A Cell-Based Screening Method for Resistance of Bcr-Abl Positive Leukemia Identifies the Mutation Pattern for an Alternative Abl Kinase Inhibitor.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 558-558
Author(s):  
Nikolas von Bubnoff ◽  
Darren R. Veach ◽  
Heiko van der Kuip ◽  
Walter E. Aulitzky ◽  
Jana Saenger ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Screening Method ◽  
Point Mutations ◽  
Kinase Domain ◽  
Screening Strategy ◽  
Culture Conditions ◽  
Cross Resistance ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
A Cell

Abstract The increasing impact of targeted cancer treatment demands strategies to identify and evaluate resistance mechanisms toward kinase inhibitors prior to their therapeutic application. Point mutations within the Bcr-Abl kinase domain constitute the major mechanism of resistance toward imatinib mesylate in Philadelphia-positive (Ph+) leukemia. Using Bcr-Abl-transformed Ba/F3 cells, we established a cell-based screening strategy for the prediction of specific kinase mutations that cause resistance toward kinase inhibitors. With imatinib at clinically relevant concentrations, we generated 368 resistant Ba/F3 sublines that were derived from resistant colonies. Thirty-two different single point mutations within the kinase domain of Bcr-Abl were identified in twenty-five per cent (liquid culture conditions) and seventy-two per cent (solid culture conditions) of these lines at known and novel positions. Using imatinib, the pattern and relative frequency of mutations reflected matters observed in patients with imatinib resistance. We then applied this screen to the pyrido-pyrimidine PD166326 (PD16), an investigational Abl kinase inihibitor. Compared to imatinib, we observed a five to seven times lower frequency of resistant colonies with equipotent concentrations of PD16. In addition, PD16 produced a distinct pattern of Bcr-Abl mutations. P-loop, A-loop and the known imatinib contact site T315 were affected with both inhibitors, whereas C-helix and SH2 contact sites were affected in imatinib resistant colonies exclusively. In contrast to imatinib, where kinase domain mutations were still widely distributed over the kinase domain even at at 4μM, mutations observed with PD16 at a concentration of 100nM narrowed to the exchange at position T315 to iseulicine. We did not detect mutations outside the kinase domain. Some resistant sublines displayed increased Bcr-Abl activity. Mutations that were derived from the screen were cloned and examined for the extent of cross-resistance to both inhibitors. The majority of mutations were effectively suppressed by PD16 at 50–500nM. In contrast, only few mutations were inhibited by imatinib at 5–10μM. However, exchanges at position F317 mediated resistance toward PD16, but were inhibited by standard concentrations of imatinib. Since this cell-based system produced results that are clinically significant, it may be used to predict resistance mutations in Bcr-Abl and other oncogenic kinases like cKit, EGFR, FIP1L1-PDGFRalpha or FLT3 towards clinically applicated and investigational drugs. Thus, this robust and simple screening strategy provides a rational basis for combinatorial and sequential treatment strategies.

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.

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.

Inhibition of T315I Bcr-Abl and Other Imatinib-Resistant Bcr-Abl Mutants by the Selective Abl Kinase Inhibitor SGX70393.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1373-1373 ◽  
Author(s):  
Thomas O’Hare ◽  
Christopher A. Eide ◽  
Jeffrey W. Tyner ◽  
Matthew J. Wong ◽  
Caitlyn A. Smith ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Imatinib Resistant ◽  
Escape Mutants ◽  
A Cell ◽  
Almost All ◽  
Proliferation Assays

Abstract Imatinib effectively inhibits the tyrosine kinase activity of Bcr-Abl, the molecular driver of CML. Emergence of imatinib resistance due to mutations within the Bcr-Abl kinase domain (KD) has prompted the development of new Abl kinase inhibitors. A particularly important target is Bcr-Abl(T315I), which accounts for 15–20% of patients with resistance. To address this unresolved need, we profiled the novel Abl kinase inhibitor SGX70393 against native and mutant Bcr-Abl. Methods: We assessed the efficacy of SGX70393 in cellular and biochemical assays against a panel of KD mutants. Cell proliferation assays and Bcr-Abl tyrosine phosphorylation immunoblot analyses were performed for parental Ba/F3 cells, Ba/F3 cells expressing unmutated Bcr-Abl, or Ba/F3 cells expressing a single Bcr-Abl KD mutation (M244V, G250E, Q252H, Y253F, Y253H, E255K, E255V, F311L, T315I, F317L, M351T, F359V, V379I, L387M, H396P, or H396R). The resistance profile of SGX70393 was also evaluated using a recently developed accelerated, cell-based mutagenesis assay (Bradeen, et al. Blood, June 2006; doi:10.1182). Results: SGX70393 inhibited growth of cells expressing Bcr-Abl(T315I) (IC50: 7.3 nM) or unmutated Bcr-Abl (IC50: 12 nM). Sensitivity of Bcr-Abl mutants to SGX70393 partitioned into three categories: high (IC50<25 nM: M244V, T315I, F359V, V379I, L387M, H396P, and H396R), medium (IC50<300 nM: Q252H, Y253H, E255K, and F311L), and low (IC50>500 nM: G250E, Y253F, E255V, and F317L). A cell-based mutagenesis screen for Bcr-Abl kinase domain escape mutants emerging in the presence of SGX70393 revealed a concentration-dependent reduction in surviving clones, with five previously reported Bcr-Abl mutations (L248M; G250E; Y253F; E255V; F317V) accounting for almost all resistance. Conclusions: (a) SGX70393 is a potent inhibitor of native and T315I mutant Bcr-Abl. (b) SGX70393 coverage extends to most clinically relevant mutants except mutations of the p-loop and F317.

The Mutation Spectrum of CML Patients Resistant to Imatinib: More Heterogeneous Than Just BCR-ABL Kinase Domain Point Mutations?.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1948-1948
Author(s):  
Tuija Lundan ◽  
Franz Gruber ◽  
Aleksandra Silye ◽  
Henrik Hjorth-Hansen ◽  
Ingvild Mikkola ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Point Mutations ◽  
Treatment Strategies ◽  
Diagnostic Procedures ◽  
Kinase Domain ◽  
Molecular Response ◽  
Imatinib Resistance ◽  
Log Reduction ◽  
Abl Kinase ◽  
Catalytic Loop

Abstract Cells harboring BCR-ABL kinase domain (KD) mutations are the major source for resistance to tyrosine kinase inhibitors (TKI) in chronic myeloid leukemia (CML). A second and third generation of TKIs are in development for inhibition of mutated forms of BCR-ABL. At present, clinical treatment strategies are based on molecular response and mutational profiling. Profiling is usually performed using unbiased methods (sequencing and D-HPLC of PCR products), but mutation specific methods are upcoming (ARMS-PCR, ASO-PCR, ligation-PCR). Here, we describe molecular findings resulting from a collaborative Nordic approach for molecular characterization of imatinib resistant Norwegian (N) and Finnish (F) CML patients. Complementary DNA derived from a total of 87 subjects (60 N and 27 F) with suboptimal or failed response to imatinib (less than 3 log reduction in tumor load after 12 months of treatment or loss of any response) were subjected to BCR-ABL KD sequencing of amino acid residues 240 to 420 (N) and 209 to 498 (F). In addition, single samples were analyzed by multiplex PCR for detection of transcript variants as well as cloning and sequencing of single clones. BCR-ABL KD mutations were found in 35 patients (20 N, 15 F, 40% of the total material). The most frequent findings were ABL KD point mutations with M351T and G250E representing the single most frequent mutations, 25 and 16%, respectively. The most frequently mutated cluster was in the catalytic loop (40% of all mutations), followed by P-loop (38%) and other regions (22%). Mutations in the gatekeeper cluster were extremely rare in our material (one case of a F317L). Additional sequence variations were observed in 6 patients: the L248V associated deletion of exon 5 (n=1), smaller insertion or deletion variants (2–4 basepairs) resulting in truncated proteins (n=2) and ABL exon 7 splicing (n=3). Finally, three cases with imatinib resistance expressed rare BCR-ABL transcript types without any evidence for KD mutations (two cases of e6a2 and one with e19a2 transcript). Imatinib resistance due to T315I BCR-ABL is extremely rare in Nordic CML patients. In addition to known hotspot mutations we identified novel sequence irregularities in association to imatinib resistance. We hypothesize that resistance-associated BCR-ABL isoforms are more heterogeneous than previously thought. Consequently, diagnostic procedures aiming in comprehensive molecular verification of TKI resistance should be designed as unbiased, since methods solely targeting known mutated BCR-ABL variants will miss many patients. However, an early detection of low-level mutated clones will require sensitive, mutation-specific analyses.

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.

Optimal Treatment for Patients after Dasatinib or Nilotinib: Comparison of Preclinical Activities of Approved and Promising Investigational Kinase Inhibitors Against BCR-ABL Kinase Domain Mutations That Confer Clinical Resistance to Dasatinib or Nilotinib.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4589-4589
Author(s):  
Corynn Kasap ◽  
Christopher Weier ◽  
Neil P. Shah
Keyword(s):  
Second Generation ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Kinase Domain ◽  
Inhibitor Therapy ◽  
Drug Resistant ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Clinical Resistance ◽  
Kinase Domain Mutations

Abstract The optimal management of patients with chronic myeloid leukemia (CML) is increasingly reliant upon molecular studies. Loss of response to imatinib in CML is most commonly associated with selection for a limited number of BCR-ABL kinase domain mutations that impair the ability of imatinib to effectively bind to BCR-ABL Molecular understanding of imatinib resistance mechanisms has led to the development of effective “second generation” BCR-ABL kinase inhibitors, such as dasatinib and nilotinib, which have clinical activity against most, but not all, drug-resistant mutations. Analysis of the BCR-ABL kinase domain in patients who develop resistance to second-generation inhibitors has implicated further selection of drug-resistant BCR-ABL kinase domain mutants in nearly all cases reported to date. Encouragingly, the number of resistant mutations capable of conferring clinical resistance to the most clinically-advanced second-generation agents, dasatinib (approved by the US FDA and EMEA) and nilotinib (approved in Mexico and Switzerland), appears to be restricted to a relatively small number of amino acid substitutions. As clinical experience with dasatinib and nilotinib grows, an understanding of the relative sensitivities of dasatinib- and nilotinib-resistant BCR-ABL mutants to other kinase inhibitors, both approved and investigational, is critical to optimize clinical outcomes in patients with resistance to dasatinib or nilotinib. At the present time, kinase inhibitor therapy options for patients with resistance to one of these agents include the investigational options bosutinib and MK-0457 (VX-680), as well as dasatinib and nilotinib (for patients not yet exposed to one of these agents) and re-exposure imatinib. It is likely that the success of therapeutic intervention in these cases can be predicted based upon the preclinical sensitivity of the mutation(s) involved with the agent chosen. We have therefore conducted a thorough biochemical and biological cross-analysis of the activities of each of these clinically-useful kinase inhibitors against mutations that confer clinical resistance to dasatinib or nilotinib. These studies provide clinicians with a useful reference for choosing an appropriate kinase inhibitor based upon the identity of the resistant BCR-ABL kinase domain mutation(s) detected at the time of relapse when faced with a patient who has lost response to dasatinib or nilotinib. It is hoped that the application of such “personalized medicine” strategies to the clinical management of CML cases will further improve outcomes in patients treated with kinase inhibitor therapy.

Screening of Mutations in BCR-ABL Kinase Domain in Chronic Myeloid Leukemia (CML) Patients Treated with Kinase Inhibitors by Denaturing High-Performance Liquid Chromatography (D-HPLC).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4580-4580
Author(s):  
Cintia C. Mascarenhas ◽  
Anderson F. Cunha ◽  
Katia B.B. Pagnano ◽  
Rosana A. Silveira ◽  
Fernando F. Costa ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Bone Marrow Transplantation ◽  
Marrow Transplantation ◽  
Kinase Inhibitors ◽  
Blast Crisis ◽  
Point Mutations ◽  
Kinase Domain ◽  
Molecular Response ◽  
Abl Kinase ◽  
Clinical Resistance

Abstract Point mutations within the ABL kinase domain are the most frequent mechanism for reactivation of kinase activity of the BCR-ABL gene and have been associated with clinical resistance to tyrosine kinases (TK) inhibitors in CML patients conferring in some of them a poor prognosis. The T315I (Treonine → Isoleucine) is a mutation described in exon 6 of BCR-ABL gene that makes the protein resistant to all kinase inhibitors most currently used for treating CML (imatinib, nilotinib and dasatinib). D-HPLC allows for high throughput mutation screening. This technique is based on heteroduplex formation by PCR products amplified from wild type and mutant alleles. Under optimized denaturing conditions, these heteroduplexes can be distinguished from homoduplex. In this study we screened mutations in exon 6 of BCR-ABL gene in patients treated with kinase inhibitors, in different phases of the disease. We evaluated 85 patients: 9 at diagnosis, 81 in chronic phase, 3 in accelerated phase, one in blast crisis. Thirty four were resistant to imatinib, 10 of them to dasatinib and three had suboptimal response to imatinib. In 9 of 85 (10,5%) samples, D-HPLC showed an abnormal elution profile suggesting the presence of nucleotide changes. Automated sequencing confirmed the presence of two point mutations: T315I (two patients) and F359V (two patients). Five patients requires sequencing confirmation. Patients with T315I mutation failed to imatinib and dasatinib. One of them relapsed after bone marrow transplantation in blast crisis. Patients with F359V mutation were resistant to imatinib. One of them has partial hematological response with dasatinib and the other is in complete molecular response after bone marrow transplantation. D-HPLC seems to be a ship and practical method for routine clinical monitoring for emergence of kinase domain mutations and may be useful for optimizing therapy in CML. Early detection of emerging mutant clones may help in decision-making of alternative treatment.

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.

Comparison of Imatinib, AMN107 and Dasatinib in an Accelerated Cell-Based Mutagenesis Screen.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 691-691 ◽  
Author(s):  
Michael W.N. Deininger ◽  
Heather Bradeen ◽  
Taiping Jia ◽  
Thomas O’Hare ◽  
Stephanie G. Willis ◽  
...  
Keyword(s):  
Structural Changes ◽  
Kinase Inhibitors ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Drug Concentrations ◽  
A Cell ◽  
Vitro Model ◽  
Proliferation Assays ◽  
Abl Kinase Inhibitors

Abstract Background. Mutations in the Bcr-Abl kinase domain (KD) are the leading cause of acquired imatinib (IM) resistance. Dasatinib (BMS354825) and AMN107 are potent alternate Abl inhibitors with activity at nanomolar levels against wild type Bcr-Abl and most KD mutants, with the exception of T315I. In a cell-line based mutagenesis assay we compared incidence and type of Bcr-Abl mutants emerging in the presence of IM, AMN107 and dasatinib. Methods. BaF3-p210Bcr-Abl cells were mutagenized by 24 hours exposure to 0.42 μM N-ethyl-N-nitrosourea (ENU), a dose with minimal cytotoxicity. After ENU washout cells were seeded at 5 x 105/well in 96-well plates and observed for growth for up to 4 weeks. Cells from wells with growth were expanded and subjected to BCR-ABL KD sequencing. Results: The frequency of wells with growth decreased with higher doses of all 3 inhibitors (table 1) and growth tended to occur later. Only isolated wells had growth without ENU exposure. At ≥2 μM IM (2-fold the IC90 in cell proliferation assays) 18 different mutations were seen, with highly resistant mutants prevailing at higher concentrations (table 2). At 50 nM AMN107 (2-fold the IC90) Y253H, G250E, F359C, E255K, L384M, L387F, E292V and T315I were detected, at 500 nM Y253H, E255V and T315I were recovered and only T315I at 2000 nM. At 5 nM dasatinib (2-fold the IC90), E255K, L284V, F317V were detected in addition to T315I, at 10 nM T315I, F317V/I and V299L were found and at 25 nM only T315I. All resistant clones growing out at ≥4 μM IM, 500 nM AMN107 or 10 nM dasatinib were KD mutant, suggesting that KD mutations were the sole cause of the observed resistance. Conclusions: (i) At drug concentrations corresponding to 2-fold IC9018 different mutations were recovered with IM, 9 with AMN107 and 6 with dasatinib, suggesting that the conformational requirements for dasatinib binding to Abl may be least stringent. If free plasma trough levels ≥25 nM dasatinib or ≥2000 nM AMN107 are achievable, the only mutant predicted to emerge clinically is T315I. (ii) No additional mutations were observed with AMN107 compared to IM, suggesting the structural changes in AMN107 compared to IM did not generate novel vulnerable sites. (iii) At least in this in vitro model, resistance to Abl kinase inhibitors is entirely dependent on Bcr-Abl, despite the fact that ENU treatment is expected to induce multiple additional mutations. Thus a T315I inhibitor combined with AMN107 or dasatinib may be effective at preventing the emergence of resistance to Abl kinase inhibitors. Table 1 Recovery of resistant clones (representative experiment) Imatinib (microM) Wells with mutations/wells sequenced/wells with growth Dasatinib (nM) Wells with mutations/wells sequenced/wells with growth AMN107 (nM) Wells with mutations/wells sequenced/wells with growth 2 62/62/82 5 8/24/96 10 0/24/96 4 74/74/74 10 38/38/56 50 20/24/96 8 27/27/42 25 22/22/24 500 45/45/46 16 12/12/12 100 20/20/21 2000 23/23/24 Table 2 Percentage of resistant clones with T315I mutations Imatinib (μM) number of different mutations/% T315I Dasatinib (nM) number of different mutations/% T315I AMN107 (nM) number of different mutations/% T315I 2 16/27.8 5 4/16.7 10 0/0.0 4 7/43.2 10 3/63.2 50 8/20.8 8 4/37.4 25 1/100 500 3/62.0 16 4/50.0 100 1/100.0 2000 1/100.0

Generation of Specific Resistance Profiles in FLT3-ITD and FIP1L1-PDGFRalpha towards Specifically Acting Tyrosine Kinase Inhibitors.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1376-1376
Author(s):  
Nikolas von Bubnoff ◽  
Silvia Thoene ◽  
Sivahari P. Gorantla ◽  
Jana Saenger ◽  
Christian Peschel ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinase Inhibitors ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Kinase Domain ◽  
Myelogenous Leukemia ◽  
Resistance Mutations ◽  
Abl Kinase ◽  
Mechanism Of Resistance

Abstract BCR-ABL kinase domain mutations constitute the major mechanism of resistance in patients with chronic myelogenous leukemia treated with the ABL kinase inhibitor imatinib. Mutations causing resistance to therapeutic kinase inhibition were also identified in other target kinases in various malignant diseases, such as FLT3-ITD in acute myelogenous leukemia, cKit in gastrointestinal stromal tumors, EGFR in patients with lung cancer, and FIP1L1-PDGFRalpha in hypereosinophilic syndrome. Thus, mutations in kinase domains seem to be a general mechanism of resistance to therapeutically applicated tyrosine kinase inhibitors. We recently developed a cell-based screening strategy that allows one to predict the pattern and relative abundance of BCR-ABL resistance mutations emerging in the presence of imatinib, and the novel ABL kinase inhibitor AMN107 (nilotinib). We therefore intended to determine, if this method would also allow the generation of resistant cell clones with other oncogeneic tyrosine kinases as targets in the presence of specifically acting kinase inhibitors. When FLT3-ITD and su5614 were used as drug/target combination in our cell-based method, the frequency of resistant clones in the presence of su5614 at 10 times the IC50 was 0.17 per million cells. In 40 per cent of resistant clones, point mutations were detected leading to amino acid exchanges within the FLT3-ITD split kinase domain. The yield of resistant clones was increased by the factor of 14 to 2.37 per million cells by adding ethyl-nitrosourea (ENU), a potent inducer of point mutations. Also, the proportion of mutant clones increased from 40 to 74 per cent. In 83 mutant clones that were examined so far, we detected eight exchanges affecting kinase domain two (TK2) of the split kinase domain within or shortly behind the FLT3-ITD activation loop (A-loop). We did not detect exchanges affecting TK1. We next examined whether resistant clones would also come up with FIP1L1-PDGFRalpha-transformed cells in the presence of imatinib. Again, the yield of resistant clones increased when cells were pretreated with ENU, and a proportion of resistant clones contained mutations in the FIP1L1-PDGFRalpha kinase domain, affecting the nucleotide-binding loop (P-loop) and A-loop. We conclude that cell-based resistance screening is a simple and powerful tool that allows prediction of resistance mutations towards kinase inhibitors in various relevant oncogeneic kinases.

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