Identification of differentially expressed proteins in imatinib mesylate-resistant chronic myelogenous cells

Resistance to imatinib mesylate (also known as Gleevec, Glivec, and STI571) often becomes a barrier to the treatment of chronic myelogenous leukemia (CML). In order to identify markers of the action of imatinib mesylate, we used a mass spectrometry approach to compare protein expression profiles in human leukemia cells (K562) and in imatinib mesylate-resistant human leukemia cells (K562-R) in the presence and absence of imatinib mesylate. We identified 118 differentially regulated proteins in these two leukemia cell-lines, with and without a 1 microM imatinib mesylate challenge. Nine proteins of unknown function were discovered. This is the first comprehensive report regarding differential protein expression in imatinib mesylate-treated CML cells.     

Role of P-glycoprotein in evolution of populations of chronic myeloid leukemia cells treated with imatinib

Imatinib mesylate (imatinib) is a new generation preparation that is now successfully used for treatment of cancer, particularly for chemotherapy of chronic myeloid leukemia (CML). Imatinib inhibits the activity of chimeric kinase BCR-ABL, which is responsible for the development of CML. The goal of this study was to investigate the role of a multidrug resistance protein, P-glycoprotein (Pgp), in the evolution of CML treated with imatinib. We demonstrate here that although imatinib is a substrate for Pgp, cultured CML cells (strain K562/i-S9), overexpressing active Pgp, do not exhibit imatinib resistance. Studies of CML patients in the accelerated phase have shown variations in the number of Pgp-positive cells (Pgp+) among individual patients treated with imatinib. During treatment of patients with imatinib for 6-12 months, the number of Pgp-positive cells significantly increased in most patients. The high number of Pgp+ cells remained in patients at least for 4.5 years and correlated with active Rhodamine 123 (Rh123) efflux. Such correlation was not found in the group of imatinib-resistant patients examined 35-60 months after onset of imatinib therapy: cells from the imatinib-resistant patients exhibited efficient Rh123 efflux irrespectively of Pgp expression. We also compared the mode of Rh123 efflux by cells from CML patients who underwent imatinib treatment for 6-24 months and the responsiveness of patients to this therapy. There were significant differences in survival of patients depending on the absence or the presence of Rh123 efflux. In addition to Pgp, patients' cells expressed other transport proteins of the ABC family. Our data suggest that treatment with imatinib causes selection of leukemic stem cells characterized by expression of Pgp and other ABC transporters.     

Targeting the kinase activity of the BCR-ABL fusion protein in patients with chronic myeloid leukemia

Imatinib mesylate is a major advance in the therapy of patients with chronic myelogenous leukemia (CML). Imatinib mesylate binds to the inactive conformation of BCR-ABL tyrosine kinase suppressing the Philadelphia chromosome positive clone in CML. Clinical studies have yielded impressive results in all phases of CML. With higher rates of complete cytogenetic response with imatinib, molecular monitoring of disease is now advisable in assessing response and determining prognosis. Emergence of resistance to imatinib may be manifest at the hematologic, cytogenetic, or molecular levels in patients who remain in chronic phase, or may be evidenced by the development of more advanced CML phases. Resistance and eventual clinical failure of imatinib occurs in most patients with blastic phase disease. Resistance may occur at the level of Bcr-Abl, with reduction or loss of imatinib effectiveness as a kinase inhibitor, or, despite retention of its inhibitory ability, with changes in the ability to deliver an effective dose at the cellular level, and/or, the leukemia becoming less dependent on Bcr-Abl. The various mechanisms underlying these differing, non-mutually exclusive, mechanisms of resistance must be understood to develop corresponding therapeutic remedies. We review the current data on imatinib in CML, the criteria for diagnosis of imatinib resistance, and the mechanisms that underlie such resistance in CML.     

Imatinib-resistant CML cells have low ENT activity but maintain sensitivity to gemcitabine

Philadelphia chromosome-positive chronic myelogenus leukemia (CML) is widely treated with imatinib mesylate (imatinib), a potent inhibitor of the Bcr-Abl tyrosine kinase. However, resistance to this compound remains a concern. Current treatment approaches include combinations of imatinib with nucleoside analogs such as gemcitabine, which requires equilibrative nucleoside transporters (ENTs) for uptake, to overcome this resistance. Here we report that imatinib treatment decreased ENT1-dependent activity and mRNA expression. Although, imatinib-resistant cells showed decreased levels of both ENT1 and ENT2 activity and expression, these cells remained sensitive to gemcitabine, suggesting that nucleoside analogs can be used as adjunctive therapy.     

Response to treatment with imatinib mesylate in previously treated chronic-phase chronic myeloid leukemia patients in a hospital in Brazil

We analyzed the results of treatment with imatinib mesylate in 70 patients with chronic-phase chronic myeloid leukemia who had previously been treated (with second-line or higher imatinib), many of them in a late chronic phase. The median follow-up period was 60.5 months (range 3-100 months). Our objective was to assess the efficacy and safety of treatment. The mean dose was 400 mg per day. The hematologic response rate was 92.1% at six months, while the cumulative rates of major and complete cytogenetic responses were 73.6 and 66.3%, respectively. Molecular response rate improved slowly and steadily over time, reaching 65.8% at 60 months, remaining stable for up to 96 months. The five-year progression-free survival and overall survival were 84 and 89%, respectively. Cytogenetic response by 12 months significantly correlated with overall survival (P = 0.0007) and progression-free survival (P = 0.0280). Sokal risk score did not differ significantly between subgroups. The medication was well tolerated, with only 16% of patients showing hematologic toxicity grades 3 and 4. At the end of the assessment, 57% of the patients were still on imatinib mesylate; most of those who discontinued treatment (17/30) did so because of unsatisfactory response. Treatment with imatinib mesylate in previously treated chronic-phase chronic myeloid leukemia induced durable responses in a high proportion of patients and was related to satisfactory long-term and event-free survival.     

Imatinib impairs CD8+ T lymphocytes specifically directed against the leukemia-associated antigen RHAMM/CD168 in vitro

The Bcr-Abl tyrosine kinase inhibitor imatinib mesylate is highly effective in the front-line treatment of chronic myeloid leukemia (CML) and is increasingly used in patients with residual disease or relapse after allogeneic stem cell transplantation (allo-SCT). Since an impairment of anti-viral CD8+ T-lymphocyte function by imatinib has been described, we question whether imatinib also affects specific anti-leukemic CD8+ T lymphocytes generated from the peripheral blood of healthy donors, and of CML patients after allo-SCT. Here, we assessed CD8+ T-cell expansion and function from healthy donors and patients with CML. The release of IFN-γ and granzyme B by CD8+ T-lymphocytes specific for R3, a recently described T-cell epitope peptide derived from a leukemia-associated antigen designated RHAMM/CD168 (receptor for hyaluronic acid mediated motility), was inhibited by imatinib in a dose-dependent fashion (range: 1–25 μM). These T cells were able to lyse cognate peptide labeled T2 cells and CD34+ CML progenitor cells. This lysis was inhibited by imatinib. The inhibitory effect was not associated with an increased rate of apoptosis of T cells and reversible after removal of imatinib. In the light of these findings, clinical administration of imatinib might result in the reduction of efficacy of the graft-versus-leukemia effect or other T-cell-based immunotherapies.     

MYC in chronic myeloid leukemia: induction of aberrant DNA synthesis and association with poor response to imatinib

Untreated chronic myeloid leukemia (CML) progresses from chronic phase to blastic crisis (BC). Increased genomic instability, deregulated proliferation, and loss of differentiation appear associated to BC, but the molecular alterations underlying the progression of CML are poorly characterized. MYC oncogene is frequently deregulated in human cancer, often associated with tumor progression. Genomic instability and induction of aberrant DNA replication are described as effects of MYC. In this report, we studied MYC activities in CML cell lines with conditional MYC expression with and without exposure to imatinib, the front-line drug in CML therapy. In cells with conditional MYC expression, MYC did not rescue the proliferation arrest mediated by imatinib but provoked aberrant DNA synthesis and accumulation of cells with 4C content. We studied MYC mRNA expression in 66 CML patients at different phases of the disease, and we found that MYC expression was higher in CML patients at diagnosis than control bone marrows or in patients responding to imatinib. Further, high MYC levels at diagnosis correlated with a poor response to imatinib. MYC expression did not directly correlate with BCR-ABL levels in patients treated with imatinib. Overall our study suggests that, as in other tumor models, MYC-induced aberrant DNA synthesis in CML cells is consistent with MYC overexpression in untreated CML patients and nonresponding patients and supports a role for MYC in CML progression, possibly through promotion of genomic instability.     

Effects of carboxyamidotriazole on in vitro models of imatinib-resistant chronic myeloid leukemia

Although imatinib mesylate (IM) has revolutionized the treatment of chronic myeloid leukemia (CML), some patients develop resistance with progression of leukemia. Alternative or additional targeting of signaling pathways deregulated in bcr-abl-driven CML cells may provide a feasible option for improving clinical response and overcoming resistance. In this study, we show that carboxyamidotriazole (CAI), an orally bioavailable calcium influx and signal transduction inhibitor, is equally effective in inhibiting the proliferation and bcr-abl dependent- and independent-signaling pathways in imatinib-resistant CML cells. CAI inhibits phosphorylation of cellular proteins including STAT5 and CrkL at concentrations that induce apoptosis in IM-resistant CML cells. The combination of imatinib and CAI also down-regulated bcr-abl protein levels. Since CAI is already available for clinical use, these results suggest that it may be an effective addition to the armamentarium of drugs for the treatment of CML.     

Rapid detection of phosphotyrosine proteins by flow cytometric analysis in Bcr-Abl-positive cells.

BACKGROUND: Constitutive tyrosine phosphorylation derived from Bcr-Abl kinase activity is the major characteristic of Bcr-Abl positive cells. In this study, we developed a method to detect the phosphotyrosine proteins by flow cytometry and we asked whether phosphorylation was affected by imatinib mesylate treatment. METHODS: Cells were treated or not with imatinib mesylate, fixed and permeabilized by PFA followed by saponin, then stained with anti-phosphotyrosine (p-tyr) monoclonal antibody and analyzed by flow cytometry. RESULTS: Optimal staining parameters were performed with p-tyr antibody using K562 and LAMA84 lines that displayed high levels of tyrosine phosphorylation as compared to the control line, HL60. Tyrosine phosphorylation was inhibited by imatinib in a dose-dependent manner, but not modified by other inhibitors demonstrating that the staining detected is specific to Bcr-Abl phosphorylation. The staining of imatinib-resistant cell lines such as the mutated BaF/Bcr-AblT315I cell line or resistant CML patient cells, showed that hyperphosphorylation was not affected by imatinib treatment. In one CML patient, our technique permitted us to detect a small hyperphosphorylated population resistant to imatinib that appeared hyperphosphorylated and amplified at the time of relapse. CONCLUSIONS: We have developed a flow cytometric technique presenting several advantages such as rapidity and sensitivity, which requires fewer cells than the Western blot.     

The role of interferon-alpha in the treatment of chronic myeloid leukemia

Biological agents have long been used in the treatment of cancer, and interferon-alpha was the first human cytokine to be widely studied in this setting. Chronic myeloid leukemia (CML) is a hematopoietic stem cell disorder for which interferon-alpha has demonstrated substantial activity. In the 1980s interferon-alpha became first-line therapy for patients with chronic-phase CML, not eligible for allogeneic stem cell transplantation. Following the discovery of the leukemic oncogene BCR/ABL and its causal association with CML, the potent BCR/ABL tyrosine kinase inhibitor imatinib mesylate was developed. Imatinib proved to be superior to interferon-alpha in all outcome measures, making imatinib the new standard of care for patients with CML. There is both clinical and laboratory evidence suggesting imatinib therapy alone is not curative in CML, whereas IFN has induced a low but reproducible curative effect in some patients. This unique activity may be the basis for the reincorporation of IFN into the management of CML. These observations may be best explained by imatinib's negligible activity against the leukemic stem cell (LSC) population. This review discusses the history of interferon-alpha in the treatment of CML, the evolution of molecularly targeted therapies, and some of the lessons we have learned from years of informative research in CML. It also explores the new challenge of managing minimal residual disease in the imatinib era, and addresses the promising role for LSC-directed therapies in the future treatment of CML.     

Strategic Treatment Interruptions During Imatinib Treatment of Chronic Myelogenous Leukemia

Although imatinib is an effective treatment for chronic myelogenous leukemia (CML), and nearly all patients treated with imatinib attain some form of remission, imatinib does not completely eliminate leukemia. Moreover, if the imatinib treatment is stopped, most patients eventually relapse (Cortes et al. in Clin. Cancer Res. 11:3425–3432, 2005). In Kim et al. (PLoS Comput. Biol. 4(6):e1000095, 2008), the authors presented a mathematical model for the dynamics of CML under imatinib treatment that incorporates the anti-leukemia immune response. We use the mathematical model in Kim et al. (PLoS Comput. Biol. 4(6):e1000095, 2008) to study and numerically simulate strategic treatment interruptions as a potential therapeutic strategy for CML patients. We present the results of numerous simulated treatment programs in which imatinib treatment is temporarily stopped to stimulate and leverage the anti-leukemia immune response to combat CML. The simulations presented in this paper imply that treatment programs that involve strategic treatment interruptions may prevent leukemia from relapsing and may prevent remission for significantly longer than continuous imatinib treatment. Moreover, in many cases, strategic treatment interruptions may completely eliminate leukemic cells from the body. Thus, strategic treatment interruptions may be a feasible clinical approach to enhancing the effects of imatinib treatment for CML. We study the effects of both the timing and the duration of the treatment interruption on the results of the treatment. We also present a sensitivity analysis of the results to the parameters in the mathematical model.     

Global proteome quantification for discovering imatinib-induced perturbation of multiple biological pathways in K562 human chronic myeloid leukemia cells

Imatinib mesylate, currently marketed by Novartis as Gleevec in the U.S., has emerged as the leading compound to treat the chronic phase of chronic myeloid leukemia (CML), through its inhibition of Bcr-Abl tyrosine kinases, and other cancers. However, resistance to imatinib develops frequently, particularly in late-stage disease. To identify new cellular pathways affected by imatinib treatment, we applied mass spectrometry together with stable isotope labeling by amino acids in cell culture (SILAC) for the comparative study of protein expression in K562 cells that were untreated or treated with a clinically relevant concentration of imatinib. Our results revealed that, among the 1344 quantified proteins, 73 had significantly altered levels of expression induced by imatinib and could be quantified in both forward and reverse SILAC labeling experiments. These included the down-regulation of thymidylate synthase, S-adenosylmethionine synthetase, and glycerol-3-phosphate dehydrogenase as well as the up-regulation of poly(ADP-ribose) polymerase 1, hemoglobins, and enzymes involved in heme biosynthesis. We also found, by assessing alteration in the acetylation level in histone H4 upon imatinib treatment, that the imatinib-induced hemoglobinization and erythroid differentiation in K562 cells are associated with global histone H4 hyperacetylation. Overall, these results provided potential biomarkers for monitoring the therapeutic intervention of CML using imatinib and offered important new knowledge for gaining insight into the molecular mechanisms of action of imatinib.     

Design and synthesis of 3-substituted benzamide derivatives as Bcr-Abl kinase inhibitors

A series of 3-substituted benzamide derivatives structurally related to STI-571 (imatinib mesylate), a Bcr-Abl tyrosine kinase inhibitor used to treat chronic myeloid leukemia (CML), was prepared and evaluated for antiproliferative activity against the Bcr-Abl-positive leukemia cell line K562. About ten 3-halogenated and 3-trifluoromethylated benzamide derivatives were identified as highly potent Bcr-Abl kinase inhibitors. One of these, NS-187 (9b), is a promising new candidate Bcr-Abl inhibitor for the therapy of STI-571-resistant chronic myeloid leukemia.     

Dasatinib treatment can overcome imatinib and nilotinib resistance in CML patient carrying F359I mutation of BCR-ABL oncogene

Point mutations of bcr-abl tyrosine kinase are the most frequent causes of imatinib resistance in chronic myeloid leukaemia (CML) patients. In most CML cases with BCR-ABL mutations leading to imatinib resistance the second generation of tyrosine kinase inhibitors (TKI- e.g. nilotinib or dasatinib) may be effective. Here, we report a case of a CML patient who during imatinib treatment did not obtain clinical and cytogenetic response within 12 months of therapy. The sequencing of BCR-ABL kinase domains was performed and revealed the presence of a F359I point mutation (TTC-to-ATC nucleotide change leading to Phe-to-Ile amino acid substitution). After 1 month of nilotinib therapy a rapid progression of clinical symptoms was observed. In the presence of the F359I point mutation only dasatinib treatment overcame imatinib and nilotinib resistance.     

Exosomes derived from imatinib-resistant chronic myeloid leukemia cells mediate a horizontal transfer of drug-resistant trait by delivering miR-365

Chronic myeloid leukemia (CML) is a malignant disorder of hematopoietic stem/progenitor cells. Majority of patients can be effectively treated with tyrosine kinase inhibitors (TKIs) such as imatinib, but a portion of patients will develop drug resistance. Accumulated evidences have identified exosomes in cancer as promoters of tumor progression. Herein, we found that exosomes derived from imatinib resistant CML cells can be internalized into sensitive CML cells and confer drug-resistance traits. We also demonstrated a significant higher level of miR-365 in exosomes derived from drug-resistant CML cells compared with those from sensitive ones using microarray and qRT-PCR. The imatinib sensitive CML cells transfected with pre-miR-365 displayed lower chemosensitivity and apoptosis rate compared with controls. We further confirmed that exosomal transfer of miR-365 induced drug resistance by inhibiting expression of pro-apoptosis protein in sensitive CML cells. In conclusion, our study reveals that exosomes mediate a horizontal transfer of drug-resistant trait in chronic myeloid leukemia cell by delivering miR-365.     

The use of isobaric tag peptide labeling (iTRAQ) and mass spectrometry to examine rare,primitive hematopoietic cells from patients with chronic myeloid leukemia

Chronic Myeloid Leukemia (CML) is a hematopoietic stem cell disease, associated with a t(9, 22) chromosomal translocation leading to formation of the BCR/ABL chimeric protein, which has an intrinsic tyrosine kinase activity. Recently, the BCR/ABL tyrosine kinase inhibitor imatinib mesylate (imatinib) has been successfully used clinically, although, disease relapse can still occur. The precise detail of the mechanism by which CML cells respond to imatinib is still unclear. We therefore systematically examined the effects of imatinib on the primitive CML cell proteome, having first established that the drug inhibits proliferation and induces increased apoptosis and differentiation. To define imatinib-induced effects on the CML proteome, we employed isobaric tag peptide labeling (iTRAQ) coupled to two-dimensional liquid chromatography/tandem mass spectrometry. Given the limited clinical material available, the isobaric tag approach identified a large population of proteins and provided relative quantification on four samples at once. Novel consequences of the action of imatinib were identified using this mass spectrometric approach. DEAD-box protein 3, heat shock protein 105 kDa, and peroxiredoxin-3 were identified as potential protein markers for response to imatinib. Electronic Supplementary Material The online version of this article (doi: ) contains supplementary material, which is available to authorized users. Stephen D. Griffiths and John Burthem contributed equally to this publication. This work is supported by The Leukaemia Research Fund (UK).     

Backbone NMR resonance assignment of the Abelson kinase domain in complex with imatinib

Imatinib (Glivec or Gleevec) potently inhibits the tyrosine kinase activity of BCR-ABL, a constitutively activated kinase, which causes chronic myelogenous leukemia (CML). Here we report the first almost complete backbone assignment of c-ABL kinase domain in complex with imatinib. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.