduced both Lyn and BCR-ABL activation in co-expressing cells, suggesting that Lyn-mediated phosphorylation plays a direct role in imatinib resistance. We conclude that dual inhibition of SRC/ABL kinases in CML cells by BMS-354825 overcomes resistance to imatinib in vitro and in vivo and induces anti-tumor effects in CML patient specimens resistant to imatinib through expression of imatinib-inactivating BCR-ABL kinase mutations as well as other resistance mechanisms.

Dasatinib (BMS-354825) Overcomes Multiple Mechanisms of Imatinib Resistance in Chronic Myeloid Leukemia (CML).

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1994-1994 ◽  
Author(s):  
Francis Y. Lee ◽  
Mei-Li Wen ◽  
Rajeev Bhide ◽  
Amy Camuso ◽  
Stephen Castenada ◽  
...  
Keyword(s):  
Cell Lines ◽  
Kinase Inhibitor ◽  
Leukemic Cell ◽  
K562 Cells ◽  
Src Family Kinases ◽  
Imatinib Resistance ◽  
Over Expression ◽  
Imatinib Resistant

Abstract Resistance to imatinib is a growing concern in CML, particularly in advanced disease. The most common cause of resistance is mutations in BCR-ABL, but other mechanisms have also been identified, including over-expression of BCR-ABL, activation of SRC family kinases and the P-glycoprotein (PGP) efflux pump (via MDR1 over-expression). Dasatinib (BMS-354825) is a novel, oral, multi-targeted tyrosine kinase inhibitor that targets BCR-ABL and SRC kinases. Dasatinib has 325-fold greater potency versus imatinib in cell lines transduced with wild-type BCR-ABL and is active against 18 out of 19 BCR-ABL mutations tested that confer imatinib resistance (Shah et al, Science305:399, 2004; O’Hare et al, Cancer Res65:4500–5, 2005), and preliminary results from a Phase I study show that it is well tolerated and has significant activity in imatinib-resistant patients in all phases of CML (Sawyers et al, J Clin Oncol23:565s, 2005; Talpaz et al, J Clin Oncol23:564s, 2005). We assessed the ability of dasatinib to overcome a variety of mechanisms of imatinib resistance. First, the leukemic-cell killing activity of dasatinib was tested in vitro in three human imatinib-resistant CML cell lines (K562/IM, MEG-01/IM and SUP-B15/IM). Based on IC50 values, dasatinib had >1000-fold more potent leukemic-cell killing activity compared with imatinib versus all three cell lines. Furthermore, in mice bearing K562/IM xenografts, dasatinib was curative at doses >5 mg/kg, while imatinib had little or no impact at doses as high as 150 mg/kg, its maximum tolerated dose. We determined that the MEG-01/IM and SUP-B15/IM cell lines carried BCR-ABL mutations known to confer imatinib resistance to imatinib clinically (Q252H and F359V, respectively). In K562/IM cells, BCR-ABL mutations or BCR-ABL over-expression were not detected, but the SRC family member FYN was over-expressed. PP2, a known inhibitor of SRC family kinases but not BCR-ABL, could reverse the imatinib resistance in these cells. Together, these data suggest that activation of FYN may be a cause of imatinib resistance in K562/IM. Based on cell proliferation IC50, we found that the anti-leukemic activity of dasatinib in K562/IM cells was 29-fold more potent compared with AMN107 (a tyrosine kinase inhibitor that inhibits BCR-ABL but not SRC family kinases). Given that the human serum protein binding of dasatinib, imatinib and AMN107 were 93, 92 and >99% respectively, the difference in potency between dasatinib and AMN107 in vivo may be far greater than the simple fold-difference in the in vitro IC50 values. Finally, in K562 cells over-expressing PGP (K562/ADM), we found that dasatinib was only 6-fold less active than in parental K562 cells. Because of the extreme potency of dasatinib in K562 cells, this reduced potency still afforded an IC50 of 3 nM, which is readily achievable in vivo. Indeed, in mice bearing K562/ADM xenografts, dasatinib was curative at 30 mg/kg, with significant anti-leukemic activity at 15 mg/kg. In conclusion, the rational design of dasatinib as a multi-targeted kinase inhibitor allows this agent to overcome a variety of mechanisms of resistance to imatinib in CML, including mechanisms that are not overcome by agents with a narrower spectrum of inhibition, such as AMN107. Dasatinib is currently in Phase II evaluation in imatinib-resistant/-intolerant patients in the ‘START’ program, and in Phase I evaluation in solid tumors.

Constitutive Activation of SRC-Family Kinases in Chronic Myelogenous Leukemia Patients Resistant to Imatinib Mesylate in the Absence of BCR-ABL Mutations: A Rationale for Use of SRC/ABL Dual Kinase Inhibitor-Based Therapy.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1087-1087 ◽  
Author(s):  
Nicholas J. Donato ◽  
Ji Wu ◽  
Ling-Yuan Kong ◽  
Feng Meng ◽  
Francis Lee ◽  
...  
Keyword(s):  
Cell Survival ◽  
Imatinib Mesylate ◽  
Src Family Kinases ◽  
Myelogenous Leukemia ◽  
Leukemic Cells ◽  
Imatinib Resistance ◽  
Kinase Activation ◽  
Abl Kinase ◽  
Lyn Kinase ◽  
Imatinib Resistant

Abstract BCR-ABL is an unregulated tyrosine kinase expressed as a consequence of a reciprocal chromosomal translocation that is common in chronic myelogenous and acute lymphocytic leukemia. BCR-ABL induces transformation of hematopoetic stem cells through tyrosine phosphorylation of multiple substrates. The src-family kinases (SFKs), Lyn and Hck, are highly activated by BCR-ABL in leukemic cells and recent studies suggest that they are substrates and essential mediators of BCR-ABL signal transduction and transformation. In cells selected for resistance to the BCR-ABL inhibitor, imatinib mesylate, Lyn kinase is overexpressed and its activation is not dependent on or regulated by BCR-ABL, suggesting that autonomous regulation of SFKs may play a role in imatinib resistant. In this report, activation of Lyn and Hck was compared in CML specimens derived from imatinib responsive and resistant patients that did not express a mutant BCR-ABL protein as their primary mediator of resistance. In imatinib sensitive cell lines and specimens derived from imatinib responsive CML patients imatinib effectively reduced activation of both BCR-ABL and SFKs. However, in multiple specimens from resistant patients, imatinib reduced BCR-ABL kinase activation but failed to reduce SFK activation. The dual ABL/SRC inhibitor, BMS-354825, blocked activation of both BCR-ABL and SFKs expressed in leukemic cells and correlated with clinical responsiveness to this agent. Animal models demonstrated that loss of imatinib-mediated inhibition of Lyn kinase activation significantly impaired its anti-tumor activity which was recovered by treatment with BMS-354825. Direct silencing of Lyn or Hck reduced CML cell survival in imatinib resistant patient specimens and cell models, suggesting a direct role for these kinases in cell survival. Our results show that SFK activation is mediated by BCR-ABL in imatinib responsive cells but these kinases escape control by BCR-ABL in CML patients that develop imatinib resistance in the absence of BCR-ABL point mutations. This form of resistance can effectively be overcome by BMS-354825 through its dual SRC and ABL kinase inhibitory activities. Dual specificity kinase inhibitors may be indicated for the treatment and prevention of imatinib resistance in CML when it is associated with constitutively activated src-family kinases.

scholarly journals Emodin Exerts an Antiapoptotic Effect on Human Chronic Myelocytic Leukemia K562 Cell Lines by Targeting the PTEN/PI3K-AKT Signaling Pathway and Deleting BCR-ABL

2016 ◽  
Vol 16 (4) ◽  
pp. 526-539 ◽  
Author(s):  
Chun-Guang Wang ◽  
Liang Zhong ◽  
Yong-Li Liu ◽  
Xue-Jun Shi ◽  
Long-Qin Shi ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
K562 Cells ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Myelocytic Leukemia ◽  
Novel Drugs ◽  
Alternative Drug ◽  
Drug Treatments

The BCR-ABL kinase inhibitor, imatinib mesylate, is the front-line treatment for chronic myeloid leukemia, but the emergence of imatinib resistance has led to the search for alternative drug treatments. There is a pressing need, therefore, to develop and test novel drugs. Natural products including plants, microorganisms, and halobios provide rich resources for discovery of anticancer drugs. In this article, we demonstrate that emodin inhibited the growth of K562 cells harboring BCR-ABL in vitro and in vivo, and induced abundant apoptosis, which was correlated with the inhibition of PETN/PI3K/Akt level and deletion of BCR-ABL. These findings suggest that emodin is a promising agent to kill K562 cells harboring BCR-ABL.

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.

The Crystal Structure of Abl Kinase with BMS-354825, a Dual SRC/ABL Kinase Inhibitor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 553-553 ◽  
Author(s):  
John S. Tokarski ◽  
John Newitt ◽  
Francis Y. Lee ◽  
Louis Lombardo ◽  
Robert Borzilleri ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Small Molecule ◽  
Kinase Inhibitor ◽  
High Rate ◽  
Dimensional Structure ◽  
Dual Function ◽  
Activation Loop ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant

Abstract Chronic myeloid leukemia (CML) is a stem cell disorder caused by a constitutively activated tyrosine kinase, the BCR-ABL oncoprotein. Imatinib (STI571, Gleevec) is a small-molecule inhibitor of this kinase that produces clinical remissions in CML patients and is now frontline therapy for this disease. While this agent has a high rate of clinical success in early phases of CML, development of resistance to this drug becomes increasingly problematic in later stages of the disease. BMS-354825, a small-molecule dual-function SRC/ABL tyrosine kinase inhibitor, appears to overcome many of the limitations associated with imatinib therapy. BMS-354825 is 500-fold more potent than imatinib against BCR-ABL and more importantly retains activity against 14 of 15 imatinib-resistant BCR-ABL mutants (Shah et al., Science, 2004;305(5682):399). In addition, BMS-354825 proved to be equally effective against several pre-clinically and clinically derived tumor models of imatinib resistance (Lee et al., Proceedings of the AACR, Volume 45, March 2004 abstract number 3937). In order to better understand the molecular basis of the relationship between inhibitor chemistry and biological activity, the three-dimensional structure of the kinase domain of Abl kinase complexed with BMS-354825 was determined by X-ray crystallography. The structure reveals that BMS-354825 binds in the ATP-binding site. A comparison with the imatinib-Abl complex (PDB entry 1IEP) reveals that the central cores of BMS-354825 and imatinib occupy overlapping regions but that these two inhibitors extend in opposite directions. The activation loop is observed to be in the active conformation in the presence of bound BMS-354825 in contrast to bound imatinib. There do not appear to be any steric clashes that would preclude BMS-354825 from also binding to the inactive conformation of the activation loop. This observation suggests that the increased binding affinity of BMS-354825 over imatinib is at least partially due to its apparent ability to recognize multiple states of the enzyme. The P-loop is partially disordered as indicated by high B-factors and broken electron density which suggests that interactions between this part of the protein and BMS-354825 are less critical for binding. Interestingly, several imatinib-resistant mutations occur in the P-loop. The structure was analyzed for the 15 imatinib-resistant BCR-ABL mutants and attempts are made to rationalize the activity of BMS-354825 against these mutants.

Activity of ABL Kinase Inhibitors in Two Distinct Models of Imatinib Resistance.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4819-4819
Author(s):  
Nicholas J. Donato ◽  
Ji Wu ◽  
William Bornmann ◽  
Moshe Talpaz
Keyword(s):  
Cell Lines ◽  
Inhibitory Activity ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
K562 Cells ◽  
Mutant Form ◽  
Imatinib Resistance ◽  
Resistant Variant ◽  
Lyn Kinase ◽  
Kinase Inhibitory Activity

Abstract The tyrosine kinase inhibitor imatinib mesylate (Gleevec) is effective in controlling BCR-ABL expressing leukemias but resistance occurs in a small subset of early stage patients and is very common in advanced stage patients. Resistance is associated with overexpression and/or mutations in the BCR-ABL gene but patients are also increasingly reported to fail imatinib therapy while retaining wild-type BCR-ABL expression. To circumvent or overcome resistance novel kinase inhibitors have been synthesized and tested clinically. However, while investigators have designed models to measure and predict activity of novel compounds in mutation-mediated imatinib resistance, other mechanisms of resistance have not been modeled or shown clinical relevance. In this report, the activity of four novel kinase inhibitors (norlotinib, dasatinib, SKI-606, ON012380) was compared in two distinct models of imatinib resistance. These models include cell lines established from natural resistant variant clones selected from imatinib sensitive cell lines and associated with a T315I BCR-ABL mutation (BV-173R) or overexpressed Lyn kinase (K562R). Kinase inhibitory activity in these models was compared to transfectant-based resistance models including BaF3 cells expressing the T315I mutant form of BCR-ABL and overexpression of Lyn kinase in K562 cells. K562R cells were completely resistant to imatinib and norlotinib but highly sensitive to Src/Abl inhibitors (dasatinib, SKI-606) but only partially sensitive to ON012380. Overexpression of Lyn in K562 cells reduced imatinib and norlotinib sensitivity (3-fold) but did not affect sensitivity to the other kinase inhibitors. BV-173R cells expressing the T315I mutant form of BCR-ABL were completely resistant to Abl-selective kinase inhibitors (imatinib, norlotinib) and ~100-fold less sensitive (IC50 ~ 2 microM) to Src/Abl-directed inhibitors (dasatinib, SKI-606) while the non-ATP competitive kinase inhibitor, ON012380, was equally effective against both BV-173 and BV-173R cells. Expression of the T315I mutant form of BCR-ABL in BaF3 cells completely blocked kinase inhibitory activity of all inhibitors except ON012380. IL-3 dependent BaF3 cells were not inhibited by imatinib, norlotinib or SKI-606 but were equally sensitive to ON012380 when compared to IL-3 independent BCR-ABL transfectants. Cellular sensitivity was associated with reduced phosphorylation of BCR-ABL, CrkL and Lyn kinase with all inhibitors except ON012380, which mediated apoptosis in the absence of alterations in tyrosine phosphorylation. Together, our results suggest that imatinib resistant cell models are useful in evaluating the activity of novel kinase inhibitors but need to be carefully interpreted and mechanistically tested. Additional mediators of imatinib resistance need to be defined and modeled so that an appropriate individualized therapy can be applied to most effectively overcome resistant disease.

Hematologic and Cytogenetic Responses in Imatinib-Resistant Chronic Phase Chronic Myeloid Leukemia Patients Treated with the Dual SRC/ABL Kinase Inhibitor BMS-354825: Results from a Phase I Dose Escalation Study.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1-1 ◽  
Author(s):  
Charles L. Sawyers ◽  
Neil P. Shah ◽  
Hagop M. Kantarjian ◽  
Nicholas Donato ◽  
John Nicoll ◽  
...  
Keyword(s):  
Phase I ◽  
Dose Escalation ◽  
Clinical Benefit ◽  
Kinase Inhibitor ◽  
Chronic Phase ◽  
Pharmacokinetic Studies ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant

Abstract Disease relapse in CML patients treated with imatinib is often associated with mutations in the BCR-ABL gene that interfere with the ability of imatinib to block BCR-ABL kinase activity. BMS-354825 is a novel, orally available, dual SRC/ABL kinase inhibitor with more than 100-fold greater potency than imatinib that has in vitro and in vivo preclinical activity against 14 of 15 imatinib resistant BCR-ABL mutants (Shah et al, Science, 305:399, 2004). Here we report the phase I clinical results of BMS-354825 in Philadelphia chromosome positive (Ph+) CML patients in chronic phase with hematologic progression or intolerance while being treated with imatinib. To date (Aug 6, 2004), 29 patients have been treated on 9 cohorts with doses ranging from 15 to 180 mg of BMS-354825 per day given in single or divided doses for 5–7 days per week, for up to 9 months. Similar to imatinib, BMS-354825 has been well tolerated in all patients, with a single episode of grade 4 thrombocytopenia as the only potential drug related adverse event. BMS-354825 is rapidly absorbed with peak concentrations achieved within 2 hours and a terminal phase half-life of about 5 hours. Serum levels well above the concentration required to block CML cell proliferation in vitro have been readily achieved without side effects. Pharmacodynamic studies demonstrate greater than 50 percent inhibition of phosphorylation of the BCR-ABL substrate CRKL and the SRC kinase Lyn, consistent with the serum concentrations observed in pharmacokinetic studies. Patients not receiving optimal clinical benefit were escalated to the next higher dose for which safety parameters were available, thereby allowing a preliminary assessment of clinical activity. To date, 26 patients (22 with imatinib resistance, 4 with imatinib intolerance; average CML duration 6.1 years) have been followed for greater than 4 weeks and are eligible for assessment of hematologic benefit. 22 patients had detectable BCR-ABL kinase domain mutations prior to starting BMS-354825. All 26 patients have been treated with doses of 35 mg per day or greater and have had clinical benefit, including 19 with complete hematologic responses (73%). Of the 7 partial responders, two subsequently had disease progression, one of whom had expansion of a CML subclone containing the imatinib-resistant T315I mutation in BCR-ABL, which also confers resistance to BMS-354825 in preclinical studies. The other 5 partial responders are now being treated with higher doses to attempt conversion to complete hematologic response. 11 of 21 patients (52%) treated for greater than 3 months have cytogenetic benefit, including 6 major (1–35% Ph+), 1 minor (36–65% Ph+) and 4 minimal (66–95% Ph+) cytogenetic responses. One patient has achieved complete cytogenetic response. Dose escalation continues, and phase II studies in chronic, accelerated and blast crisis CML are currently being initiated. These data provide compelling evidence supporting the safety and efficacy of BMS-354825 in imatinib-resistant chronic phase CML.

Loss of Response to Imatinib: Mechanisms and Management

Hematology ◽  
2005 ◽  
Vol 2005 (1) ◽  
pp. 183-187 ◽  
Author(s):  
Neil P. Shah
Keyword(s):  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Imatinib Resistance ◽  
Early Clinical Trial ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Trial Experience ◽  
Highly Effective ◽  
Mutant Forms

AbstractThe treatment of chronic myeloid leukemia (CML) has been revolutionized by the small molecule BCR-ABL-selective kinase inhibitor imatinib. Although imatinib is highly effective initially and generally well-tolerated, relapse is increasingly encountered clinically. Until recently, for the majority of CML patients with disease no longer responsive to imatinib, as well as for patients with imatinib intolerance, few effective therapeutic options existed. Our understanding of the major mechanisms of imatinib resistance has led to the clinical development of two novel BCR-ABL inhibitors that harbor significant therapeutic promise in early clinical trial experience. These agents, dasatinib (BMS-354825) and AMN107, are more potent inhibitors of BCR-ABL than imatinib, and moreover, harbor activity against nearly all imatinib-resistant BCR-ABL kinase domain mutant forms tested in vitro. Notably, neither of these compounds is effective against the imatinib-resistant BCR-ABL/T315I mutation. The potential availability of highly effective medications for the treatment of imatinib-resistant and intolerant cases of CML is expected to further complicate the timing of other effective therapies, such as allogeneic stem cell transplantation. Additionally, periodic genotyping of the BCR-ABL kinase domain to screen for drug-resistant mutations may play an increasingly important role in the future management of CML cases.

Sign in / Sign up

Export Citation Format

Share Document