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.

Hematologic and Cytogenetic Responses in Imatinib-Resistant Accelerated and Blast Phase Chronic Myeloid Leukemia (CML) 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. 20-20 ◽  
Author(s):  
Moshe Talpaz ◽  
Hagop Kantarjian ◽  
Neil P. Shah ◽  
Nicholas Donato ◽  
John Nicoll ◽  
...  
Keyword(s):  
Phase I ◽  
Dose Escalation ◽  
Kinase Inhibitor ◽  
Cytogenetic Response ◽  
Blast Phase ◽  
Abl Kinase ◽  
Hematologic Response ◽  
Imatinib Resistant ◽  
A Minor

Abstract BMS-354825 is a novel, orally available, dual SRC/ABL kinase inhibitor with 100-fold greater potency to inhibit BCR-ABL in vitro than imatinib and 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 accelerated phase (AP) and blast phase (BP) CML patients who had hematologic progression or intolerance while being treated with imatinib. As of Aug 6, 2004, 17 patients (6 with AP; 11 with BP) have been treated in 3 cohorts with doses ranging from 35 mg BID (1 patient) to 70 mg BID of BMS-354825. BMS-354825 is rapidly absorbed with peak concentrations achieved within 2 hours and a terminal phase half-life of about 5 hours. Consistent, rapid and sustained inhibition of LYN kinase, a member of the SRC family of tyrosine kinases, has been demonstrated. Of the 11 BP patients, 7 have had hematologic response: 3 complete hematologic response (CHR), 2 ‘no evidence of leukemia’ (NEL), and 2 ‘return to chronic phase’ (RTC). Three additional patients have had significant hematologic improvement despite being on treatment only a short period of time (10–23 days). One patient with extramedullary disease was stable. Cytogenetic data is available for 8 of the 11 BP patients. Four patients had major cytogenetic response, 2 patients had a minor cytogenetic response and 2 patients had no response. BCR-ABL mutation data is available for 2 patients: one patient did not have a mutation and one patient who had a non-sustained CHR was found to have a E355G mutation. Three of 6 AP patients have had hematologic response: 2 CHRs and 1 NEL. Two patients are too early to assess. One patient demonstrated resistance to BMS-354825 due to a T315I mutation in BCR-ABL found in 8 of 10 clones. This mutation confers resistance to BMS-354825 in preclinical studies. BCR-ABL mutation status is available for 3 additional AP patients: 2 patients had no mutations identified and 1 patient in CHR had M351T/A imatinib-resistant mutations. Of 3 patients for whom early cytogenetic data is available, 1 had a minor cytogenetic response (40% Ph+). To date BMS-354825 has been very well tolerated. Two patients in blast phase had evidence of mild tumor lysis syndrome. Dose escalation is continuing and phase II studies in chronic, accelerated and blast phase CML are currently being initiated. Further studies are required to establish LYN’s potential role in imatinib-resistant CML. The clinical data demonstrate that BMS-354825 can frequently override imatinib resistance in advanced CML, and provide compelling evidence supporting the safety and efficacy of BMS-354825 in imatinib-resistant accelerated and blast 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.

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.

Sequences of topotecan and cisplatin: phase I, pharmacologic, and in vitro studies to examine sequence dependence.

1996 ◽  
Vol 14 (12) ◽  
pp. 3074-3084 ◽  
Author(s):  
E K Rowinsky ◽  
S H Kaufmann ◽  
S D Baker ◽  
L B Grochow ◽  
T L Chen ◽  
...  
Keyword(s):  
Phase I ◽  
Dose Escalation ◽  
Topoisomerase I ◽  
Single Agent ◽  
Maximum Tolerated Dose ◽  
In Vitro Studies ◽  
Pharmacokinetic Studies ◽  
Sequence Dependence ◽  
Renal Tubular

PURPOSE A phase I and pharmacologic study was performed to evaluate the feasibility of administering the topoisomerase I (topo I) inhibitor topotecan (TPT) in combination with cisplatin (CDDP) in minimally pretreated adults with solid tumors. The study was designed to evaluate the magnitude of the toxicologic and pharmacologic differences between the two sequences of drug administration. MATERIALS AND METHODS TPT was administered as a 30-minute infusion daily for 5 days and CDDP was given either before TPT on day 1 or after TPT on day 5. Each patient was treated with both schedules on an alternating basis every 3 weeks. Sequential dose escalation of TPT or CDDP resulted in three dosage permutation of TPT/CDDP (mg/m2): 0.75/50, 1/50, and 0.75/75. After the maximum-tolerated dose (MTD) level was achieved, the feasibility of using granulocyte colony-stimulating factor (G-CSF) to permit further dose escalation was studied. To examine the interaction of TPT and CDDP in vitro, human A549 lung cancer cells were exposed to these agents concurrently and sequentially. RESULTS Dose-limiting neutropenia and thrombocytopenia resulted after the doses of TPT or CDDP were increased to greater than 0.75 and 50 mg/m2, respectively, without and with G-CSF. The sequence of CDDP before TPT induced significantly worse neutropenia and thrombocytopenia than the alternate sequence. In vitro studies failed to provide any evidence for the differences in the cytotoxicity of these two sequences. Instead, pharmacokinetic studies suggested that the differences in toxicity were due, in part, to lower TPT clearance and exposure when CDDP preceeds TPT, possibly due to subclinical renal tubular toxicity induced by CDDP. CONCLUSION The sequence of CDDP before TPT at doses of 50 and 0.75 mg/m2, respectively, is recommended for subsequent clinical trials in tumor types in which both agents have significant single-agent activity. The potential for sequence-dependent cytotoxic, toxicologic, and pharmacologic effects should be evaluated in concurrent clinical and laboratory studies in the course of developing combination chemotherapy regimens that consist of topo I-targeting agents and other antineoplastic agents, particularly DNA-damaging agents.

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.

Monitoring of Phosphorylated CRKL in Imatinib Resistant Patients with BCR-ABL Positive Leukemias Expressing BCR-ABL Mutants Treated with AMN107.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 497-497
Author(s):  
Paul La Rosée ◽  
Susanne Holm-Eriksen ◽  
Thomas Ernst ◽  
Heiko König ◽  
Philipp Erben ◽  
...  
Keyword(s):  
Phase I ◽  
Direct Sequencing ◽  
Lymphoblastic Leukemia ◽  
Significant Proportion ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Complete Suppression ◽  
Molecular Response ◽  
Imatinib Resistance ◽  
Imatinib Resistant

Abstract AMN107 is a new, highly potent and selective BCR-ABL inhibitor currently in clinical development for the treatment of imatinib-resistant chronic myelogenous leukemia (CML) or Philadelphia positive acute lymphoblastic leukemia ALL (Ph+ALL). Pre-clinical testing has revealed AMN107 to inhibit all but one (T315I) BCR-ABL mutants which have been associated with imatinib resistance. We sought to determine the pharmacodynamic activity of AMN107 by measuring the proportion of phosphorylated CrkL (CrkL-P) as a surrogate of BCR-ABL activity in vivo. Assay validation revealed a CV-value of 13%, which was defined as cut-off value for significant modulation of the Crkl-P/CrkL ratio. A total of 34 patients (median age 61 years, range 35–80) diagnosed with imatinib resistant Ph+ ALL (n=10), CML in chronic phase (n=1), accelerated phase (n=13), myeloid (n=7), or lymphoid blast crisis (n=3) were investigated in a phase I study permitting individual dose escalation (50–1200 mg/day). Proportion of CrkL-P (Crkl-P/total Crkl) was determined by Western blot, ratio BCR-ABL/ABL by quantitative RT-PCR, and mutation status by direct sequencing in 73 peripheral blood or bone marrow samples from baseline and during treatment with AMN107. Median follow up was 89 days (range 13–386). Patients expressed e1a2 (n=7), b2a2 (n=12), b3a2 (n=14), and b2a2&b3a2 (n=1) BCR-ABL transcripts. At baseline, 18 pts exhibited BCR-ABL mutations (P-loop, n=4; T315I, n=3; others, n=11), in 4 pts two different mutations were found in parallel. Prior to treatment with AMN107, the median proportion of CrkL-P indicating BCR-ABL activity was 47% (range 0–69%). Significant reductions of the proportion of CrkL-P were observed from a dose level of 200 mg AMN107/day. CRKL-P (0%) became undetectable during treatment with AMN107 indicating complete suppression of BCR-ABL in 16 pts starting at AMN107 dose levels of 200 (n=1), 400 (n=2), 600 (n=4), 800 (n=8) or 1200 mg/d (n=1). At baseline, patients had unmutated BCR-ABL (n=8), M244V, Y253H, E255K, T315I, M351T, L384M/H396P, A217V/F311L, L324Q/A350V (n=1 each). Undetectability of CrkL-P, correlated with a good molecular response (ratio BCR-ABL/ABL <2%) in 3 pts. We conclude that a minimum of 200 mg of AMN107 is required to induce effective BCR-ABL inhibition in patients. Effectively repressed CrkL phosphorylation in patients lacking molecular response indicates multifactorial resistance mechanisms. Even in patients with BCR-ABL mutations, BCR-ABL may be inactive suggesting alternative signaling pathways that stimulate proliferation. However, treatment with AMN107 is associated with a reduction of the proportion of CrkL-P indicating suppression of BCR-ABL activity in a significant proportion of patients after imatinib resistance. The CrkL phosphorylation status may help to determine alternative treatment strategies including dose optimization in phase I studies.

Acquired Extramedullary Resistance to Dasatinib Due to Selection of Philadelphia-Positive Lymphoblast Clone Harboring a T315I BCR-ABL Gene Mutation: Reversal by Dose Escalation and Hydroxyurea.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4579-4579
Author(s):  
Tuija Lundan ◽  
Franz Gruber ◽  
Martin Hoglund ◽  
Bengt Simonsson ◽  
Sakari Knuutila ◽  
...  
Keyword(s):  
Dose Escalation ◽  
Low Dose ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Mutant Cell ◽  
Skin Lesions ◽  
Fish Analysis ◽  
Imatinib Resistance

Abstract Most patients with advanced Philadelphia-positive (Ph+) hematologic malignancies develop resistance to imatinib. Acquired resistance to imatinib is commonly a result of selection for subclones bearing point-mutations in the catalytic kinase domain of BCR-ABL. Dasatinib (BMS-354825), a dual-specific SRC/ABL kinase inhibitor, has shown activity in imatinib-resistant Ph+ diseases both in vitro and in vivo. Preliminary data also indicate efficacy in patients. Based on laboratory evidence, dasatinib appears to inhibit all known BCR-ABL mutant clones, with the exception of T315I, a gatekeeper mutation conferring resistance to several kinase inhibitors. Here we describe a Ph+ ALL patient, who initially developed imatinib resistance (hematologic) possibly due to BCR-ABL amplification (FISH). His disease relapsed as extensive extramedullary tumors bearing wild-type BCR-ABL. He received dasatinib 70 mg BID as part of the BMS CA180–015 study and achieved a very good partial remission. After 5 months of therapy, the disease relapsed as a solitary axillary tumor and several small palmar skin lesions. He also had blasts in the CSF indicative of neuroleukemia. Bone marrow remained in cytogenetic remission. FISH analysis of the tumor revealed 2–3 copies of BCR-ABL as previously. A highly sensitive, quantitative, mutation-specific PCR (Gruber F, ASH 2004) showed the presence of the T315I mutation, which was confirmed by sequencing. A very low level of T315I transcript was also detected in the blood. Dasatinib dose was escalated to 100 mg BID, and low-dose hydroxyurea 500 mg BID was initiated to putatively enhance the access of dasatinib in the CSF sanctuary. He also received two doses of i.t. therapy (methotrexate, cytarabine). Patient’s symptoms (confusion, headache) related to neuroleukemia resolved rapidly, skin lesions disappeared and axillary tumor decreased in size. He is currently symptom-free and has no signs of active ALL. The favorable response to dasatinib dose escalation and low-dose hydroxyurea was unexpected. Preclinical data on T315I mutant cell lines would argue against a significant concentration dependence in kinase inhibition by dasatinib. Putatively, targets other than BCR-ABL may be of importance in particular in Ph+ ALL (e.g. Src, Lyn), and this effect may account for the response. Similar off-target activity of hydroxyurea is utilized in clinical trials to overcome resistance to multidrug HIV therapy - a setting resembling current treatment of Ph+ malignancies with kinase inhibitors.

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.

Correlation of Clinical Response to Nilotinib with BCR-ABL Mutation Status in Advanced Phase Chronic Myelogenous Leukemia (CML-AP) Patients with Imatinib-Resistance or Intolerance.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1940-1940 ◽  
Author(s):  
Giuseppe Saglio ◽  
Dong-Wook Kim ◽  
Andreas Hochhaus ◽  
Simona Soverini ◽  
P. Erben ◽  
...  
Keyword(s):  
Disease Progression ◽  
Cytogenetic Response ◽  
Kinase Domain ◽  
Mutant Clone ◽  
Imatinib Resistance ◽  
Mutation Status ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Kinase Domain Mutations

Abstract The 2nd-generation bcr-abl inhibitor nilotinib is more potent than imatinib (IC50 <30 nM) against unmutated bcr-abl and active against 32/33 imatinib-resistant BCR-ABL mutants in vitro. We investigated the in vivo activity of nilotinib stratified by the baseline BCR-ABL mutation status in 127 imatinib-resistant or -intolerant CML-AP patients (pts) enrolled in an open-label phase II trial of nilotinib. Eighty-five pts (85/127, 67%) were screened prior to nilotinib therapy for BCR-ABL kinase domain mutations by direct sequencing. Of the 85 pts, 75 (88%) were resistant to imatinib and 10 (12%) were intolerant using standard published criteria. Twenty-two different baseline mutations involving 19 amino acids were identified in 50 (59%) pts analyzed. Other 35 (41%) pts did not have a baseline mutation. The most frequent mutation types identified included M351T (8 pts), G250E (7 pts), Y253H (6 pts), M244V (5 pts), F359V (5 pts) and T315I (5 pts). Twenty-two percent of pts with baseline mutations (11/50) showed more than one mutation (9 with two, 1 with three, and 1 with four mutations). All baseline mutations occurred in imatinib-resistant pts but none in intolerant pts. After 12 months of therapy, confirmed (confirmed in two consecutive analyses 4 week apart) hematologic response (HR) was achieved in 48% (21/50), major cytogenetic response (MCR) in 20% (10/50), and complete cytogenetic response (CCR) in 16% (8/50) of imatinib-resistant pts with baseline mutation versus 44% (12/25), 40% (10/25), and 20% (2/25) of imatinib-resistant pts without baseline mutation, respectively. Responses appeared to be affected by the in vitro sensitivity of the mutant clone against nilotinib. Pts with less sensitive mutation (cellular IC50 of >200nM: Y253H, E255K, E255V, F359C) representing 13% (11/85) of all patients assessed for baseline mutation, showed 13% (1/11) HR and 13% (1/11) MCyR compared to 74% (17/28) and 18% (5/28) respectively in the mutant group with IC50 of ≤200 nM. The nilotinib resistant T315I mutation occurred in 5 pts. Only one of these 5 pts who had T315I and G250E dual mutation achieved HR conceivably reflecting the sensitivity of G250E or non-mutant clone to nilotinib. At the time of data analyses, 50% of pts with baseline mutation were free of disease progression versus 62% of pts without baseline mutation. Rate of progression was 64% (7/11) in the group with less sensitive mutations and 60% (3/5) in pts. with T315I. However, the mutants most frequently associated with progression were F359V and M244V both having 4/5 pts (80%) progressed. In summary, BCR-ABL kinase domain mutations were identified at baseline in 59% of all pts in this cohort and in 67% of pts with imatinib resistance. Responses were observed across a broad spectrum of mutant genotypes. The rate of responses and disease progression may be affected by the baseline mutation types, although a larger data set with longer follow up is needed to further establish the correlation.

To Study Resistant Mechanisms and Reversal In An Imatinib Resistant Ph+ Positive Acute Lymphoblastic Leukemia Cell Line

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 2135-2135
Author(s):  
Hongyun Xing ◽  
Yuping Gong ◽  
Ting Liu
Keyword(s):  
Cell Signaling ◽  
Cell Line ◽  
Lymphoblastic Leukemia ◽  
Kinase Domain ◽  
Rt Pcr ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Cell Signaling Pathways

Abstract Abstract 2135 Objective To establish an imatinib resistant Bcr-Abl positive acute lymphoblastic leukemia (ALL) cell line in vitro and to study imatibin resistance in Ph+ ALL. The reversal of the imatinib resistance by rapamycin, the second generation tyrosine kinase inhibitor and proteasome inhibitor was studied. Methods Ph(+) ALL SUP-B15 cell line was cultured in gradually increasing concentrations of imatinib to generate the imatinib resistant cell line at 6 μM imatinib. The cytotoxic effect of imatinib and other drugs was analyzed by MTT assay. RT-PCR, flow cytometry, Western blot analyses of proteins, DNA sequence analysis of ABL kinase domain were used to clarify the possible mechanisms of the imatinib resistance in the SUP-B15/RI cell line. Results We established the imatinib resistant Ph+ ALL cell line. The fusion bcr-abl gene was 6.1 times as high as that of the parental sensitive cell, and the mdr1 gene also increased 1.7 times in SUP-B15/RI cell line by the RT-PCR detection. However, the expression of hoct1 Abcl–2 and topoIIα gene were no difference between two cell lines by the RT-PCR detection. A K362S point mutation in the Abl kinase domain of SUP-B15/RI was found. The detection of cell signaling pathway of PI3K/AKT/mTOR, RAS/RAF, NF-κBA JNK and STAT showed the expression of PTEN and 4EBP-1 was down-regulated, AKT, mTOR and P70S6K was up-regulated and the expression of other cell signaling pathways in SUP-B15/RI was similar to its parental sensitive cell line. Dasatinib, nilotinib, and bortezomib could inhibit proliferation of SUP-B15/RI cells at nM concentration. SUP-B15/RI cell line also showed partial resistance to dasatinib and nilotinib, but not bortezomib. The combination of imatinib with rapamycin had synergistic effect to the resistance cell line. Conclusion In vitro, we establish imatinib resistant Ph + ALL cell line. Overexpression of bcr-abl and mdr1 gene, K362S point mutation in ABL kinase domain and up-regulation of the cell signaling pathways of PI3K/AKT/mTOR, RAS/RAF in SUP-B15/RI cell line were involved in the resistance mechanisms. The SUP-B15/RI cell line was also resistant to the second generation tyrosine kinaeses dasatinib and nilotinib,not bortezomib in vitro. However, the combination of imatinib with rapamycin can partially overcome the resistance. Blockade of the ubiquitin-proteasome could be a promising pathway to overcome resistance to imatinib. Disclosures: No relevant conflicts of interest to declare.

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