Comparative Analysis of Two BCR-ABL Small Molecule Inhibitors Reveals Overlapping but Distinct Mechanisms of Resistance.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 552-552 ◽  
Author(s):  
Michael R. Burgess ◽  
Neil P. Shah ◽  
Brian J. Skaggs ◽  
Francis Y. Lee ◽  
Charles L. Sawyers
Keyword(s):  
Small Molecule ◽  
Small Molecule Inhibitors ◽  
Point Mutations ◽  
Kinase Domain ◽  
Drug Binding ◽  
Saturation Mutagenesis ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant

Abstract A novel dual SRC/ABL kinase inhibitor, BMS-354825, is showing promise for the treatment of imatinib-resistant chronic myeloid leukemia not only in vitro (Shah NP, et al., Science 305:399), but also in a phase I clinical trial (ASH abstract: Sawyers CL, et al.) Resistance to imatinib is increasingly found in patients due to point mutations in the BCR-ABL kinase domain that do not impair kinase activity but prevent drug binding. BMS-354825 is more potent than imatinib and retains activity against 14 of 15 imatinib-resistant BCR-ABL mutants in vitro. The compound’s ability to inhibit imatinib-resistant forms of BCR-ABL is presumed to be due to its relaxed binding requirements, whereas imatinib requires the adoption of a closed conformation of the kinase to bind. We addressed the hypothesis that the relaxed binding requirements of BMS-354825 would limit the range of BCR-ABL mutations that confer drug resistance. To address this question, we employed a saturation mutagenesis experiment as described by others (Azam M, et al., Cell 112:831) and found that the spectrum of BMS-354825-resistant mutants was reduced compared to that of imatinib. In a series of such screens, mutations at only four amino acids have been isolated, two of which account for the vast majority of resistant clones. In contrast, Azam et al. isolated over 20 mutations in a screen for imatinib resistance, a finding which has been generally reproduced in our lab. All four BMS-354825-resistant mutations map to known BMS-354825 contact residues as shown by co-crystallographic studies (ASH abstract: Tokarski JS et al., Bristol-Myers Squibb). Mutations at L248, T315, and F317 show BMS-354825 resistance and have been previously reported to confer imatinib resistance. Mutation at V299 represents a novel mode of resistance. Interestingly, some point mutations conferring BMS-354825 resistance were at positions known to be mutated in cases of imatinib resistance, but the mutated residues differed. Furthermore, the identity of the mutated residue was crucial in conferring sensitivity or resistance to an individual drug as shown by comparison of cellular IC50’s (see table). For example, F317L was shown previously to confer imatinib resistance. F317V, on the other hand, demonstrates relative BMS-354825-resistance but is still exquisitely sensitive to imatinib. In a screen for mutants simultaneously resistant to both drugs, we consistently recover 30–50 fold fewer mutant clones compared to single drug treatment. All such clones isolated to date encode for T315I. Kinase domain point mutation is becoming an increasingly encountered clinical problem in diseases treated with small molecule inhibitors. Our findings suggest that combination therapy with imatinib and BMS-354825 may be of clinical utility in CML, particularly by delaying the development of resistance. IC50 for growth (nM) Baf3 Clone imatinib BMS-354825 p210 wt < 1,000 < 5 T315I > 10,000 > 500 T315A 1,000 100 F317L 2,000 10 F317V < 1,000 60 V299L 1,000 20 L248R > 10,000 20

BCR-ABL P-Loop Deletion Detected in Imatinib-Resistant CML Patients Corresponds to Splice Variant Associated with L248V Point Mutation, Retains BCR-ABL Kinase Activity, and Responds to Dasatinib Treatment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2168-2168
Author(s):  
Nikolas von Bubnoff ◽  
Philipp Erben ◽  
Martin Müller ◽  
Tanja Lahaye ◽  
Susanne Schnittger ◽  
...  
Keyword(s):  
Splice Variant ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Point Mutations ◽  
Kinase Domain ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Exon 4

Abstract Clonal selection of cells harboring point mutations of the BCR-ABL kinase domain are considered a major cause of resistance to imatinib. More than 40 different point mutations have been described that cause a variable degree of imatinib resistance, and display a differential response to alternative kinase inhibitors, like dasatinib or nilotinib. Here, we describe three cases (2 m, 1 f) with imatinib resistant chronic myelogenous leukemia (CML) associated with a specific deletion of 81 bp of ABL exon 4. Patients were diagnosed with chronic phase (CP) CML at the age of 52, 54, and 68 years. After initial interferon alpha based therapies for 32, 60, and 71 mo, imatinib therapy was initiated at dosages between 400–800 mg per day. After 18, 24, and 29 mo patients lost hematologic response in CP CML (n=2) or progressed to lymphoid blast crisis (BC, n=1). Molecular analysis of the ABL kinase domain revealed a deletion of a 81 bp fragment associated with a loss of amino acids 248–274 in all cases. In one patient, an additional M351T mutation was found. In the two cases with CP CML, dasatinib was commenced for imatinib resistance, resulting in a partial hematologic and minor cytogenetic response (60 and 70% Ph+ metaphases, respectively) after 14 mo of therapy. The patient with lymphoid BC was treated with vincristine and prednisone and died 24 mo after appearance of imatinib resistance. In two cases, sequencing of genomic DNA revealed an underlying CTG/GTG mutation associated with a L248V amino acid switch. The point mutation activated a cryptic splice site within ABL exon 4 leading to an in-frame splice variant characterized by the loss of a 81 bp 3′ portion of exon 4. We sought to evaluate the BCR-ABL kinase activity of the splice variant and the response to tyrosine kinase inhibitors in vitro. The 81 bp deletion of p210 BCR-ABL was cloned using cDNA from one of the patients. Using this construct, retrovirally transduced Ba/F3 cells were transformed upon growth factor withdrawal. These cells expressed BCR-ABL at the transcript and protein levels. Presence of the 81 bp deletion was confirmed by sequencing. Despite the presence of the corresponding 27 amino acid P-loop deletion (Δ248–274), Western blot indicated strong autophosphorylation of BCR-ABL, which decreased in the presence of imatinib to non-detectable levels at concentrations of 1.25μM and above. In the presence of imatinib/nilotinib/dasatinib, the growth of BCR-ABL expressing Ba/F3 cells was shifted from an IC50 of 125/30/0.5nM for wild-type BCR-ABL to 470/185/1.9nM for Δ248–274 cells. Thus, in vitro data demonstrate that deletion of almost the entire P-loop does not abrogate BCR-ABL kinase activity and results in only marginal resistance towards ABL kinase inhibitors. We conclude that deletions of BCR-ABL may be the result of alternative splicing generated by point mutations associated with resistance to imatinib. The Δ248–274 splice variant retains BCR-ABL kinase activity and sensitivity to imatinib, nilotinib, and dasatinib.

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 &lt;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 &gt;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.

BCR-ABL Kinase Dynamics and Drug Resistance.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1996-1996 ◽  
Author(s):  
Mohammad Azam ◽  
Valentina Nardi ◽  
William C. Shakespear ◽  
Robert R. Latek ◽  
Darren Veach ◽  
...  
Keyword(s):  
Kinase Domain ◽  
Drug Binding ◽  
Hinge Region ◽  
Activation Loop ◽  
In Vitro Mutagenesis ◽  
Resistance Mutations ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Clinical Resistance

Abstract The aberrant signaling behavior caused by the expression of BCR-ABL is necessary and sufficient to cause chronic myeloid leukemia (CML), an observation which paved the way for the development of imatinib (GleevecTM), a small molecule inhibitor of the BCR-ABL kinase. Enthusiasm for the remarkable efficacy of imatinib has been tempered by the development of clinical resistance. The most common mechanisms for resistance are the development of kinase domain mutations and/or overexpression of the BCR-ABL gene, with mutations in the kinase accounting for ~90 % of all cases. The resistance-conferring lesions are found in regions of the kinase that are critical to its autoregulation, such as P-loop, C-helix, gatekeeper area, activation loop and the SH2-C-lobe interface. Mechanistically, these mutations effect either a steric blockade or a change in the dynamic equilibrium that favors the active kinase conformation that precludes imatinib binding. We have analyzed two dual Src-Abl kinase inhibitors, AP23464 and PD166326, against 58 BCR-ABL kinase variants conferring imatinib resistance. PD166326 binds to the Abl kinase domain in the open although enzymatically inactive conformation, while AP23464 targets the active conformation. Both of these compounds have effectively suppressed the cell growth of imatinib resistance variants, except for a recurrent mutation in the gatekeeper residue (T315I). The P-loop variants are more sensitive to AP23464 than PD166326. Interestingly, the imatinib resistant variants from the C-helix, hinge region, activation loop and SH2-C-lobe region, are hypersensitive to both compounds, as compared to native BCR-ABL. The BCR-ABL variants in the C-helix, gatekeeper area, and the activation loop are more sensitive to AP23464 than PD166326, while variants from the hinge region and the SH2-C-lobe interface are hypersensitive to PD166326. Altogether, these results define a differential requirement for a specific ABL conformation for drug binding of AP23464 and PD166326. In order to better understand their structure activity relationships and the patterns of resistance, we carried out an in-vitro mutagenesis-screen using different concentration of the drug either alone or in combination with imatinib. AP23464 mediates 2–3 time less resistance than PD166326. A higher concentration of all three compounds suppresses all resistance mutations, save for the notable exceptions, T315I and F317L/VandC. Resistance conferring mutations selected at 10–20 fold higher IC50 values are different. AP23464 efficiently suppresses the mutations from the P-loop (except E255K) and two mutations from the activation loop, while PD166326 remains refractory to the mutations in the C-helix and SH2-C-lobe interface. In combination with imatinib, AP23464 and PD166326 suppressed the emergence of most resistance mutations, with the notable exception of T315I. These in-vitro studies demonstrate that the combination of two or three different conformation specific inhibitors is needed to suppress the emergence of resistance. We are characterizing variants of AP23464 that we predict will show activity against the most challenging imatinib resistance mutant T315I.

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.

scholarly journals In vitro Activity of Bcr-Abl Inhibitors AMN107 and BMS-354825 against Clinically Relevant Imatinib-Resistant Abl Kinase Domain Mutants

2005 ◽  
Vol 65 (11) ◽  
pp. 4500-4505 ◽  
Author(s):  
Thomas O'Hare ◽  
Denise K. Walters ◽  
Eric P. Stoffregen ◽  
Taiping Jia ◽  
Paul W. Manley ◽  
...  
Keyword(s):  
Kinase Domain ◽  
Vitro Activity ◽  
In Vitro Activity ◽  
Abl Kinase ◽  
Imatinib Resistant

scholarly journals Mutation in Abl kinase with altered drug-binding kinetics indicates a novel mechanism of imatinib resistance

2021 ◽  
Vol 118 (46) ◽  
pp. e2111451118
Author(s):  
Agatha Lyczek ◽  
Benedict-Tilman Berger ◽  
Aziz M. Rangwala ◽  
YiTing Paung ◽  
Jessica Tom ◽  
...  
Keyword(s):  
Binding Site ◽  
Kinase Domain ◽  
Protein Kinase Inhibitors ◽  
Drug Binding ◽  
The Body ◽  
Binding Kinetics ◽  
Wild Type ◽  
Resistance Mutations ◽  
Imatinib Resistance ◽  
Abl Kinase

Protein kinase inhibitors are potent anticancer therapeutics. For example, the Bcr-Abl kinase inhibitor imatinib decreases mortality for chronic myeloid leukemia by 80%, but 22 to 41% of patients acquire resistance to imatinib. About 70% of relapsed patients harbor mutations in the Bcr-Abl kinase domain, where more than a hundred different mutations have been identified. Some mutations are located near the imatinib-binding site and cause resistance through altered interactions with the drug. However, many resistance mutations are located far from the drug-binding site, and it remains unclear how these mutations confer resistance. Additionally, earlier studies on small sets of patient-derived imatinib resistance mutations indicated that some of these mutant proteins were in fact sensitive to imatinib in cellular and biochemical studies. Here, we surveyed the resistance of 94 patient-derived Abl kinase domain mutations annotated as disease relevant or resistance causing using an engagement assay in live cells. We found that only two-thirds of mutations weaken imatinib affinity by more than twofold compared to Abl wild type. Surprisingly, one-third of mutations in the Abl kinase domain still remain sensitive to imatinib and bind with similar or higher affinity than wild type. Intriguingly, we identified three clinical Abl mutations that bind imatinib with wild type–like affinity but dissociate from imatinib considerably faster. Given the relevance of residence time for drug efficacy, mutations that alter binding kinetics could cause resistance in the nonequilibrium environment of the body where drug export and clearance play critical roles.

High Sensitive Detection of Imatinib-Resistance Associated Mutations Based on ARMS-PCR.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2943-2943
Author(s):  
Franz X.E. Gruber ◽  
Mikchail Soevershaev ◽  
Marita Olsen ◽  
Bjoern Skogen
Keyword(s):  
Kinase Inhibitors ◽  
Point Mutations ◽  
High Sensitivity ◽  
Kinase Domain ◽  
Single Step ◽  
Sensitive Detection ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Loop Region ◽  
Highly Sensitive Detection

Abstract Background: Point mutations in the Abl kinase domain are associated with resistance against imatinib. Strategies to overcome resistance include dose escalation, combination treatment using imatinib with conventional or other developmental agents or, in the future, imatinib may be replaced by other tyrosine kinase inhibitors which work effectively against mutated clones. Mutational profiling of the BCR-ABL kinase domain will in this scenario become an important analysis as a supplement to BCR-ABL quantitation and may provide the rational basis for therapy, once resistance is diagnosed. Our group reported recently a sensitive, single step PCR assay for quantitation of mutated clones based on the ARMS principle. Aim: We describe an optimized, two step analysis for high sensitivity screening of mutated clones associated with resistance against imatinib targeting all P-Loop mutations, the T315I and M351T. Methods: In a first conventional PCR-reaction a cDNA-region spanning the BCR-ABL breakpoint is amplified resulting in an isolation of the BCR-ABL kinase domain for further analysis. An aliquot is then analysed in a second PCR step, conducted on the real time PCR Taqman platform. Selectivity for the mutated clone is conferred by the amplification refractoriness of non complementary primer 3′-ends (ARMS principle). By introducing potent nucleotide-mismatches in position n-2, selectivity of the assay could be further increased. Even in the P-Loop region, which is known to be a difficult PCR template, misannealing could be reduced to an acceptable level. Results: Assays targeting all P-Loop mutations inclusive the T315I and M351T were tested by analysis of patient samples diluted in normal cDNA and non-mutated BCR-ABL and plasmid dilutions, containing the targeted mutation in a background of wildtype plasmids. Generally a 1:1000 dilution of mutated templates could be detected (sensitivity 0.1%). For some mutations even higher sensitivity could be achieved (0.01%). The level of sensitivity is generally higher than reported for other methods described before. The first PCR step can be conducted in parallel to other PCR-based detection strategies. The second step can be run simultanously to Taqman based BCR-ABL quantitation. This makes the described assay the ideal supplement to general mutation detection approaches like D-HPLC or sequencing strategies. Compared to the single step assay we desribed before, the two step approach increases sensitivity with one or two log factors. Conclusion: The described assay may be suitable for highly sensitive detection of mutated clones in resistant CML patients as a supplement to less sensitive general screening approaches and BCR-ABL quantitation.

Leukemic Stem Cells of Chronic Phase CML Patients Possess Uniquely Elevated BCR-ABL Kinase Activity and Acquire Spontaneous BCR-ABL Kinase Domain Mutations at a High Frequency.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 438-438 ◽  
Author(s):  
Xiaoyan Jiang ◽  
Kyi Min Saw ◽  
Allen Eaves ◽  
Connie Eaves
Keyword(s):  
Stem Cells ◽  
Tyrosine Kinase ◽  
Kinase Activity ◽  
Point Mutations ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Tyrosine Kinase Activity ◽  
Abl Kinase ◽  
Kinase Domain Mutations

Abstract Growing evidence indicates that the therapeutic potential of imatinib mesylate (IM) for the treatment of CML may be limited initially by a relative innate resistance of the leukemic stem cells and eventually by an accumulation of cells with BCR-ABL tyrosine kinase domain mutations. We now show that the amount and tyrosine kinase activity of p210-BCR-ABL in the most primitive and relatively IM-unresponsive lin−CD34+CD38− CML cells is 3 to 10-fold higher than in the majority of the lin−CD34+CD38+ CML progenitors (n=3). These results confirm previous BCR-ABL transcript data and identify elevated p210-BCR-ABL expression to be a likely important factor in the characteristic IM-insensitivity of very primitive CML cells. To determine whether in vivo, CML stem cells also accumulate gene mutations affecting the BCR-ABL kinase domain, cDNAs were prepared from RNA extracts of purified lin−CD34+CD38− cells isolated from 3 chronic phase patients that had not received IM therapy. Bidirectional sequencing of individually cloned cDNAs from these samples revealed BCR-ABL kinase domain mutations in 2 of the 3 patients at frequencies of 10% (1/10), 20% (2*/10,*identical mutations). Incubation of these lin−CD34+CD38− cells in vitro for 2–3 wk ± a high concentration of IM (up to 10 μM, which was sufficient to reduce the tyrosine kinase activity in the input cells by 70±12% and in their 2 wk progeny by 10±5%) selected a subpopulation of more differentiated and completely IM-resistant cells. This was shown in Western blots by the inability of 10 μM IM to reduce either their p210-BCR-ABL tyrosine kinase activity or CrkL phosphorylation and in methylcellulose assays ±5 μM IM. As predicted, IM-selected cells showed a higher frequency of kinase domain mutations (13–20% vs 0–20% of cDNA clones analyzed from 3 wk cells cultured ±IM). Analysis of individual colonies produced from CFCs in the cultured cells showed all (21/21) colonies from IM-selected cells had mutations vs 50% (5/10) in those cultured without IM. The total frequency of mutant cDNAs detected was also increased in the IM-resistant cells (35–55% vs 10–25% mutant cDNAs in selected vs control cells). Interestingly, in most cases, both wild-type and mutant cDNAs were identified in the same colony, indicating de novo generation of mutations in vitro. Overall, >50 different mutations were identified. These included 10 point mutations previously associated with clinical IM resistance (including G250 and T315), another 13 point mutations previously identified in a comprehensive mutational screen, and >20 previously undescribed mutations. Several of the latter affect the critical region of the P loop, the c-helix and the activation loop and would be predicted to confer significant IM resistance. To investigate the possibility that the observed genomic instability of very primitive CML cells might be related to their elevated innate p210-BCR-ABL activity, BCR-ABL transcript levels in individual IM-selected, fully resistant and control (similarly treated but no IM exposure) colonies were compared. This showed that BCR-ABL transcripts were ~20-fold higher (P<0.05) in the resistant colonies (30 assessed from 3 patients). These findings suggest that the increased BCR-ABL expression and activity that uniquely characterizes the most primitive CML cells may contribute not only to their innate insensitivity to IM but also to a deregulation of genomic stability leading to the emergence of IM-resistant mutants and other subclones associated with disease progression.

Overcoming Resistance in Chronic Myelogenous Leukemia

2013 ◽  
pp. 306-312
Author(s):  
Michael J. Mauro
Keyword(s):  
Chronic Myelogenous Leukemia ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Kinase Domain ◽  
Drug Binding ◽  
Myelogenous Leukemia ◽  
Novel Therapy ◽  
Abl Kinase ◽  
Clinical Resistance

Resistance in chronic myelogenous leukemia is an issue that has developed in parallel to the availability of rationally designed small molecule tyrosine kinase inhibitors to treat the disease. A significant fraction of patients with clinical resistance are recognized to harbor point mutations/substitutions in the Abl kinase domain, which limit or preclude drug binding and activity. Recent data suggest that compound mutations may develop as well. Proper identification of clinical resistance and prudent screening for all causes of resistance, ranging from adherence to therapy to Abl kinase mutations, is crucial to success with kinase inhibitor therapy. There is currently an array of Abl kinase inhibitors with unique toxicity and activity profiles available, allowing for individualizing therapy beginning with initial choice at diagnosis and as well informed choice of subsequent therapy in the face of toxicity or resistance, with or without Abl kinase domain mutations. Recent studies continue to highlight the merits of increasingly aggressive initial therapy to subvert resistance and importance of early response to identify need for change in therapy. Proper knowledge and navigation amongst novel therapy options and consideration of drug toxicities, individual patient characteristics, disease response, and vigilance for development of resistance are necessary elements of optimized care for the patient with chronic myelogenous leukemia.

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