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 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.

Several Bcr-Abl kinase domain mutants associated with imatinib mesylate resistance remain sensitive to imatinib

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4611-4614 ◽  
Author(s):  
Amie S. Corbin ◽  
Paul La Rosée ◽  
Eric P. Stoffregen ◽  
Brian J. Druker ◽  
Michael W. Deininger
Keyword(s):  
Imatinib Mesylate ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Myelogenous Leukemia ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Cellular Assays ◽  
Kinase Domain Mutations

Abstract Imatinib mesylate is a selective Bcr-Abl kinase inhibitor, effective in the treatment of chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Kinase domain mutations are the most commonly identified mechanism associated with relapse. Many of these mutations decrease the sensitivity of the Abl kinase to imatinib, thus accounting for resistance to imatinib. The role of other mutations in the emergence of resistance has not been established. Using biochemical and cellular assays, we analyzed the sensitivity of several mutants (Met244Val, Phe311Leu, Phe317Leu, Glu355Gly, Phe359Val, Val379Ile, Leu387Met, and His396Pro/Arg) to imatinib mesylate to better understand their role in mediating resistance.While some Abl mutations lead to imatinib resistance, many others are significantly, and some fully, inhibited. This study highlights the need for biochemical and biologic characterization, before a resistant phenotype can be ascribed to a mutant.

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.

A Novel Mechanism for Imatinib Mesylate (STI571) Resistance in CML Cells: Contribution of TC-PTP to Modulating Signals Down-Stream from the BCR-ABL Fusion Protein.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1984-1984
Author(s):  
Takatsune Shimizu ◽  
Yoshitaka Miyakawa ◽  
Satoshi Iwata ◽  
Akiko Kuribara ◽  
Tony Tiganis ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Imatinib Mesylate ◽  
Acquired Resistance ◽  
Myelogenous Leukemia ◽  
Cell Protein ◽  
Imatinib Treatment ◽  
Apoptotic Cells ◽  
Imatinib Resistance ◽  
Imatinib Resistant ◽  
Proliferation Assays

Abstract Acquired resistance to imatinib mesylate (STI571) in chronic myelogenous leukemia (CML) patients has become a serious problem. To adress the novel molecular mechanism for imatinib-resistance in CML, we previously established imatinib-resistant sublines (designated KTR cells) from the CML cell line KT-1. We have analyzed p-glycoprotein expression, the number of bcr-abl fusion gene and the sequence of ATP binding site of ABL kinase domain. However, these were not responsible for imatinib-resistance in KTR cells. Interestingly, T-cell protein tyrosine phosphatase (TC-PTP) protein levels were markedly down-regulated in all KTR cells as compared to parental KT-1 cells. Therefore, we examined whether the suppression of TC-PTP expression might contribute to imatinib-resistance in KTR cells. We transduced the nuclear isoform of TC-PTP (TC45) and catalytically inactive TC45-D182A cDNA into KTR cells by retroviral gene transfer. Subsequently, we analyzed the sensitivity to imatinib by MTT proliferation assays. We also studied the signaling pathways in all transduced cells by Western blottings. KTR cells successfully expressed TC45 and TC45-D182A protein (designated KTR-TC45 and KTR-D182A cells, respectively). In MTT proliferation assays, the proliferation of KTR-TC45 cells restored their sensitivity to imatinib, but not in KTR-mock or KTR-D182A cells, indicating that transduced catalytically active TC45 restored the sensitivity to imatinib in KTR cells. In KTR2-mock cells, the percentage of annexin V positive apoptotic cells was 8% in the control and was increased to 25% upon imatinib treatment. In KTR-TC45 cells, the percentage of apoptotic cells was increased from 12% to 56% by the treatment with imatinib, suggesting that TC45 expression in KTR cells restored the susceptibility to apoptosis by imatinib mesylate. Taken together, these results indicate that the sensitivity to imatinib in KTR cells can be modulated by TC-PTP expression. In parental KT-1 cells, phosphorylation of STAT5 was abolished with the treatment of 0.5 μM imatinib for 1 hour. In contrast, STAT5 phosphorylation in KTR cells was stronger than that of KT-1 cells and only slightly suppressed upon exposure to 0.5 μM imatinib. In KTR-mock and KTR-D182A transduced cells, STAT5 phosphorylation was augmented compared to KTR-TC45 transduced cells. Upon treatment with 0.5 μM imatinib for 1 hour, phosphorylation of STAT5 was abolished in KTR2-TC45 cells whereas it remained elevated in KTR-mock and KTR-D182A cells. The expression of TC-PTP had no effect on the phosphorylation of the JAK2 or BCR-ABL in KTR cells. Besides, expression of TC-PTP did not alter protein kinase PKB/AKT or mitogen-activated protein kinase signaling in KTR cells. These results indicate that the loss of TC-PTP could enhance tyrosine phosphorylation of STAT5 and was involved in the acquired resistance to imatinib in KTR cells. In conclusion, we demonstrated that reconstitution of TC-PTP in imatinib-resistant KTR cells restored the sensitivity to imatinib. Although it will be necessary to ascertain the relevance of our studies in primary samples, we would like to propose that the loss of TC-PTP may represent a novel mechanism by which CML cells can acquire imatinib-resistance.

Detection of BCR-ABL kinase mutations in CD34+ cells from chronic myelogenous leukemia patients in complete cytogenetic remission on imatinib mesylate treatment

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 2093-2098 ◽  
Author(s):  
Su Chu ◽  
Helen Xu ◽  
Neil P. Shah ◽  
David S. Snyder ◽  
Stephen J. Forman ◽  
...  
Keyword(s):  
Imatinib Mesylate ◽  
Chronic Myelogenous Leukemia ◽  
Drug Binding ◽  
Myelogenous Leukemia ◽  
In Vitro Mutagenesis ◽  
Mrna Levels ◽  
Imatinib Resistance ◽  
Secondary Resistance ◽  
Abl Kinase ◽  
Cd34 Cells

AbstractThe BCR-ABL kinase inhibitor imatinib mesylate induces complete cytogenetic response (CCR) in a high proportion of chronic myelogenous leukemia (CML) patients. However, patients in CCR usually demonstrate evidence of residual BCR-ABL–positive progenitors. The mechanisms underlying persistence of small numbers of malignant progenitors in imatinib-sensitive patients are unclear. BCR-ABL kinase domain mutations affecting drug binding can lead to secondary resistance to imatinib. We show here that kinase mutations could be detected in CD34+ cells isolated from CML patients in CCR on imatinib. Most mutations seen have not been reported in previous clinical studies. Interestingly, several of the involved amino acid positions have been implicated in an in vitro mutagenesis screen. These BCR-ABL mutations were associated with varying levels of imatinib resistance. Two of 5 patients in whom mutations were detected on initial evaluation have relapsed. In addition, 4 patients in whom mutations were not initially detected, but with rising BCR-ABL mRNA levels on quantitative polymerase chain reaction (Q-PCR) analysis, had mutations detected on follow-up evaluation. We conclude that BCR-ABL kinase mutations can be detected in CD34+ cells from CML patients in CCR on imatinib, may contribute to persistence of small populations of malignant progenitors, and could be a potential source of relapse.

AMD107: Efficacy as a Selective Inhibitor of the Tyrosine Kinase Activity of BCR-ABL in Murine Leukemia Models.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 551-551 ◽  
Author(s):  
James D. Griffin ◽  
Doriano Fabbro ◽  
Gabriele Fendrich ◽  
Brian Huntly ◽  
Sandra Jacob ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Rational Drug Design ◽  
Leukemic Cells ◽  
In Vivo Studies ◽  
Imatinib Treatment ◽  
Imatinib Resistance ◽  
Active Inhibitor ◽  
Abl Kinase ◽  
Contact Points ◽  
Imatinib Resistant

Abstract Patient relapse due to the emergence of imatinib-resistance in advanced chronic myelogenous and Ph+ acute lymphocytic leukemias has prompted the search for improved Bcr-Abl kinase inhibitors. Up to 40% imatinib-resistant patients express mutant forms of the Bcr-Abl kinase that reduce binding of imatinib without significantly decreasing kinase activity. However, some mutants are only partially resistant, since higher concentrations of imatinib may still block kinase activity and have a beneficial clinical effect. More potent Bcr-Abl inhibitors or inhibitors with different contact points with the kinase domain are predicted to be useful for the treatment of imatinib-resistant disease. Rational drug design based upon the crystal structure of an Abl-imatinib complex, together with medicinal chemistry paradigms, resulted in the discovery of AMN107. AMN107 is a low molecular weight compound that selectively inhibits the c-Abl/Bcr-Abl, imatinib-resistant mutants of Bcr-Abl, PDGFR, and c-Kit tyrosine kinases, with IC50 values for the inibition of autophosphorylation (cell capture ELISA) of 23 nM (32D cells transfected with p210 Bcr-Abl), 83 nM (A31 cells) and 192 nM (GIST cells), respectively. Following oral administration to mice (20 mg/kg in NMP-PEG300 10:90 v/v), AMN107 was well absorbed, with a mean plasma level after 2 h in the range of 6.0-12.1 M, which corresponds to >100-fold that required to inhibit Bcr-Abl autophosphorylation in 32D.p210 cells. Following injection of 32D.p210-luciferase cells into Balb/c mice, serial imaging of the leukemic clone in live mice indicated that AMN107 could substantially reduce the accumulation of leukemic cells in the marrow, nodes, liver, and spleen, compared to a vehicle control. Further, administration of AMN107 (75 mg/kg/day p.o.) to BALB/c mice over an 18 day period, commencing 3 days after the injection of 32 D.p210 cells, resulted in the protection of 15/20 animals over 100 days observation, whereas 19/20 vehicle treated animals developed a lethal leukemic disease(all deaths occurred within 36 days of cell injection). The spleen sizes of the surviving animals in the treated group were in the normal range (0.142±0.043 g; n = 17), whereas those of the vehicle treated animals were 0.50±0.045 (n = 20). These results were confirmed in a bone marrow transplant assay, where AMN107 was found to prolong survival of mice transplanted with marrow cells infected with a p210Bcr/Abl retrovirus. AMN107 also prolonged survival of mice transplanted with a Bcr/Abl mutant (E255V) associated with imatinib resistance in patients, while imatinib treatment was unsuccessful. These in vivo studies, along with in vitro studies reported separately, indicate that AMN107 is a highly active inhibitor of Bcr/Abl that may have clinical utility in patients with Bcr/Abl+ leukemias.

Effects of Adaphostin, a Novel Tyrphostin Inhibitor, in Diverse Models of Imatinib Mesylate Resistance.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2097-2097 ◽  
Author(s):  
Joya Chandra ◽  
Jeannette Tracy ◽  
Mercedes Gorre ◽  
Moshe Talpaz ◽  
James Griffin ◽  
...  
Keyword(s):  
Imatinib Mesylate ◽  
Cell Lines ◽  
Reactive Oxygen Species Generation ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Ros Generation ◽  
Polymorphonuclear Cells ◽  
Similar Data ◽  
Imatinib Resistance ◽  
Imatinib Resistant

Abstract The impetus for kinase inhibition as a therapeutic endpoint in leukemia research has been driven largely by the success of imatinib mesylate in chronic myelogenous leukemia. However, while imatinib mesylate has been extremely effective in the treatment of chronic phase CML, resistant disease often emerges in both Philadelphia positive ALL and aggressive phase CML patients. Adaphostin is a tyrphostin inhibitor originally developed to target the bcr/abl kinase and demonstrates selectivity in CML patient isolates. Here we show that in vitro, adaphostin is cytotoxic in numerous models that are resistant to imatinib. Features of adaphostin action in bcr/abl containing cells include apoptosis induction via an oxidant dependent mechanism and degradation of bcr/abl protein. Cell lines containing point mutations in p210 bcr/abl which confer imatinib resistance exhibit intracellular peroxide production and DNA fragmentation in response to adaphostin. Bcr/abl degradation also occurs but can be dissociated from peroxide production and subsequent apoptosis signaling. Similar data has also been obtained in cell lines containing p190 bcr/abl. Studies extending these observations into mononuclear and polymorphonuclear cells from imatinib resistant CML patients and Ph+ ALL patients confirm the same mechanism in primary clinical isolates. Collectively, these data suggest that reactive oxygen species generation by adaphostin bypasses imatinib resistance in CML and Ph+ ALL. Further studies dissecting the mechanism of ROS generation by adaphostin in imatinib resistant cells may lead to the identification of new therapeutic targets.

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.

Bcr-Abl Resistance Screening Predicts a Limited Spectrum of Point Mutations To Be Associated with Clinical Resistance to the Abl Kinase Inhibitor AMN107.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1983-1983 ◽  
Author(s):  
Nikolas von Bubnoff ◽  
Jana Saenger ◽  
Paul W. Manley ◽  
Juergen Mestan ◽  
Christian Peschel ◽  
...  
Keyword(s):  
Clinical Trials ◽  
Imatinib Mesylate ◽  
Kinase Inhibitors ◽  
Point Mutations ◽  
Imatinib Resistance ◽  
Resistance Profile ◽  
Abl Kinase ◽  
Clinical Resistance ◽  
Limited Spectrum ◽  
Abl Kinase Inhibitors

Abstract In advanced-phase CML, resistance to imatinib mesylate is frequently associated with point mutations in the Bcr-Abl kinase domain. New, highly potent Abl kinase inhibitors such as AMN107 and BMS-354824, have recently entered clinical trials. Data from analyses of resistant patients will be available not before a large number of resistant patients will have been treated within clinical trials. Therefore, it will be important to generate specific resistance profiles for each compound prior to its therapeutic application. Using a cell-based screening method for resistance of Bcr-Abl positive leukemia to Abl kinase inhibitors, we generated a resistance profile for AMN107 and compared it to the resistance profile of imatinib mesylate. In contrast to imatinib, resistance to AMN107 was associated with a very limited spectrum of Bcr-Abl kinase mutations. While 26 exchanges at 21 positions occured with imatinib, the AMN107 screen revealed eight different exchanges at seven amino acid positions, with four exchanges affecting the P-loop. Novel mutations which have never been observed with imatinib, either in vitro or in resistant patients, emerged in the presence of AMN107 including an F359 exchange to isoleucine and a Q252H/S349L double mutant. In contrast to imatinib, the frequency of resistant colonies dramatically decreased with increasing AMN107 concentrations. Rarely emerging resistant colonies at a concentration of 400 nM AMN107 exclusively contained T315I. With the exception of T315I, all mutations that were identified were effectively suppressed when AMN107 was increased to 2000 nM, a concentration which is achieved in plasma in treated patients. Thus, in this system, increasing the AMN107 concentration to 400 nM prevented the emergence of resistant colonies, with the exception of T315I. Our findings suggest that AMN107 might be superior to imatinib in terms of the development of resistance. Also, AMN107 at clinically relevant concentrations may overcome imatinib resistant disease, including cases with expression of P-loop mutations. However, our study indicates that clinical resistance to AMN107 may be associated with the predominant emergence of T315I. Using this or similar approaches, it will be possible to provide information that translates into combinatorial and sequential treatment strategies and to determine critical plasma concentrations for mutations that might occur during treatment.

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