The Immunogenicity of BCR/ABL+ Cells Is Dominated by BCR/ABL-Regulated Antigens and Is Impaired by the Inhibition of BCR/ABL Kinase Activity.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 2200-2200
Author(s):  
Florian Scheich ◽  
Christian Peschel ◽  
Helga Bernhard
Keyword(s):  
T Cell ◽  
Fusion Protein ◽  
Kinase Activity ◽  
Kinase Domain ◽  
Cell Populations ◽  
Target Cells ◽  
Abl Kinase ◽  
Long Term Treatment ◽  
Region Segment ◽  
Constitutively Active

Abstract The inhibition of BCR/ABL kinase activity by imatinib mesylate (IM, STI571, Gleevec®) is the standard therapy for patients with Philadelphia chromosome+ (Ph+) chronic myeloid leukemia (CML). However, the long term treatment with IM or other BCR/ABL kinase inhibitors may be limited due to the development of resistant disease and accumulating side effects. Immunotherapeutical approaches directed against Ph+ CML may overcome these problems. So far, the research to develop an immunotherapy against Ph+ leukemia focused on the BCR-ABL fusion protein, giving the promising opportunity to stimulate cytotoxic T lymphocytes against the joining region segment of p210 BCR/ABL. However, only a limited number of peptides spanning the fusion region is endogenously processed and presented in context with HLA class I molecules. In contrast, the constitutively active BCR/ABL kinase leads to the upregulation and activation of multiple proteins, which may subsequently result in the expression of different, probably leukemia-associated antigens on the cell surface of BCR/ABL-positive malignant cells. In this study, we investigated the immunogenicity of antigens upregulated by BCR-ABL kinase activity, and verified the capacity of these antigens to induce an anti-leukemia T cell response in vitro. We performed CD8+ T-cell stimulations with dendritic cells transfected with mRNA coding BCR/ABL wild-type (WT). Following two stimulations, the proliferating T-cell populations were analyzed for cytokine secretion (IFN-γ) in response to target cells expressing either BCR/ABL WT or a kinase deficient (KD, K1172R) variant of BCR/ABL. With this experimental setting it was possible to compare the immunogenic potential of antigens upregulated by BCR/ABL with the immunogenicity of the BCR/ABL protein itself. We were not able to activate T-cell populations directed against either the breakpoint-region of BCR/ABL or the BCR- or ABL part of the fusion protein. In contrast, we show here that the constitutively active kinase domain of BCR/ABL has a key role in enhancing the immunogenicity of BCR/ABL+ cells. A broad, HLA-dependent T-cell immune response specifically directed against BCR/ABL regulated antigens was detected. The inhibition of BCR/ABL kinase activity by IM significantly impaired the immunogenicity of BCR/ABL+ cells. We found the same T-cell reactivity pattern in several healthy donors and a CML patient. This is the first study demonstrating the major contribution of the BCR/ABL kinase domain to the immunogenicity of BCR/ABL+ cells as T-cell responses against these cells are dominated by BCR/ABL regulated antigens, and not by BCR/ABL itself. These results may contribute to the design of clinical vaccination trials for the treatment CML patients with minimal residual disease, e.g. following successful induction therapy with BCR/ABL inhibitors.

scholarly journals A Superantigen-Antibody Fusion Protein for T-Cell Immunotherapy of Human B-Lineage Malignancies

Blood ◽  
1997 ◽  
Vol 89 (6) ◽  
pp. 2089-2097 ◽  
Author(s):  
Cecilia Gidlöf ◽  
Mikael Dohlsten ◽  
Peter Lando ◽  
Terje Kalland ◽  
Christer Sundström ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
B Cell ◽  
Binding Affinity ◽  
Mhc Class Ii ◽  
Class Ii ◽  
Target Cells ◽  
Cell Based Therapy ◽  
B Lineage

Abstract The bacterial superantigen staphylococcal enterotoxin A (SEA) is an efficient activator of cytotoxic T cells when presented on major histocompatibility complex (MHC) class II molecules of target cells. Our previous studies showed that such SEA-directed T cells efficiently lysed chronic B-lymphocytic leukemia (B-CLL) cells. Next, we made a mutated SEA–protein A (SEAm-PA) fusion protein with more than 1,000-fold reduced binding affinity for MHC class II compared with native SEA. The fusion protein was successfully used to direct T cells to B-CLL cells coated with different B lineage–directed monoclonal antibodies (MoAbs). In this communication, we constructed a recombinant anti-CD19-Fab-SEAm fusion protein. The MHC class II binding capacity of the SEA part was drastically reduced by a D227A point mutation, whereas the T-cell activation properties were retained. The Fab part of the fusion protein displayed a binding affinity for CD19+ cells in the nanomolar range. The anti-CD19-Fab-SEAm molecule mediated effective, specific, rapid, and perforin-like T-cell lysis of B-CLL cells at low effector to target cell ratios. Normal CD19+ B cells were sensitive to lysis, whereas CD34+ progenitor cells and monocytes/macrophages were resistant. A panel of CD19+ B-cell lines representing different B-cell developmental stages were efficiently lysed, and the sensitivity correlated with surface ICAM-1 expression. The anti-CD19-Fab-SEAm fusion protein mediated highly effective killing of tumor biopsy cells representing several types of B-cell non-Hodgkin's lymphoma (B-NHL). Humanized severe combined immune deficiency (SCID) mice carrying Daudi lymphoma cells were used as an in vivo therapy model for evaluation of the anti-CD19-Fab-SEAm fusion protein. Greater than 90% reduction in tumor weight was recorded in anti-CD19-Fab-SEAm–treated animals compared with control animals receiving an irrelevant Fab-SEAm fusion protein. The present results indicate that MoAb-targeted superantigens (SAgs) may represent a promising approach for T-cell–based therapy of CD19+ B-cell malignancies.

scholarly journals Direct demonstration of the clonogenic potential of every human peripheral blood T cell. Clonal analysis of HLA-DR expression and cytolytic activity.

1983 ◽  
Vol 157 (2) ◽  
pp. 743-754 ◽  
Author(s):  
A Moretta ◽  
G Pantaleo ◽  
L Moretta ◽  
J C Cerottini ◽  
M C Mingari
Keyword(s):  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Human Peripheral Blood ◽  
Cell Populations ◽  
Target Cells ◽  
Flow Cytofluorometry ◽  
Hla Dr ◽  
Clonogenic Potential ◽  
Precursor Frequency

In an attempt to determine the clonogenic properties of human peripheral blood T cells, we have developed a limiting dilution microculture system using phytohemagglutinin (PHA) as T cell activator and supernatant from PHA-stimulated spleen cultures as a source of T cell growth factors. The frequencies of cells capable of extensive proliferation under these culture conditions were 0.52-0.73, 0.98-1.11, and less than 0.02 in peripheral blood mononuclear, E-rosette-positive, and E-rosette-negative cell populations, respectively. The clonogenic potential of virtually all T cells was confirmed in experiments using single cells isolated by micromanipulation. Clone size ranged between 5 and 30 X 10(4) cells on day 14 of culture. The same microculture system was used to determine the precursor frequency of all cytolytic T lymphocytes (CTL-P). As assessed by a lectin-dependent 51Cr release assay, the CTL-P frequency in purified T cell populations ranged between 0.30 and 0.34. In comparison, the precursor frequency of T cells capable of lysing K562 target cells was ranging between 0.14 and 0.16. Parallel analysis of individual clonal cultures for both lytic activities showed that 50% of the clones exhibiting lectin-dependent lysis were also active against K562 target cells. All of the proliferating clones expressed HLA-DR antigens, although to a varying degree as assessed by flow cytofluorometry. Given the high cloning efficiency of this culture system, it appears now possible to determine the precursor frequencies of the various classes of functional cells in T cell populations.

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.

ITK-SYK Induces T-Cell Lymphoma with Accompanying Inflammatory Disease in Mice.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 693-693
Author(s):  
Christine Dierks ◽  
Hong Ma ◽  
Hendrik Veelken ◽  
Markus Warmuth ◽  
Francisco Adrian
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
T Cell ◽  
Fusion Protein ◽  
Cd8 T Cells ◽  
Point Mutations ◽  
Kinase Domain ◽  
Pleckstrin Homology Domain ◽  
T Cell Lymphomas ◽  
Malignant T Cells

Abstract The ITK-SYK fusion protein was previously described to be present in 17% of unspecified peripheral T-cell lymphomas. Here we demonstrate that expression of ITK-SYK in the bone marrow of Balb\c mice causes T-cell lymphomas in mice with a latency of only 3–4 weeks. The disease is characterized by infiltration of the spleen, lymph nodes, bone marrow and the skin with malignant T-cells and progredient destruction of these organs. The mice die about 2 months after the transplantation due to dramatic weight loss caused by infiltration of T-cells into the colon and because of progredient anemia end neutropenia due to progredient infiltration of the bone marrow. The malignant T-cells were characterized as a mixed population of CD3+, CD4+, CD8- T cells and CD3+, CD4-, CD8- T-cells. The malignant disease was accompanied by a generalized inflammatory reaction including upregulation of the inflammatory cytokines IL-5 and INF-γ. Modulation of the membrane binding of ITK-SYK or its binding to Cbl by point mutations in the pleckstrin homology domain of ITK could alter the transforming activity of ITK-SYK. The intact kinase domain was essential for the transformation process and the disease could be reversed by treatment of diseased mice with the Syk-inhibitor Curcumine. Our results demonstrate that the fusion protein ITK-SYK causes T-cell lymphomas in mice and mimics the human disease. Therefore pharmacological inhibition of Syk in patients with U-PTCL carrying the ITK-SYK fusion protein might be a new and effective treatment strategy.

Enhanced SH3:Linker Interaction Suppresses Activating Mutations of the c-Abl Protein-Tyrosine Kinase.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1208-1208
Author(s):  
Shoghag Panjarian ◽  
Shugui Chen ◽  
John Engen ◽  
Thomas Smithgall
Keyword(s):  
Tyrosine Kinase ◽  
Myristic Acid ◽  
Protein Tyrosine Kinase ◽  
Kinase Activity ◽  
Binding Pocket ◽  
Sh3 Domain ◽  
Kinase Domain ◽  
Abl Kinase ◽  
Defective Mutant ◽  
T315i Mutation

Abstract Abstract 1208 Bcr-Abl, the chimeric protein-tyrosine kinase expressed as a result of the Philadelphia chromosome translocation, plays a pivotal role in the initiation and maintenance of chronic myelogenous leukemia (CML). Imatinib (Gleevec) is an ATP-competitive Bcr-Abl inhibitor that selectively kills Bcr-Abl+ CML cells. Despite its clinical success, imatinib is less effective in the advanced stages of CML due to the emergence of drug resistance caused by point mutations in the Abl kinase domain. Second generation Bcr-Abl inhibitors such as dasatinib and nilotinib are active against most imatinib-resistant forms of Bcr-Abl, with the exception of the T315I “gatekeeper” mutant. The Abl gatekeeper residue (Thr315) is located between the ATP-binding site and an adjacent hydrophobic pocket, and forms a key hydrogen bond with imatinib. Additionally, the T315I mutation produces a strong activating effect on the downregulated c-Abl “core,” consisting of the myristoylated N-terminal Ncap, tandem SH3 and SH2 regulatory domains, the SH2-kinase linker, which forms a polyproline type II helix for internal SH3 docking, and the tyrosine kinase domain. Using hydrogen-exchange mass spectrometry, we recently found that the T315I mutation not only induced conformational changes in the Abl kinase domain as expected, but also at a distance in the RT-loop of the SH3 domain. Such changes may allosterically contribute to kinase domain activation by disturbing the negative regulatory influence of SH3:linker interaction. Recently, a new class of allosteric Bcr-Abl inhibitors has been reported that targets the myristate-binding pocket of Abl, which localizes to C-lobe of the kinase domain and away from the active site. Together with our finding that the T315I mutation perturbs SH3:linker interaction, these inhibitors support the existence of an extensive network of allosteric interactions that work together to regulate Abl kinase activity. In this project, we investigated whether enhanced SH3:linker interaction can allosterically reverse the activating effects of the T315I imatinib resistance mutation as well as mutations of the N-terminal myristoylation site and myristic acid binding pocket. We created modified versions of Abl [High Affinity Linker proteins (HALs)] by mutating multiple residues within the SH2-kinase linker to proline, thereby enhancing the SH3 domain binding affinity. Using mammalian cell-based expression assays and immunoblotting with phosphospecific antibodies, we identified five of eleven Abl-HAL proteins that did not exhibit changes in basal kinase activity. The Abl-HAL protein with the greatest enhancement of SH3:linker interaction was then combined with the T315I mutation, a myristoylation-defective mutant, and a myristic acid binding pocket mutation. Remarkably, this HAL substitution completely reversed the activating effect of the myristic acid binding pocket mutation, while substantially suppressing the activity of Abl T315I and the myristoylation-defective mutant. These results indicate that stabilization of SH3:linker interaction allosterically represses Abl activation by a wide variety of mechanisms, and suggests a new approach to allosteric control of Bcr-Abl kinase activity. Disclosures: No relevant conflicts of interest to declare.

The Role of c-Abl in Jak2 Activation in IL-3-Dependent Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1672-1672
Author(s):  
Wenjing Tao ◽  
Xiaohong Leng ◽  
Ralph B. Arlinghaus
Keyword(s):  
Cell Survival ◽  
Kinase Activity ◽  
Conflicts Of Interest ◽  
Hematopoietic Cells ◽  
Chromosomal Translocation ◽  
Kinase Domain ◽  
Bimolecular Fluorescence Complementation ◽  
Abl Kinase ◽  
A Minor

Abstract Abstract 1672 The reciprocal chromosomal translocation of Abl and Bcr locus [t(9: 22)] is present in 95% of chronic myeloid leukemia (CML) patients. The resulting Bcr-Abl oncoprotein contains a persistently activated tyrosine kinase activity that activates Jak2/Stat5 signaling pathways. Little is known about the molecular mechanism of Jak2 activation in Bcr-Abl positive CML cells, except that the IL-3 receptor is required (Tao et al., Oncogene 2008). We found that the Jak2 activity (measured by pY1007/1008) in 32D mouse hematopoietic cells steadily diminished immediately upon IL-3 withdrawal. However, expression of kinase-inactive form of Bcr-Abl (p210K1172R) in 32D cells maintained Jak2 activity for up to 8 hrs after IL-3 withdrawal. Our previous studies have shown that the C-terminal region (CT-4) of c-Abl binds to Jak2 as does the kinase domain of c-Abl (Xie et al. Oncogene, 2001). We found that Jak2 activation depends on its binding to the CT-4 region of c-Abl using Bimolecular fluorescence complementation assays. In order to examine the role of c-Abl in Jak2 activation, we expressed c-Abl in both 32D cells (32D-Abl) and 32D cells expressing p210K1172R (32D-p210K1172R+Abl). We found that unlike 32D-Abl cells which remained cytokine-dependent, a minor population (∼7%) of 32D- p210K1172R+Abl cells gained growth independency of IL-3. Compared to 32D-Abl cells in which the level of Jak2 activity was barely detected by pY1007/1008 antibody, 32D-p210K1172R+Abl cells showed a dramatic elevation of Jak2 activation, indicating that c-Abl alone is unable to induce Jak2 activation in hematopoietic cells. Phosphorylation on p210Y177 in 32D-p210K1172R+Abl cells was also strongly increased, indicative of activated Jak2 activity (Samanta et al., Leukemia 2011). We found that 32D-p210K1172R+Abl cells were sensitive to Imatinib Mesylate (IM), as 80% of 32D-p210K1172R+Abl cells were apoptotic after treatment with 5μM IM for 24hrs, indicating that the cell survival depends on the activated c-Abl kinase. The apoptosis induced by IM in 32D-p210K1172R+Abl cells could be effectively rescued by addition of IL-3, indicating the importance of Jak2 activation through IL-3 pathway in maintaining cell survival. The above results suggest that a higher level of c-Abl enables cells expressing a Bcr-Abl kinase defective protein to acquire cytokine-independent growth. The elevation of Jak2 activity in 32D-p210K1172R+ABL cells correlated with the increased c-Abl kinase activity. We propose that the c-Abl kinase plays two crucial roles in these Bcr-Abl kinase mutant cells: 1) making cells cytokine-independent for growth, and 2) promoting persistent Jak2 activation. These results lead us to propose that the Abl kinase domain within Bcr-Abl promotes Jak2 activation by binding to the Jak2 kinase. As our recent findings indicate that Jak2 is a dominant player in CML (Samanta et al., Leukemia 2011) and particularly in later stages of Bcr-Abl positive CML, we propose that the inhibition of both Jak2 and Bcr-Abl kinase activities will result in a near complete elimination of leukemia cells including CD34+CML progenitor cells. Disclosures: No relevant conflicts of interest to declare.

A Novel BCR-ABL Mutation Predicts Resistance to Tyrosine Kinase Inhibitor (TKI) Therapy by a Unique Mechanism in Patients (pts) with Philadelphia-Positive (Ph+) Leukemia.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4773-4773
Author(s):  
Alfonso Quintas-Cardama ◽  
Jorge Cortes ◽  
Hagop M. Kantarjian ◽  
Moshe Talpaz ◽  
Ji Wu ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Lymphoblastic Leukemia ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Abl Kinase ◽  
High Level ◽  
A Minor ◽  
Unique Mechanism

Abstract Albeit most pts with chronic myeloid leukemia (CML) treated with imatinib (IM) have a favorable outcome, some will acquire resistance, mainly due to the development of Abl kinase domain mutations, which confer varying levels of TKI resistance. We describe a novel V304D mutation in pts with Ph+ leukemia who failed TKI therapy. Expression of V304D mutation in BCR-ABL failed to induce cytokine-independence in Ba/F3 cells. Studies in Cos-7 cells demonstrated that this mutant did not induce autophosphorylation and was deficient in kinase activity. We detected V304D mutation in 13 (18%) of 70 IM-resistant pts screened (12 CML, 1 Ph+ acute lymphoblastic leukemia [ALL]), and it was present in a median of 37% (range, 20% to 80%) resistant clones. Median age was 60 years (range, 30 to 81) and median time from diagnosis to IM therapy was 39 months (range, 1 to 91). Eleven (92%) of 12 pts with CML were in chronic phase (CP) at IM start and 1 was in blast phase (BP). Pts received IM for a median of 35 months (range, 2 to 66). Nine pts with CML had failed interferon and 2 (1 CML, 1 Ph+ALL) allogeneic stem cell transplantation prior to IM. Ten (83%) of 12 pts started IM at 400 mg/d but all eventually received ≥600 mg/d. Six pts with CML achieved a complete hematologic response (CHR), 1 BP returned to chronic phase (RCP), and 6 (5 CML, 1 Ph+ALL) had primary hematologic resistance (HR). No cytogenetic (CG) responses were observed and 7 pts with CML CP progressed (4 to AP and 3 to BP) after IM discontinuation. Four pts with CML (1 CP, 2 AP, 1 BP) received nilotinib after IM failure for a median of 2 months (range, 1 to 3.5). Two pts (1 CP, 1 AP) showed primary HR, 1 AP progressed to BP, and 1 BP (on 600 mg twice daily) had a transient (6 weeks) CHR before showing secondary HR. Twelve pts (11 CML, 1 Ph+ALL) received dasatinib: 7 at 70 mg twice daily, 1 at 90 mg daily, 1 at 140 mg daily, 1 at 180 mg daily, 1 at 90 mg twice daily, and 1 at 120 mg twice daily. Dasatinib was administered for a median of 8 months (range, 1 to 23). Two pts achieved CHR and a minor CG response in 1 analysis (75% and 65% Ph+ cells, respectively), 1 RCP, 1 no evidence of leukemia, and 8 (67%) primary HR. One of 4 pts who started dasatinib in CP progressed to AP. Responders to dasatinib had V304D mutation in 20%, 20%, and 25% of clones, respectively. Four pts exhibited concomitant Abl kinase mutations developed prior to dasatinib therapy: 3 with F317L and 1 with G250E. One pt had a 6 base pair in-frame insertion in the TK domain. T315I mutation evolved in 1 pt after dasatinib discontinuation. Eight pts discontinued dasatinib due to disease progression (7 died), 2 were lost to follow-up, and 2 remain on CHR after 17+ and 23+ months on dasatinib. In vitro studies of cells from one pt in CP with V304D mutation (50% of clones) failed to detect CrkL phosphorylation despite detectable expression of the Bcr-Abl protein. In summary, the V304D mutation in the Abl kinase domain results in kinase inactivation and is associated with high-level resistance to TKI therapy, transformation to AP/BP in CML and a particularly poor prognosis. Loss of kinase activity by mutation represents a very unique mechanism of kinase inhibitor resistance and predicts acquisition of other transforming events that support CML cell survival.

New CFSE-based assay to determine susceptibility to lysis by cytotoxic T cells of leukemic precursor cells within a heterogeneous target cell population

Blood ◽  
2004 ◽  
Vol 103 (7) ◽  
pp. 2677-2682 ◽  
Author(s):  
Inge Jedema ◽  
Nicole M. van der Werff ◽  
Renée M. Y. Barge ◽  
Roel Willemze ◽  
J. H. Frederik Falkenburg
Keyword(s):  
T Cells ◽  
Cell Death ◽  
T Cell ◽  
Cell Population ◽  
Target Cell ◽  
Precursor Cells ◽  
Leukemic Cells ◽  
Cell Populations ◽  
Target Cells ◽  
Specific Cell

Abstract For the clinical evaluation of the efficacy of cellular immunotherapy it is necessary to analyze the effector functions of T cells against primary leukemic target cell populations which are usually considerably heterogeneous caused by differential maturation stages of the leukemic cells. An appropriate assay should not only allow the quantitative analysis of rapid cell death induction as measured by the conventional 51Cr release assay but also of the more slowly executing pathways of T-cell-induced apoptosis occurring within days instead of hours which cannot be measured using this method. Furthermore, it should dissect the differential susceptibility to T-cell-induced cell death of various target cell subpopulations and characterize the malignant precursor cells capable of producing malignant progeny. To fulfill these requirements we developed a new assay based on carboxyfluorescein diacetate succinimidyl ester (CFSE) labeling of the target cell population combined with antibody staining of specific cell populations and addition of fluorescent microbeads to quantitatively monitor target cell death occurring within a longer time frame up to at least 5 days. This new assay facilitates the analysis of differential recognition of distinct cell types within a heterogeneous target cell population and allows simultaneously evaluation of the proliferative status of surviving target cells in response to relevant cytokines. (Blood. 2004;103: 2677-2682)

scholarly journals Kinase Domain Mutants of Bcr-Abl Exhibit Altered Transformation Potency, Kinase Activity, and Substrate Utilization, Irrespective of Sensitivity to Imatinib

2006 ◽  
Vol 26 (16) ◽  
pp. 6082-6093 ◽  
Author(s):  
Ian J. Griswold ◽  
Mary MacPartlin ◽  
Thomas Bumm ◽  
Valerie L. Goss ◽  
Thomas O'Hare ◽  
...  
Keyword(s):  
Kinase Activity ◽  
Kinase Inhibitors ◽  
Philadelphia Chromosome ◽  
Kinase Domain ◽  
Atp Binding ◽  
Loss Of Function ◽  
Imatinib Resistance ◽  
Major Mechanism ◽  
Abl Kinase ◽  
Binding Loop

ABSTRACT Kinase domain (KD) mutations of Bcr-Abl interfering with imatinib binding are the major mechanism of acquired imatinib resistance in patients with Philadelphia chromosome-positive leukemia. Mutations of the ATP binding loop (p-loop) have been associated with a poor prognosis. We compared the transformation potency of five common KD mutants in various biological assays. Relative to unmutated (native) Bcr-Abl, the ATP binding loop mutants Y253F and E255K exhibited increased transformation potency, M351T and H396P were less potent, and the performance of T315I was assay dependent. The transformation potency of Y253F and M351T correlated with intrinsic Bcr-Abl kinase activity, whereas the kinase activity of E255K, H396P, and T315I did not correlate with transforming capabilities, suggesting that additional factors influence transformation potency. Analysis of the phosphotyrosine proteome by mass spectroscopy showed differential phosphorylation among the mutants, a finding consistent with altered substrate specificity and pathway activation. Mutations in the KD of Bcr-Abl influence kinase activity and signaling in a complex fashion, leading to gain- or loss-of-function variants. The drug resistance and transformation potency of mutants may determine the outcome of patients on therapy with Abl kinase inhibitors.

Sign in / Sign up

Export Citation Format

Share Document