Identification of Bcr/Abl Point Mutations Conferring Resistance to the Abl Kinase Inhibitor AMN107 by a Random Mutagenesis Study.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 494-494 ◽  
Author(s):  
Arghya Ray ◽  
Sandra Cowan-Jacob ◽  
Paul W. Manley ◽  
Jurgen Mestan ◽  
James D. Griffin
Keyword(s):  
Tyrosine Kinase ◽  
Rank Order ◽  
Point Mutations ◽  
Random Mutagenesis ◽  
Kinase Domain ◽  
Multiple Point ◽  
Tyrosine Kinase Domain ◽  
Clinical Activity ◽  
Imatinib Resistant ◽  
Mutagenesis Study

Abstract The development of kinase inhibitors such as imatinib that block the Bcr/Abl tyrosine kinase has significantly improved chronic myeloid leukemia (CML) therapy. However, patients with advanced disease often develop resistance to imatinib due to the emergence of clones with point mutations in the tyrosine kinase domain. AMN107, a novel second generation inhibitor of Bcr/Abl (Weisberg et al., Cancer Cell, 7:129, 2005) is currently in Phase 2 clinical trials and shows significant clinical activity in some patients with imatinib-resistant CML. However, it is possible that resistance to AMN107 could occur through the emergence of new Bcr/Abl point mutations, and here we report the results of a random mutagenesis study to identify Bcr/Abl mutants selected for resistance to AMN107. A library of mutations was generated in the target gene by propagating a native BCR-ABL-GFP retroviral construct through a bacterial strain deficient in a DNA repair pathway. Murine Ba/F3 cells were then transfected/infected with the mutated vector and subsequently the cells were selected for the ability to proliferate in the presence of AMN107 (0.125–0.5 μM). The Ba/F3 cells expressing native Bcr-Abl did not grow under these conditions. Single cell clones were expanded and a total of 60 individual colonies were isolated for which BCR/ABL was sequenced. Twenty colonies had single point mutations located in the kinase domain of the BCR-ABL gene. The rest had multiple point mutations and were not considered for further analysis. The point mutants identified in this way were all validated by preparing the corresponding Bcr-Abl cDNA using site-directed mutagenesis, generating a new mutant-Bcr-Abl/Ba/F3 cell line and testing for resistance to both AMN107 and imatinib. The mutant cell lines confer varying degrees of resistance to AMN107, from 5- to 400-fold. The mutant variants identified in this study included 15 novel mutations and 5 known imatinib-resistant mutations that have previously been identified in CML patients. These latter mutations included, T315I, which similar to imatinib, showed maximum resistance against AMN107 (~50% survival at 10 μM concentration). Interestingly, the majority of novel AMN107-resitant mutants were also found to be resistant to imatinib and the rank order was highly correlated to the rank order of resistance to AMN107. These data may be helpful in providing insights into the mechanism of acquired resistance of Bcr-Abl to small molecule inhibitors and are likely to predict some of the resistance mutations that may be observed in the clinic.

Identification of BCR-ABL point mutations conferring resistance to the Abl kinase inhibitor AMN107 (nilotinib) by a random mutagenesis study

Blood ◽  
2007 ◽  
Vol 109 (11) ◽  
pp. 5011-5015 ◽  
Author(s):  
Arghya Ray ◽  
Sandra W. Cowan-Jacob ◽  
Paul W. Manley ◽  
Jürgen Mestan ◽  
James D. Griffin
Keyword(s):  
Imatinib Mesylate ◽  
Kinase Inhibitor ◽  
Single Point ◽  
Point Mutations ◽  
Random Mutagenesis ◽  
Kinase Domain ◽  
Site Directed Mutagenesis ◽  
Multiple Point ◽  
Resistance Mutations ◽  
Mutagenesis Study

Abstract Patients with advanced stages of chronic myeloid leukemia (CML) often manifest imatinib mesylate resistance associated with point mutations in BCR-ABL. AMN107 is a new higher-potency inhibitor of BCR-ABL. To identify mutations in BCR-ABL that could result in resistance to AMN107, a cDNA library of BCR-ABL mutants was introduced into Ba/F3 cells followed by selection in AMN107 (0.125-0.5 μM). A total of 86 individual, drug-resistant colonies were recovered, and the SH3, SH2, and kinase domains of BCR-ABL were sequenced. A total of 46 colonies had single point mutations in BCR-ABL, with a total of 17 different mutations, all within the kinase domain. The other 40 colonies had multiple point mutations and were not analyzed further. Each of the 17 single point mutants were reconstructed by site-directed mutagenesis of native BCR-ABL and found to be approximately 2.5- to 800-fold more resistant to AMN107 than native BCR-ABL. The mutations included 6 known imatinib mesylate–resistant mutations, including T315I, which showed complete resistance to AMN107. Interestingly, most AMN107-resistant mutants were also resistant to imatinib mesylate. These results may predict some of the resistance mutations that will be detected in clinical trials with this kinase inhibitor.

Point Mutation and Alternative Splicing in the ABL Kinase Domain as a Mechanism for Imatinib Resistance.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4814-4814
Author(s):  
Rong Chen ◽  
Steven Potts ◽  
Wanlong Ma ◽  
Hagop Kantarjian ◽  
Francis Giles ◽  
...  
Keyword(s):  
Alternative Splicing ◽  
Tyrosine Kinase ◽  
Point Mutations ◽  
Sr Proteins ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Imatinib Resistance ◽  
Exon Junction ◽  
Abl Tyrosine Kinase ◽  
Imatinib Resistant

Abstract Missense point mutations in the region encoding the ABL tyrosine kinase domain have been reported in approximately 35% of patients with imatinib-resistant chronic myeloid leukemia (CML). The reported mutations result in reactivation of the BCR-ABL tyrosine kinase. Screening patients with imatinib-resistant CML, we identified 42 different mutations in the ABL tyrosine kinase domain-encoding region, 2 of which were silent (no amino acid change): A864G and G909A. The A864G mutation was associated with a 54-nucleotide reduction in the length of the mRNA transcript, representing a loss of nucleotides 1089-1143; the G909A mutation was associated with a normal-length transcript. The nt1089-1143 transcript deletion represents a partial exon deletion in which the first half of exon 8 is skipped, suggesting that A864G leads to abnormal splicing. Splicing is regulated by 6- to 8-nucleotide exonic splicing enhancer (ESE) and exonic splicing silencer (ESS) motifs recognized by the SR proteins (a family of splicing factors). We therefore used ESEfinder to examine whether A864G or G909A alter ESE motifs, which could block the ability of SR proteins to recognize and bind. This search showed that A864G is at the 7th position of an AGCTGCAG ESE motif, a binding site for SR35, and is within 35 bp of the intron-exon junction. In total, ESEfinder predicted 18 putative SR35-binding ESEs within 50 bp of the intron-exon junction, covering 20% of the kinase domain. The AGCTGCAG motif is conserved in primates (chimpanzees and monkeys) but not in rodents, while A864G is found in mice Although similar links cannot be made with the G909A mutation, these data suggest that imatinib resistance may develop in some patients through alternative splicing and the expression of a truncated (or potentially elongated) protein.

scholarly journals 4558 Investigating the functional consequences of anaplastic lymphoma kinase (ALK) mutations arising upon Lorlatinib treatment

2020 ◽  
Vol 4 (s1) ◽  
pp. 9-10
Author(s):  
Gabriela Maria Witek ◽  
Whelton Miller ◽  
David Slochower ◽  
Esther Berko ◽  
Yael Mossé ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Anaplastic Lymphoma Kinase ◽  
De Novo ◽  
Point Mutations ◽  
Md Simulations ◽  
Competitive Inhibitor ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Anaplastic Lymphoma ◽  
Mechanism Of Resistance

OBJECTIVES/GOALS: Neuroblastoma (NB) is an embryonal cancer of the sympathetic nervous system that affects mostly infants and young children. The complex genetic background present across NB patients results in diverse clinical response and difficulty in individualizing therapy. Currently, NB patients undergo a regimen of genotoxic chemotherapeutics, radiation therapy, and new immunotherapy that, while effective, has significant side effects, including excruciating pain. One promising avenue for targeted therapy in neuroblastoma focuses on anaplastic lymphoma kinase (ALK), a cell surface neural receptor tyrosine kinase. We previously identified activating point mutations within the tyrosine kinase domain of ALK as the primary cause of hereditary NB, and we and others subsequently showed that these same alterations are the most common somatic single-nucleotide mutations in the sporadic forms of the disease. Crizotinib, a first-generation small molecule ATP-competitive inhibitor of the ALK tyrosine kinase, showed limited anti-tumor activity in patients with relapsed NB harboring ALK F1174 and F1245 mutations. We have demonstrated that lorlatinib, a novel ATP-competitive ALK inhibitor, overcomes this de novo resistance in preclinical models of ALK-driven NB. Recent clinical trials with lorlatinib in patients with non-small cell lung cancer harboring an ALK fusion, and in patients with NB harboring ALK mutations show the emergence of multiple or compound ALK mutations as a mechanism of resistance. We postulate that these compound mutations disrupt the interaction between and ALK and cause resistance. In this study, we employ a computational approach to model mutated ALK in complex with lorlatinib as well as ATP to understand whether the new mutations alter the affinity or mode of lorlatinib/ATP binding to ALK, and thus cause suboptimal ALK inhibition. METHODS/STUDY POPULATION: We employ methods in computational structural biology and drug design, primarily based on molecular modeling, molecular dynamics (MD), and molecular docking. Based on existing crystal structures of wildtype ALK, we model the mutations and perform MD simulations in order to characterize the activation state of the protein as well as perform ensemble docking calculations to assess the binding affinities and modes in ALK-lorlatinib and ALK-ATP complexes. RESULTS/ANTICIPATED RESULTS: We expect that the compound mutations cause resistance to lorlatinib either by lowering protein affinity for the drug or increasing the affinity for ATP. Alternatively, the compound mutations may disrupt the protein activation state, in which case ALK may no longer be active, and another protein/pathway could be driving the resistance. DISCUSSION/SIGNIFICANCE OF IMPACT: The results of this study will enable the understanding of the mechanism of resistance to lorlatinib and facilitate the design of new ALK inhibitors, or help develop more optimal and mechanism-guided therapies aimed to overcome the resistance.

BMS-354825 Is a SRC/ABL Inhibitor with High Nanomolar Activity Against the Kit D816v Mutation, Which Drives Systemic Mastocytosis and Is Imatinib-Resistant.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 797-797 ◽  
Author(s):  
Neil P. Shah ◽  
Francis Y. Lee ◽  
Charles L. Sawyers ◽  
Cem Akin
Keyword(s):  
Tyrosine Kinase ◽  
Ex Vivo ◽  
Systemic Mastocytosis ◽  
Kinase Domain ◽  
Pharmacokinetic Analysis ◽  
Tyrosine Kinase Domain ◽  
Independent Manner ◽  
Kit Mutation ◽  
Imatinib Resistant

Abstract The vast majority of systemic mastocytosis cases are associated with a somatic KIT oncoprotein point mutation which substitutes a valine for aspartic acid (D816V), resulting in KIT receptor auto-phosphorylation in a ligand-independent manner. Previous reports have demonstrated that this mutation is inherently imatinib-resistant. Although interferon-alpha has some activity against aggressive systemic mastocytosis, major responses are uncommon, and the drug is associated with significant toxicity. To date, there remains no effective therapy for systemic mastocytosis. We recently described BMS-354825, a novel orally bioavailable SRC/ABL inhibitor that has activity against multiple imatinib-resistant BCR-ABL isoforms in vitro (Shah et al, Science 305:399, 2004). BMS-354825 is presently undergoing evaluation in a phase I clinical trial of imatinib-resistant CML patients, and is showing signs of clinical efficacy. Pharmacokinetic analysis suggests that high nanomolar concentrations of the compound can be safely achieved in humans (see Sawyers et al, Talpaz et al, abstracts submitted for this meeting). To determine if this compound warrants study in other human hematologic conditions, we tested BMS-354825 for activity against human mastocytosis cell lines HMC-1560 and HMC-1560,816, carrying an activating c-kit mutation in juxtamembrane domain (codon 560) with or without a second mutation in tyrosine kinase domain (codon 816), respectively. While 1 um imatinib failed to inhibit the growth of HMC-1560,816 cells carrying the tyrosine kinase domain c-kit mutation, BMS-354825 led to an almost complete growth inhibition at the same concentration, with an IC50 of 0.1–1 uM. In addition, growth of HMC-1560 cells carrying the juxtamembrane c-kit mutation alone was more effectively inhibited by BMS-354825 as compared to imatinib (IC50 of <0.01 vs 0.01–0.1 micromolars respectively). Significantly, detection of phospho-KIT by Western blot analysis was significantly reduced in the presence of BMS-354825 at nanomolar concentrations. An ex vivo assessment of D816V-harboring mast cell sensitivity using a flow cytometric method in systemic mastocytosis bone marrow samples is ongoing. Our findings suggest that studies to evaluate BMS-354825 for the treatment of systemic mastocytosis are warranted. Additionally, the compound may harbor activity in other disease settings that contain activating KIT mutations.

Rapid Identification Of Compound Mutations In Patients With Ph-Positive Leukemias By Long-Range Next Generation Sequencing

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1373-1373
Author(s):  
Sandra Preuner ◽  
Renate Kastner ◽  
Agnes Zopf ◽  
Proell Johannes ◽  
Pierre Foskett ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Long Range ◽  
Sanger Sequencing ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Insertions And Deletions ◽  
Dna Molecule ◽  
Generation Sequencing

Abstract Mutations in the BCR-ABL1 tyrosine kinase domain (TKD) are regarded as the most important mechanism of resistance to tyrosine kinase inhibitors (TKIs) in patients with Ph-positive leukemias. The occurrence of two or more mutations on the same DNA molecule, the so-called compound mutations, can be associated with particularly high resistance to multiple TKIs. Recent reports indicate that the frequency of compound mutations is rather high, thus rendering their reliable detection an important diagnostic challenge 1,2. Analysis of PCR amplicons of the BCR-ABL1 TKD by next generation sequencing (NGS) has become the method of choice for sensitive detection of compound mutations. This approach is, however, hampered by the requirement of 3-4 overlapping amplicons to cover the entire TKD due to the limited read length offered by most current NGS technologies. This prevents the assignment of nucleotide substitutions located on different amplicons to the same TKD/DNA molecule, and therefore requires additional laborious steps to facilitate unequivocal identification of such constellations. To overcome this limitation, we have established a long-range NGS approach on the FLX instrument (Roche) permitting the coverage of the entire TKD length of ∼0.9 kb in a single read. By testing a series of individual and consecutive specimens derived from five patients with chronic myeloid leukemia, we demonstrate that long-range NGS analysis readily permits the identification of mutations and their assignment to the same or to separate subclones at a limit of sensitivity comparable to NGS-based sequencing of shorter amplicons. In addition to the detection of individual and compound mutations, this approach also facilitates an interpretable documentation of insertions and deletions in the TKD. To address the possibility of artifacts inherent in the technique that could lead to incorrect identification of single and compound mutations, the NGS findings were reevaluated by independent technical approaches. Point mutations were confirmed by Sanger sequencing, LD-PCR 3 and pyrosequencing 4. In select cases, PCR amplicons of the BCR-ABL1 TKD derived from individual specimens were subcloned into pGEM®T easy plasmids, and >100 clones were subjected to analysis by Sanger sequencing. The observations made by NGS analysis including various single mutations (e.g. G250E, Y253H, T315A, F317I, Q252H, T315I), compound mutations (e.g. G250E/Y253H, G250E/T315A, G250E/F317I), and combinations of point mutations with small insertions or deletions (e.g. E459K/C475fs, Q252H/R362fs, T315I/R362fs) as well as large deletions involving multiple exons, could be confirmed in individual clones by Sanger sequencing, thereby documenting the reliability of the long-range NGS technology. The technical advancement presented therefore provides an economic approach to the identification of compound mutations and other genetic alterations in the entire BCR-ABL1 TKD, thus extending the diagnostic armamentarium for rapid assessment of impending resistant disease. 1. Khorashad JS, Kelley TW, Szankasi P, et al. BCR-ABL1 compound mutations in tyrosine kinase inhibitor-resistant CML: frequency and clonal relationships. Blood. 2013;121(3):489-498. 2. Soverini S, De Benedittis C, Machova Polakova K, et al. Unraveling the complexity of tyrosine kinase inhibitor-resistant populations by ultra-deep sequencing of the BCR-ABL kinase domain. Blood. 2013. 3. Preuner S, Denk D, Frommlet F, Nesslboeck M, Lion T. Quantitative monitoring of cell clones carrying point mutations in the BCR-ABL tyrosine kinase domain by ligation-dependent polymerase chain reaction (LD-PCR). Leukemia. 2008;22(10):1956-1961. 4. Alikian M, Gerrard G, Subramanian PG, et al. BCR-ABL1 kinase domain mutations: methodology and clinical evaluation. Am J Hematol. 2012;87(3):298-304. Figure Strategy of long-range NGS analysis for the detection of single and compound mutations, insertions and deletions in the BCR-ABL1 TKD. Figure. Strategy of long-range NGS analysis for the detection of single and compound mutations, insertions and deletions in the BCR-ABL1 TKD. Disclosures: Valent: Novartis: Honoraria, Research Funding. Lion:Novartis, Bristol-Myers- Squibb, Pfizer: Honoraria, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

scholarly journals SRC is a signaling mediator in FLT3-ITD– but not in FLT3-TKD–positive AML

Blood ◽  
2012 ◽  
Vol 119 (17) ◽  
pp. 4026-4033 ◽  
Author(s):  
Hannes Leischner ◽  
Corinna Albers ◽  
Rebekka Grundler ◽  
Elena Razumovskaya ◽  
Karsten Spiekermann ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Point Mutations ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Src Family Kinases ◽  
Tyrosine Kinase Domain ◽  
Molecular Abnormalities ◽  
Flt3 Mutations ◽  
Worse Prognosis ◽  
Tandem Duplications

Abstract Mutations of Fms-like tyrosine kinase 3 (FLT3) are among the most frequently detected molecular abnormalities in AML patients. Internal tandem duplications (ITDs) are found in approximately 25% and point mutations within the second tyrosine kinase domain (TKD) in approximately 7% of AML patients. Patients carrying the FLT3-ITD but not the FLT3-TKD mutation have a significantly worse prognosis. Therefore, both FLT3 mutations seem to exert different biologic functions. FLT3-ITD but not FLT3-TKD has been shown to induce robust activation of the STAT5 signaling pathway. In the present study, we investigated the mechanisms leading to differential STAT5 activation and show that FLT3-ITD but not FLT3-TKD uses SRC to activate STAT5. Coimmunoprecipitation and pull-down experiments revealed an exclusive interaction between SRC but not other Src family kinases and FLT3-ITD, which is mediated by the SRC SH2 domain. We identified tyrosines 589 and 591 of FLT3-ITD to be essential for SRC binding and subsequent STAT5 activation. Using site-specific Abs, we found that both residues were significantly more strongly phosphorylated in FLT3-ITD compared with FLT3-TKD. SRC inhibition and knock-down blocked STAT5 activation and proliferation induced by FLT3-ITD but not by FLT3-TKD. We conclude that SRC might be a therapeutic target in FLT3-ITD+ AML.

FLT3 ITD and FLT3 Tyrosine Kinase Domain Mutants Induce Different Phenotypes in a Murine Bone Marrow Transplant Model.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3371-3371
Author(s):  
Rebekka Grundler ◽  
Cornelius Miething ◽  
Christian Thiede ◽  
Christian Peschel ◽  
Justus Duyster
Keyword(s):  
Bone Marrow ◽  
Clinical Outcome ◽  
Tyrosine Kinase ◽  
Lymph Nodes ◽  
Point Mutations ◽  
Marrow Transplant ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Myeloproliferative Disease ◽  
Transplant Model

Abstract Activating mutations of FLT3 are frequent in patients with acute myeloid leukemia (AML). Two distinct types of FLT3 mutations are most common: Internal tandem duplication (ITD) of the juxtamembrane domain-coding sequence in approximately 30% of patients with AML and point mutations within the second tyrosine kinase domain (TKD) in about 7% of AML patients. Patients carrying the FLT3 ITD mutation seem to have a significantly worse prognosis, whereas the impact of TKD mutations on clinical outcome has not yet been determined. Recently, point mutations within the activation loop of FLT3 were also found in a significant percentage of infant and childhood acute lymhoblastic leukemia (ALL). Previous studies demonstrate that mice receiving transplants of bone marrow retrovirally infected with FLT3 ITD develop a myeloproliferative disease. The effect of FLT3 TKD mutations in vivo has not yet been investigated. To examine the transforming properties of FLT3 TKD mutants in primary hematopoietic cells, we used a bone marrow transplant model (BMT). Therefore we transduced bone marrow with retrovirus expressing either FLT3 D835Y or FLT3 I836M+R and transplanted it to lethally irradiated syngeneic recipient mice. As control we also transplanted mice with FLT3 WT and ITD infected bone marrow, respectively. We found that mice transplanted with FLT3 ITD developed a myeloproliferative disorder in mice, as previously described. In contrast, mice transplanted with FLT3 TKD mutants developed a lymphoid disease with distinct hematologic manifestation. Most recipients of FLT3 TKD transduced bone marrow developed T lymphoma syndrome, characterized by massive enlargement of thymus and lymph nodes. Some mice developed a B lymphoid leukemia with splenomegaly and enlarged lymph nodes. Interestingly, the disease latency of 53 to 183 days (median 102 days) of FLT3 TKD mutants contrasted with FLT3 ITD mice, which succumbed myeloproliferative disease within 53 to 70 days (median 58 days). The lymphoid manifestation and longer latency of FLT3 TKD in a murine BMT model together with the absent influence of FLT3 TKD mutations on clinical outcome of AML patients suggest differences in cell signaling between FLT3 TKD mutants and FLT3 ITD. The TKD mutants seem to require lymphoid cell context for full malignant transformation, whereas FLT3 ITDs transform myeloid cells.

Activating FLT3 Mutations in CD4+/CD8− Pediatric T-Cell Acute Lymphoblastic Leukemias.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1465-1465
Author(s):  
Pieter Van Vlierberghe ◽  
Jules P.P. Meijerink ◽  
Ronald W. Stam ◽  
Wendy van der Smissen ◽  
Elisabeth R. van Wering ◽  
...  
Keyword(s):  
T Cell ◽  
Tyrosine Kinase ◽  
Point Mutations ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Activating Mutations ◽  
Activated State ◽  
Flt3 Mutations ◽  
Genetic Aberration ◽  
High Level

Abstract Activating mutations in the FMS-like tyrosine kinase 3 gene (FLT3) are the most common genetic aberration in acute myeloid leukemia (AML). Internal tandem duplications (ITD) in the juxtamembrane (JM) domain, or point mutations (PM) in the activation loop of the tyrosine kinase domain lead to a constitutive activated state of the FLT3 tyrosine kinase. Recently, FLT3 mutations were identified in a cohort of 69 adult T-ALL patients, showing that this genetic abnormality is not only restricted to myeloid leukemias. To validate the incidence of FLT3 mutations in pediatric T-ALL and investigate its relation to outcome and other clinical and immunophenotypical parameters, we screened 72 diagnostic pediatric T-ALL samples. FLT3/ITD mutations were identified in 2/72 pediatric T-ALLs (2.7%), whereas 0/72 showed point mutations in the kinase domain. Immunophenotypic analysis revealed a similar profile for both FLT3 mutated patient samples, i.e. TdT+, CD2+, CD5+, CD7+, CD4+/CD8−, cytoplasmic CD3+, surface CD3− and CD10−. Although representing early T-cell differentiation stages for both patient samples, these cases seem to have a more advanced immunophenotype compared to the FLT3 mutated adult T-ALL cases, previously described (CD34+, CD4−/CD8−). Both FLT3 mutated patients showed high level LYL1 and LMO2 expression. In addition, both pediatric samples contained a HOX11L2 translocation, which was not present in the FLT3 mutated adult T-ALL cases. The first FLT3 mutated patient suffered a relapse 13 months after initial diagnosis, whereas the other is still in continued complete remission for 61+ months. Interestingly, the relapse material showed no FLT3/ITD mutation, indicating that the FLT3 mutated T-ALL subclone seems to be effectively eradicated by current chemotherapy. These data suggest that the application of FLT3 inhibitors for FLT3-mutated T-ALLs, as recently suggested in literature, may not further improve treatment outcome in pediatric T-ALL.

The K247R Substitution in ABL Tyrosine Kinase Domain Is Not a Mutation Leading to Imatinib Resistance but a Polymorphism Rarely Present in CML Patients and in Normal Subjects.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4831-4831
Author(s):  
Kaddour Chabane ◽  
Franck Nicolini ◽  
Jean-Michel Cayuela ◽  
Philippe Rousselot ◽  
Xavier Thomas ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tyrosine Kinase ◽  
Disease Progression ◽  
Direct Sequencing ◽  
Point Mutations ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Molecular Response ◽  
Imatinib Resistance ◽  
Abl Tyrosine Kinase

Abstract The major mechanism for resistance to imatinib mesylate (IM) is the onset of ABL point mutations altering functional inhibition of the tyrosine kinase activity by IM. Biochemical, cellular assays and clinical studies have demonstrated that different BCR-ABL mutations exhibit various degrees of resistance, and mutations occurring in the ATP-binding loop may be correlated with subsequent disease progression. In this study, we investigated the status of the K247R ABL polymorphism and correlated it to disease outcome. Patients and methods: Two patients (P1 and P2) were diagnosed with CML based on peripheral blood findings, karyotyping analysis (showing t(9;22)(q34;q11)) and molecular analysis for BCR-ABL (indicating M-BCR fusion transcripts) in 2,000 and 1,994, respectively. P1, a 54 years old male, enrolled in the Novartis IRIS study, was randomly assigned to the interferon + AraC arm after informed consent. The patient achieved a complete hematologic remission (CHR) at 3 months and a complete cytogenetic response (CCR) at 24 months. At time of cytogenetic relapse (2 years later), P1 had never received IM which was introduced in April 2,005. P2 received IM (400mg to 600mg/day) during 82 months and had shown only CHR during 14 months with no MCR. Epithelial cells were collected in the mouth through sterile foam tipped applicators for both patients. DNA was obtained from blood samples of 232 individuals, including 124 patients with CML, 72 patients with acute myeloid leukemias (AML) and 36 normal healthy donors. The K247R change was studied by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique (AluI restriction enzymatic site abolished) and was confirmed by direct sequencing. Results: Both patients, showing the K247R substitution located close to the P-loop, were retrospectively investigated during disease progression. We found that this change accounted for 100% of the BCR-ABL transcripts at CML diagnosis (and in all the further samples) for P1 (no material was yet available from P2 at this time) and for 50% of the ABL gene in extracted DNA from epithelial cells in the 2 patients. P1, despite of the K247R substitution received IM (400 mg/day) and achieved a CHR, a CCR and a good molecular response (one log reduction after two months of IM). IM resistance observed in P2 was probably due to the presence of an additional F317L mutation, known to induce a very strong resistance to IM. The F317L mutation accounted for 100% of the BCR-ABL transcripts at the resistance, was absent in DNA from epithelial cells. The screening showed the K247R exchange in 1 normal subject (heterozygote), in 2 CML patients (P1 and P2: at heterozygous state in normal cells and at homozygous state in BCR-ABL transcripts) and in none AML samples. According to that, the incidence of this polymorphism seems to reach nearly 1 to 1.5%. Conclusion: This study demonstrates that the K247R substitution in ABL tyrosine kinase domain is not a mutation leading to IM resistance but only a rare polymorphism. Detailed analysis of this polymorphism status will be reported.

AML-associated Flt3 kinase domain mutations show signal transduction differences compared with Flt3 ITD mutations

Blood ◽  
2005 ◽  
Vol 106 (1) ◽  
pp. 265-273 ◽  
Author(s):  
Chunaram Choudhary ◽  
Joachim Schwäble ◽  
Christian Brandts ◽  
Lara Tickenbrock ◽  
Bülent Sargin ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Target Genes ◽  
Kinase Inhibitor ◽  
Point Mutations ◽  
Kinase Domain ◽  
Tyrosine Kinase Domain ◽  
Activating Mutations ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells ◽  
Kinase Domain Mutations

Activating mutations of Flt3 are found in approximately one third of patients with acute myeloid leukemia (AML) and are an attractive drug target. Two classes of Flt3 mutations occur: internal tandem duplications (ITDs) in the juxtamembrane and point mutations in the tyrosine kinase domain (TKD). We and others have shown that Flt3-ITD induced aberrant signaling including strong activation of signal transducer and activator of transcription 5 (STAT5) and repression of CCAAT/estradiol-binding protein α (c/EBPα) and Pu.1. Here, we compared the signaling properties of Flt3-ITD versus Flt3-TKD in myeloid progenitor cells. We demonstrate that Flt3-TKD mutations induced autonomous growth of 32D cells in suspension cultures. However, in contrast to Flt3-ITD and similar to wild-type Flt3 (Flt3-WT), Flt3-TKD cannot support colony formation in semisolid media. Also, in contrast to Flt3-ITD, neither Flt3-WT nor Flt3-TKD induced activation or induction of STAT5 target genes. Flt3-TKD also failed to repress c/EBPα and Pu.1. No significant differences were observed in receptor autophosphorylation and the phosphorylation of Erk-1 and -2, Akt, and Shc. Importantly, TKD but not ITD mutations were a log power more sensitive toward the tyrosine kinase inhibitor protein kinase C 412 (PKC412) than Flt3-WT. In conclusion, Flt3-ITD and Flt3-TKD mutations display differences in their signaling properties that could have important implications for their transforming capacity and for the design of mutation-specific therapeutic approaches.

Sign in / Sign up

Export Citation Format

Share Document