BCR/ABL kinase inhibition by imatinib mesylate enhances MAP kinase activity in chronic myelogenous leukemia CD34+ cells

Blood ◽  
2004 ◽  
Vol 103 (8) ◽  
pp. 3167-3174 ◽  
Author(s):  
Su Chu ◽  
Melissa Holtz ◽  
Mamta Gupta ◽  
Ravi Bhatia
Keyword(s):  
Growth Factor ◽  
Imatinib Mesylate ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Combined Treatment ◽  
Myelogenous Leukemia ◽  
Imatinib Treatment ◽  
Abl Kinase ◽  
Mapk Activity ◽  
Cd34 Cells

Abstract Chronic myelogenous leukemia (CML) results from malignant transformation of a primitive hematopoietic cell by the BCR/ABL oncogene. The breakpoint cluster region/ABL (BCR/ABL) tyrosine kinase inhibitor imatinib mesylate (imatinib) is highly effective in inducing remissions in CML. However, the effects of imatinib on intracellular signaling in primary progenitor cells are not well described. We show that imatinib exposure resulted in a significant dose-responsive reduction in BCR/ABL kinase activity in CML CD34+ cells. However, imatinib treatment resulted in an increase in activity of p42/44 mitogen-activated protein kinase (MAPK), an important downstream effector of BCR/ABL. Increased MAPK activity was growth factor dependent. Pharmacologic inhibition of MAPK using MAPK/extracellular signal–regulated kinase kinase–1/2 (MEK-1/2) inhibitors significantly reduced CML progenitor proliferation. Combined treatment with a MEK-1/2 inhibitor and imatinib significantly increased suppression of CML progenitors compared with either inhibitor alone. In contrast, imatinib treatment resulted in a small reduction in AKT activity. Combined treatment with a phosphatidylinositol-3 (PI-3) kinase inhibitor and imatinib significantly increased suppression of CML progenitor growth compared with either inhibitor alone. We conclude that inhibition of BCR/ABL kinase activity in CML progenitors by imatinib results in a growth factor-dependent compensatory increase in MAPK activity and in only partial inhibition of PI-3 kinase activity. These mechanisms may contribute to incomplete elimination of CML progenitors by imatinib. (Blood. 2004;103:3167-3174)

The Dual Src/Abl Kinase Inhibitor SKI-606 Effectively Inhibits Bcr-Abl Kinase Activity and Reduces Proliferation of CML Primitive Progenitor Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1370-1370
Author(s):  
Heiko Konig ◽  
Simran K. Sindhu ◽  
Frank Boschelli ◽  
Tessa L. Holyoake ◽  
Stephen J. Forman ◽  
...  
Keyword(s):  
Stem Cells ◽  
Flow Cytometry ◽  
Drug Exposure ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Abl Kinase ◽  
Mapk Activity ◽  
Cd34 Cells ◽  
Ic50 Values ◽  
Dose Dependent

Abstract Imatinib mesylate (IM) is highly effective in the treatment of CML. However resistance to IM can develop in a subset of patients. In addition CML stem cells appear to be relatively resistant to elimination by IM. Incomplete elimination of malignant progenitors may be related to incomplete Bcr-Abl kinase inhibition, persistent signaling through MAPK or other growth stimulatory pathways, or Bcr-Abl kinase mutations resulting in IM resistance. The dual Src/Abl kinase inhibitor SKI-606 has been reported to exert potent antiproliferative activity against CML cell lines in vitro and in xenograft models, and is currently being investigated in phase1/2 clinical trials. Here, we investigated whether SKI-606 could effectively target CML primary progenitors. Flow cytometry selected CD34+CD38− primitive progenitor (PPC) and CD34+CD38+ committed progenitor cells (CPC) from untreated CML patients were cultured for 96h in growth factor (GF) supplemented medium in a range of concentrations of SKI-606 (0–0.5μM), and with 5μM IM for comparison. Cells were labelled with CFSE-prior to culture and with Annexin-V at culmination of culture to allow flow cytometry assessment of the effects of drug exposure on cell proliferation and apoptosis. In addition CD34+ cells were similarly incubated with SKI-606 and subsequently plated in methylcellulose progenitor culture to assess effects on colony forming cell (CFC) growth. CFSE assays indicated significant dose dependent antiproliferative activity of SKI-606 with IC50-values of 0.2μM for CML PPC. SKI-606 resulted in moderate induction of apoptosis of CML PPC (from 22±7.2% [control] to 49±16.4% [0.5μM], n=3, ns). CFC-assays consistently revealed significant dose dependent growth inhibitory effects of SKI-606 with IC50-values of 0.1μM SKI-606. The effect of SKI-606 on Bcr-Abl-kinase activity was assessed by Western blotting with anti-P-CrkL antibodies after overnight drug exposure of CML CD34+ cells. Importantly, 0.1μM and 0.5μM SKI-606 significantly suppressed phospho-CrkL levels (from 94.7±3.5% [control] to 14.9±4.8%, n=4, p=.0001, and to 6.8±2.5%, n=4, p=.00003, respectively), whereas a higher concentration of IM (5μM) was needed to achieve a similar degree (13.1±5.0%, n=4, p=.0000007). Whereas treatment of CML CD34+ cells with IM was associated with increased p42/44 MAPK activity (n=3, p=.038), a significant increase in MAPK activity was not observed when the same samples were treated with SKI-606 (n=3, ns). In conclusion, SKI-606 significantly inhibits CML progenitor proliferation and moderately induces apoptosis at lower concentrations than previously observed with IM, although the maximum growth suppression effects observed were not greater than those observed with high concentrations of IM. SKI-606 is significantly more potent than IM in inhibiting Bcr-Abl TK in CML progenitors. Unlike IM, SKI-606 treatment was not associated with significant compensatory increase in p42/44 MAPK signaling, which could potentially be beneficial in targeting malignant stem cells. Further studies investigating the effects of SKI-606 on CML stem cells as a single agent or in combination with other compounds are warranted.

Several Bcr-Abl kinase domain mutants associated with imatinib mesylate resistance remain sensitive to imatinib

Blood ◽  
2003 ◽  
Vol 101 (11) ◽  
pp. 4611-4614 ◽  
Author(s):  
Amie S. Corbin ◽  
Paul La Rosée ◽  
Eric P. Stoffregen ◽  
Brian J. Druker ◽  
Michael W. Deininger
Keyword(s):  
Imatinib Mesylate ◽  
Kinase Inhibitor ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Kinase Domain ◽  
Myelogenous Leukemia ◽  
Imatinib Resistance ◽  
Abl Kinase ◽  
Cellular Assays ◽  
Kinase Domain Mutations

Abstract Imatinib mesylate is a selective Bcr-Abl kinase inhibitor, effective in the treatment of chronic myelogenous leukemia. Most patients in chronic phase maintain durable responses; however, many in blast crisis fail to respond, or relapse quickly. Kinase domain mutations are the most commonly identified mechanism associated with relapse. Many of these mutations decrease the sensitivity of the Abl kinase to imatinib, thus accounting for resistance to imatinib. The role of other mutations in the emergence of resistance has not been established. Using biochemical and cellular assays, we analyzed the sensitivity of several mutants (Met244Val, Phe311Leu, Phe317Leu, Glu355Gly, Phe359Val, Val379Ile, Leu387Met, and His396Pro/Arg) to imatinib mesylate to better understand their role in mediating resistance.While some Abl mutations lead to imatinib resistance, many others are significantly, and some fully, inhibited. This study highlights the need for biochemical and biologic characterization, before a resistant phenotype can be ascribed to a mutant.

Relationship between Clinical/Hematological Response and Increase of Plamacells in the Bone Marrow of Patients with Chronic Myelogenous Leukemia Imatinib Mesylate Treatment (631).

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4552-4552
Author(s):  
Alessandro Poggi ◽  
Ivana Pierri ◽  
Silvia Catellani ◽  
Francesca Olcese ◽  
Antonella Marasco ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Tyrosine Kinase ◽  
Imatinib Mesylate ◽  
B Lymphocytes ◽  
Chronic Myelogenous Leukemia ◽  
Peripheral Blood ◽  
Kinase Inhibitor ◽  
Myelogenous Leukemia ◽  
Response To Treatment ◽  
Imatinib Treatment

Abstract Tyrosine kinase inhibitors, such as imatinib mesylate (Gleevec, Novartis, formerly known as STI571) are the first line treatment of Chronic Myelogenous Leukemia (CML) and of a rare form of gastroenteric stromal cancer. It has been recently reported that in the latter case, tumor cells are refractory to imatinib antiproliferative effect in vitro and the response to the drug in vivo is due to immunocompetent cells, able to produce cytokines with antineoplastic activity. In this study, 20 CML patients, prior and during treatment with imatinib mesylate, underwent bone marrow (BM) aspirates every 6 months, including: morphologic and phenotypic analysis, cytogenetic and biomolecular evaluation, compared to peripheral blood. Plasma from BM and peripheral blood was also recovered for cytokyne-chemokine dosage. We report that in 12 out of 20 CML patients a significant increase in the percentage of BM lymphoplasmocytoid cells was observed upon treatment with imatinib mesylate, with >10% (range 10–16%) of CD20+CD126+cells. Among this population, two third of cells coexpressed IgM and one third was IgD+, while a smaller fraction of IgM+CD126+CD20– (3–4%) or IgD+CD126+CD20- (2–3%) cells was also found. The lasting 8 patients had<5% of CD20 +CD126+ lymphocytes (range2–4%), 2/3 coexpressing IgM and 1/3 coexpressing IgD. All patients with increased number of CD126+ B lymphocytes underwent hematologic remission, 7 of them with complete molecular and cytogenetic remission. On the other hand, among the patients with low or undetectable CD20+CD126+cells, only 4 underwent hemathological remission and none of them displayed stable cytogenetyc and molecular remission. In two patients relapsed after six months of treatment, the fraction of BM CD20+CD126+ lymphocytes decreased from 16% and 11% to 7 and 5%, respectively, with undetectable IgM+ CD126+CD20- or IgD+ CD126+CD20- cells. These data suggest that this population of lymphoplasmocytoid B cells depends on or contribute to the pharmacological response; by the way, this phenomenon might help in monitoring the outcome of disease and the response to treatment. To check this item and understand the biochemical mechanisms substaining the observed increase in BM lymphoplasmocitoid cells on imatinib treatment, we wonder if the production of cytokines able to induce B lymphocytes differentiation, such as interleukin (IL)-4, IL-6 (whose receptor is CD126), IL-3, IL10 or IL-21 was affected by imatinib administration. To this aim, both soluble cytokines (by ELISPOT) and their mRNA (by real time polymerase chain reaction) were evaluated in the BM of these patients: moreover, the expression of MCP-1, SDF-1, IP-10 and IL-8 were also measured, to verify whether the increse in BM CD20+ CD126+ lymphocytes was due to a redistribution rather than to “in situ” differentiation. Preliminary results seem to indicate that the latter hypothesis is unlikely; in addition, when CD20+ CD126+ were increased in the BM, they also raised in the peripheral blood. These immunological events might have a role in the response to tyrosine kinase inhibitor and need further investigations.

scholarly journals Effective and selective inhibition of chronic myeloid leukemia primitive hematopoietic progenitors by the dual Src/Abl kinase inhibitor SKI-606

Blood ◽  
2008 ◽  
Vol 111 (4) ◽  
pp. 2329-2338 ◽  
Author(s):  
Heiko Konig ◽  
Tessa L. Holyoake ◽  
Ravi Bhatia
Keyword(s):  
Chronic Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Kinase Activity ◽  
Kinase Inhibitor ◽  
Src Kinase ◽  
Selective Inhibition ◽  
Curative Therapy ◽  
Abl Kinase ◽  
Mapk Activity ◽  
High Concentrations

Imatinib mesylate (imatinib) is highly effective in the treatment of chronic myeloid leukemia (CML) but is less effective in eliminating CML stem cells. We investigated whether SKI-606, a potent Bcr-Abl and Src kinase inhibitor without anti-PDGF or c-Kit activity, could effectively target primitive CML progenitors. CML and normal progenitors were cultured with SKI-606 or imatinib. SKI-606 effectively inhibited Bcr-Abl kinase activity in CML CD34+ cells and inhibited Src phosphorylation more potently than imatinib. However, SKI-606 and imatinib resulted in similar suppression of CML primitive and committed progenitor proliferation and growth in CFC and LTC-IC assays. Exposure to either agent alone or in combination resulted in only modest increase in apoptosis. Evaluation of downstream signaling pathways indicated that Akt and STAT5 activity was not changed, but a delayed increase in MAPK activity was seen at high concentrations of SKI-606. SKI-606 inhibited normal progenitor proliferation to a lesser extent than imatinib. SKI-606 effectively inhibits Bcr-Abl and Src kinase activity and inhibits CML progenitor growth with relatively little effect on normal progenitors. However, SKI-606 does not demonstrate increased ability to eliminate primitive CML progenitors by apoptosis compared with imatinib, emphasizing the need for additional strategies besides Bcr-Abl kinase inhibition for curative therapy of CML.

Detection of BCR-ABL kinase mutations in CD34+ cells from chronic myelogenous leukemia patients in complete cytogenetic remission on imatinib mesylate treatment

Blood ◽  
2005 ◽  
Vol 105 (5) ◽  
pp. 2093-2098 ◽  
Author(s):  
Su Chu ◽  
Helen Xu ◽  
Neil P. Shah ◽  
David S. Snyder ◽  
Stephen J. Forman ◽  
...  
Keyword(s):  
Imatinib Mesylate ◽  
Chronic Myelogenous Leukemia ◽  
Drug Binding ◽  
Myelogenous Leukemia ◽  
In Vitro Mutagenesis ◽  
Mrna Levels ◽  
Imatinib Resistance ◽  
Secondary Resistance ◽  
Abl Kinase ◽  
Cd34 Cells

AbstractThe BCR-ABL kinase inhibitor imatinib mesylate induces complete cytogenetic response (CCR) in a high proportion of chronic myelogenous leukemia (CML) patients. However, patients in CCR usually demonstrate evidence of residual BCR-ABL–positive progenitors. The mechanisms underlying persistence of small numbers of malignant progenitors in imatinib-sensitive patients are unclear. BCR-ABL kinase domain mutations affecting drug binding can lead to secondary resistance to imatinib. We show here that kinase mutations could be detected in CD34+ cells isolated from CML patients in CCR on imatinib. Most mutations seen have not been reported in previous clinical studies. Interestingly, several of the involved amino acid positions have been implicated in an in vitro mutagenesis screen. These BCR-ABL mutations were associated with varying levels of imatinib resistance. Two of 5 patients in whom mutations were detected on initial evaluation have relapsed. In addition, 4 patients in whom mutations were not initially detected, but with rising BCR-ABL mRNA levels on quantitative polymerase chain reaction (Q-PCR) analysis, had mutations detected on follow-up evaluation. We conclude that BCR-ABL kinase mutations can be detected in CD34+ cells from CML patients in CCR on imatinib, may contribute to persistence of small populations of malignant progenitors, and could be a potential source of relapse.

Identification of BCR-ABL Dependent Gene Regulation by Using a Phosphoproteomics Approach.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2944-2944
Author(s):  
Patrick Ziegler ◽  
Stefan Balabanov ◽  
Ulrike Hartmann ◽  
Winfried Kammer Kammer ◽  
Alfred Nordheim ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Large Scale ◽  
Protein Identification ◽  
Combined Treatment ◽  
K562 Cells ◽  
Peptide Sequencing ◽  
Myelogenous Leukemia ◽  
Imatinib Treatment ◽  
Type Ii ◽  
Cd34 Cells

Abstract Introduction: The selective tyrosine kinase inhibitor imatinib (formerly STI571, Glivecâ) has been shown to block phosphorylation of tyrosine residues by occupying the ATP binding site of the Abl tyrosine kinases Bcr-Abl, c-Abl, v-Abl and Abl-related gene (ARG), as well as platelet-derived growth factor receptor (PDGF) alpha and beta and of the receptor for human stem cell factor (SCF) c-kit. We chose a large scale phospho-proteomics approach to identify novel downstream targets of imatinib which could possibly be utilized for combined treatment strategies. Material and Methods: Phospho-proteomics was performed by comparison of large scale phosphotyrosine-immunoprecipitation of imatinib (10 mM/2 hours) versus DMSO treated K562 cells separated by one-dimensional polyacrylamide gelelectrophoresis. In addition enriched CD34+ cells (&gt;70%) of a newly diagnosed Bcr-Abl positive CML patient were used immediately after purification and treated in the same way as described above. Resulting differentially immuno-precipitated proteins were analyzed using matrix-assisted laser desorption/ionization - time of flight mass spectrometry (MALDI-TOF) and nano electrospray ionization tandem mass spectrometry (ESI-MS/MS). Protein identification via peptide mass-fingerprinting and peptide sequencing was performed using Mascot search tool and NCBI nr database. Differential phosphorylation was confirmed by combined immunoprecipitation and western blot analysis of selected candidate proteins. Results: Phospho-proteomics of K562 cells revealed 8 differentially phosphorylated proteins after a two hour treatment with imatinib including the recently identified c-cbl, and Bcr-Abl itself, the latter confirming autophosphorylation. Ship2 which was originally identified as beeing constitutively phosphorylated in chronic myelogenous leukemia progenitor cells showed reduced, imatinib sensitive phosphorylation. Remaining candidates could be classified as being involved in protein folding or in ATPase activities associated with a variety of functions (type II AAA). The analysis of primary CD34+ cells from a CML patient showed a predominance of the nonmuscular myosin heavy chain protein in different molecular weight forms. Discussion: We detected significant imatinib-dependent differences in protein phosphotyrosine-immunoreactivity of the Bcr-Abl -dependent cell line K562. Previously identified down-stream targets of Bcr-Abl could be confirmed and novel candidate proteins were identified. Phosphorylation of Ship2, a previously identified down-stream target of Bcr-Abl, was found to be inhibited by imatinib treatment. Ongoing studies are aimed at the characterization of the role of the identified phospho-proteins, particularly type II AAAs for Bcr-Abl induced signal transduction as well as for the development of resistance to imatinib.

scholarly journals Synergistic interactions between imatinib mesylate and the novel phosphoinositide-dependent kinase-1 inhibitor OSU-03012 in overcoming imatinib mesylate resistance

Blood ◽  
2005 ◽  
Vol 105 (10) ◽  
pp. 4021-4027 ◽  
Author(s):  
Ping-Hui Tseng ◽  
Ho-Pi Lin ◽  
Jiuxiang Zhu ◽  
Kuen-Feng Chen ◽  
Erinn M. Hade ◽  
...  
Keyword(s):  
Imatinib Mesylate ◽  
Kinase Inhibitor ◽  
Mutant Cell ◽  
Point Mutations ◽  
Myelogenous Leukemia ◽  
Cross Resistance ◽  
Abl Kinase ◽  
Induced Apoptosis ◽  
Mutant Cells ◽  
Phosphoinositide Dependent Kinase

AbstractResistance to the Ableson protein tyrosine (Abl) kinase inhibitor imatinib mesylate has become a critical issue for patients in advanced phases of chronic myelogenous leukemia. Imatinib-resistant tumor cells develop, in part, as a result of point mutations within the Abl kinase domain. As protein kinase B (Akt) plays a pivotal role in Abl oncogene-mediated cell survival, we hypothesize that concurrent inhibition of Akt will sensitize resistant cells to the residual apoptotic activity of imatinib mesylate, thereby overcoming the resistance. Here, we examined the effect of OSU-03012, a celecoxib-derived phosphoinositide-dependent kinase-1 (PDK-1) inhibitor, on imatinib mesylate-induced apoptosis in 2 clinically relevant breakpoint cluster region (Bcr)-Abl mutant cell lines, Ba/F3p210E255K and Ba/F3p210T315I. The 50% inhibitory concentration (IC50) values of imatinib mesylate to inhibit the proliferation of Ba/F3p210E255K and Ba/F3p210T315I were 14 ± 4 and 30 ± 2 μM, respectively. There was no cross-resistance to OSU-03012 in these mutant cells with an IC50 of 5 μM irrespective of mutations. Nevertheless, in the presence of OSU-03012 the susceptibility of these mutant cells to imatinib-induced apoptosis was significantly enhanced. This synergistic action was, at least in part, mediated through the concerted effect on phospho-Akt. Together these data provide a novel therapeutic strategy to overcome imatinib mesylate resistance, especially with the Abl mutant T315I.

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