Mutations in the BCR/ABL Kinase Encoding the Resistance to Imatinib Mesylate Do Not Modify the Sensitivity to Genotoxic Treatment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2194-2194
Author(s):  
Milene Bargaoanu ◽  
Tomasz Skorski
Keyword(s):  
Imatinib Mesylate ◽  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Point Mutations ◽  
Kinase Domain ◽  
Dna Lesions ◽  
Leukemia Cells ◽  
Activation Loop ◽  
Γ Irradiation ◽  
Abl Kinase

Abstract Imatinib mesylate (IM), a selective inhibitor of ABL kinase activity, revolutionized the treatment of BCR/ABL-positive leukemias. Unfortunately, clinical and experimental observations reveal that resistance to IM is a rising problem, which obscures an otherwise very successful oncogene-targeted therapy. IM resistance can be achieved by point mutations in the kinase domain of BCR/ABL and have been detected in 50–90% of patients with acquired resistance to IM, including ~23% of the IM-naive patients. Strategies to enhance the effect of IM and eventually overcome the resistance are dose escalation, addition of a growth factor, and combinations with novel tyrosine kinase inhibitors like AMN107 and dasatinib, or with inhibitors targeting downstream BCR/ABL effectors, e.g. PI-3k. Unfortunately, resistance to other small molecule inhibitors is likely to appear, as well. Therefore, we tested the sensitivity of leukemia cells expressing IM-resistant BCR/ABL mutants to genotoxic agents. Baf3 cells expressing similar levels of p210BCR/ABL wild-type (WT) and Y253F, Y253H, E255K, E255V, T315I, M351T, and H396P mutants were established. These mutants were selected since they represent several functionally distinct ABL kinase domain regions, including P-loop (Y253F/H and E255K/V), the site of a hydrogen bond with IM (T315I), the activation loop hinge (M351T), and the activation loop (H396P). In addition, they exhibit altered transformation potency, kinase activity, and substrate utilization, irrespective of sensitivity to IM. Western analysis demonstrated similarity, but also differences in the patterns of tyrosine phosphorylated proteins in total cell lysates. As expected, these clones displayed different level of sensitivity to IM: T315I>Y253H>E255V>E255K=Y253F>M251T=H396P>WT. However, leukemia cells expressing the WT and IM-resistant BCR/ABL kinase mutants demonstrated similar sensitivity to cytotoxic drugs such as hydroxyurea (HU), mitomycin C (MMC), and N-Methyl N’-nitro-N-nitrosoguanidine (MNNG). Surprisingly, clones expressing IM-resistant BCR/ABL kinase mutants were more sensitive to γ-irradiation than their WT counterparts. γ-H2AX (histone H2AX phosphorylated on S139) nuclear foci, which serve as marker of DNA double-strand breaks (DSBs), the most lethal DNA lesions, were detected by immunofluorescence. Leukemia cells expressing WT and IM-resistant p210BCR/ABL proteins accumulated similar numbers of DSBs after MMC treatment and γ-irradiation. This observation suggests that leukemia cells expressing IM-resistant p210BCR/ABL mutants may be eliminated by genotoxic treatment, especially γ-irradiation. In addition, IM-resistant BCR/ABL kinase mutants, in contrast to the WT kinase may not be able to modulate the mechanisms protecting from apoptosis induced by γ-irradiation.

Bcr-Abl resistance screening predicts a limited spectrum of point mutations to be associated with clinical resistance to the Abl kinase inhibitor nilotinib (AMN107)

Blood ◽  
2006 ◽  
Vol 108 (4) ◽  
pp. 1328-1333 ◽  
Author(s):  
Nikolas von Bubnoff ◽  
Paul W. Manley ◽  
Jurgen Mestan ◽  
Jana Sanger ◽  
Christian Peschel ◽  
...  
Keyword(s):  
Imatinib Mesylate ◽  
Kinase Inhibitors ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Treatment Strategies ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Advanced Phase ◽  
Clinical Resistance ◽  
Limited Spectrum

Abstract In advanced-phase chronic myeloid leukemia (CML), resistance to imatinib mesylate is associated with point mutations in the BCR-ABL kinase domain. A new generation of potent ABL kinase inhibitors is undergoing clinical evaluation. It is important to generate specific resistance profiles for each of these compounds, which could translate into combinatorial and sequential treatment strategies. Having characterized nilotinib (AMN107) against a large panel of imatinib mesylate–resistant Bcr-Abl mutants, we investigated which mutants might arise under nilotinib therapy using a cell-based resistance screen. In contrast to imatinib mesylate, resistance to nilotinib was associated with a limited spectrum of Bcr-Abl kinase mutations. Among these were mutations affecting the P-loop and T315I. Rarely emerging resistant colonies at a concentration of 400 nM nilotinib exclusively expressed the T315I mutation. With the exception of T315I, all of the mutations that were identified were effectively suppressed when the nilotinib concentration was increased to 2000 nM, which falls within the peak-trough range in plasma levels (3.6-1.7 μM) measured in patients treated with 400 mg twice daily. Our findings suggest that nilotinib might be superior to imatinib mesylate in terms of the development of resistance. However, our study indicates that clinical resistance to nilotinib may be associated with the predominant emergence of T315I.

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.

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 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.

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.

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.

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.

A Subset of Chronic Myeloid Leukemia (CML) Patients on ABL Kinase Inhibitor Therapy Develop Point Mutations outside the BCR-ABL Kinase Domain That Decrease Drug Sensitivity and May Have a Role in Disease Progression.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2188-2188
Author(s):  
Daniel W. Sherbenou ◽  
Oliver Hantschel ◽  
Stephanie Willis ◽  
Richard D. Press ◽  
Brian J. Druker ◽  
...  
Keyword(s):  
Disease Progression ◽  
Drug Sensitivity ◽  
Kinase Inhibitors ◽  
Point Mutations ◽  
Kinase Domain ◽  
Disease Stage ◽  
Common Mechanism ◽  
Current Response ◽  
Novel Mutations ◽  
Abl Kinase

Abstract Background : Point mutations in the kinase domain (KD) of BCR-ABL are the most common mechanism of drug resistance in CML patients treated with kinase inhibitors, including imatinib (IM), dasatinib and nilotinib. It has also been shown through in vitro mutagenesis screening that mutations outside the KD in the neighboring linker, SH2, SH3 and Cap domains can confer IM resistance (Azam et al. Cell, 2003). These domains in c-ABL have an autoinhibitory function on the KD and mutations result in an activated enzyme (Hantschel et al. Cell, 2003). Despite these observations, mutations in these regions have not yet been reported in CML patients being treated with ABL kinase inhibitors. Methods: Bone marrow and peripheral blood samples were obtained following informed consent from CML patients in chronic-phase (CP, n=43), accelerated-phase (AP, n=18) and blast crisis (BC, n=8). Likewise, patients with all degrees of IM response were studied, including 29 with a complete cytogenetic response (CCR), 22 never achieving CCR (nonresponders) and 18 after sustained loss of CCR (relapse). Nested RT-PCR for BCR-ABL was performed using 2 sets of primers from the breakpoint to the end of the KD. Sequencing covered the Cap, SH3, SH2, linker and KD. Mutations found outside the KD were introduced into Ba/F3 cells for testing of drug sensitivity. Results: Seven novel mutations outside the KD were discovered in 5 of 69 (7%) patients tested, 4 of which also had KD mutations. By comparison, KD mutations were found in 22 (32%). Novel mutations were in SH2 (T212R, S154N and A196V), 2 in the linker (N231D and N231I), 1 in SH3 (K84N) and 1 in the Cap (R47C). Of 2 tested thus far, T212R and N231D both confer resistance to IM, nilotinib and dasatinib in cell proliferation assays. Based on modeling of the amino acid changes in the crystal structure of c-ABL, T212R is hypothesized to act by stabilizing the active conformation of the kinase, whereas N231D is hypothesized to destabilize the autoinhibited form. Conclusions: (1) We demonstrate that mutations in the Cap, SH3, SH2 and linker domains of BCR-ABL occur in a subset of patients on IM. (2) In each case these were associated with a second mutation (inside or outside the KD), suggesting potential cooperativity. (3) Preliminary data suggest that non-KD mutations may generate a ’super-activated’ BCR-ABL kinase, conferring decreased sensitivity to inhibitors and increased intrinsic activity that may contribute to disease progression. Characteristics of patients with mutations outside the KD. Sex/Age Disease Stage (diagnosis-study) IM Response (at study) Time on IM (months) Current Response KD Mutation Result Non-KD Mutation Result Fold Increase in IM Cellular IC50 NA: Not available. Percentages indicate the mutated/total sequence. 52/F CP CCR 18 CCR C305S(15%) N231D(100%) 1.6(N231D) 84/F CP CCR 40 Relapse WT T212R(100%),
 S154N(50%) 4.1(T212R) 38/F CP CCR 42 CCR H396R(100%) A196V(40%) NA 72/M BC Relapse 67 NA H396P(100%),
 Q346R(50%) K84N(50%),
 R47C(50%) NA 54/F AP CCR 20 CCR E292V(100%) N231I(60%) NA

Activity Of Omacetaxine Mepesuccinate Against Ponatinib Resistant Philadelphia Chromosome Positive Leukemia Cells

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3840-3840
Author(s):  
Seiichi Okabe ◽  
Tetsuzo Tauchi ◽  
Yuko Tanaka ◽  
Seiichiro Katagiri ◽  
Toshihiko Kitahara ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Myeloid Leukemia ◽  
Philadelphia Chromosome ◽  
Point Mutations ◽  
Kinase Domain ◽  
Leukemia Cells ◽  
Primary Cells ◽  
Abl Kinase ◽  
Omacetaxine Mepesuccinate ◽  
High Level

Abstract Chronic myeloid leukemia (CML) is characterized by cytogenetic aberration (Philadelphia chromosome: Ph) and chimeric tyrosine kinase BCR-ABL. ABL tyrosine kinase inhibitor (TKI) therapy (e.g. imatinib, nilotinib and dasatinib) has improved the survival of Ph-positive leukemia patients. However, despite the impressive efficacy of these agents, disease relapse has been observed in clinically. Mutations in the BCR-ABL kinase domain can cause of ABL TKI resistance. In particular, one of the BCR-ABL kinase domain mutations (e.g. T315I) is associated with a high level of resistance to all available ABL TKIs. Ponatinib (formally, AP24534) is a multi-target TKI. Recently, in the PACE (Ponatinib Ph+ acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (CML) Evaluation) trial, ponatinib showed significant efficacy against Ph-positive leukemia in patients with multi-resistant T315I mutations. However, in some patients, especially those with Ph-positive ALL, ponatinib resistant clones were identified. Omacetaxine mepesuccinate, formally known as homoharringtonine is a natural alkaloid obtained from various Cephalotaxus species. Omacetaxine is a first-in-class cephalotaxine in clinical development as anti-leukemic therapy. Omacetaxine acts by binding to the A-site cleft of ribosomes and thereby transiently inhibits protein synthesis. Omacetaxine was approved for the treatment adult patients with chronic or the accelerated phase of CML resistant to other therapies. We investigated the efficacy of omacetaxine against ponatinib resistant Ph-positive cells. Ba/F3 ponatinib resistant cells (Ba/F3 ponatinib-R) have three BCR-ABL point mutations (Y253H, E255K and T315I: data not shown). With 72 h omacetaxine treatment, the cell growth of Ba/F3 ponatinib-R and Ph-positive ALL cell line was significantly reduced even at a low concentration and it is also effective to the other hematological malignancies such as acute myeloid leukemia. In contrast, Ba/F3 ponatinib-R was resistant to ponatinib. With 48 h treatment, omacetaxine dependent apoptosis was increased. Although anti-apoptotic proteins were not increased in this cell line compared to parental cells, as compound mutations such as E255V/T315I confer high-level resistance to ponatinib, these three point mutant was associated with vitro resistance to ponatinib. We also examined intracellular signaling. The phosphorylations of BCR-ABL and a down-stream molecule, Crk-L, were decreased. Protein expressions of BCR-ABL and Crk-L were also decreased. However, caspase-3 and cleaved Poly (ADP-ribose) polymerase (PARP) levels were significantly increased in low concentration. In a previous study, omacetaxine was shown to induce apoptosis in leukemic cells due to a selective decrease in short-lived proteins. We found that omacetaxine reduced the expression of BCR-ABL and heat shock protein 90(HSP90) which is stabilize BCR-ABL protein. We also found that omacetaxine reduced the expression of anti-apoptotic protein, Bcl-2. The protein expression of c-myc was also reduced. We next examined a ponatinib resistant primary Ph+ ALL and chronic phase CML samples. The ponatinib resistant primary cells have several BCR-ABL point mutations (e.g. Q252H, E255K/V, and T315I). We found the growth of primary cells was resistant to ponatinib but to be reduced after omacetaxine treatment and similar signaling events were occurred in OM-treated primary ALL cells. Omacetaxine is an inhibitor of protein synthesis. Because omacetaxine inhibits the BCR-ABL, Bcl-2 and HSP90 pathways in BCR-ABL positive leukemia cells through reduced the levels of these proteins, Omacetaxine has anti-tumor activity and promotes apoptosis. Our findings suggest that omacetaxine may benefit patients with leukemic BCR-ABL mutant cells, possibly allowing ponatinib resistant clones to be overcome. Disclosures: Ohyashiki: Novartis: Honoraria, Research Funding.

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