Identification of Annexin A1 interacting proteins in chronic myeloid leukemia KCL22 cells

In the present study, we used a functional proteomic approach to identify Annexin A1 (Anxa1) interacting proteins in the Philadelphia‐positive KCL22 cell line. We focused on Anxa1 because it is one of the major proteins upregulated in imatinib‐sensitive KCL22S cells versus imatinib‐resistant KCL22R. Our proteomic strategy revealed 21 interactors. Bioinformatic analysis showed that most of these proteins are involved in cell death processes. Among the proteins identified, we studied the interaction of Anxa1 with two phosphatases, Shp1 and Shp2, which were recently identified as biomarkers of imatinib sensitivity in patients affected by chronic myeloid leukemia. Our data open new perspectives in the search for annexin‐mediated signaling pathways and may shed light on mechanisms of resistance to imatinib that are unrelated to Bcr‐Abl activity. All mass spectrometry data have been deposited in the ProteomeXchange with identifier PXD000030.     

Hemin reduces cellular sensitivity to imatinib and anthracyclins via Nrf2

Heme plays an important biomodulating role in various cell functions. In this study, we examined the effects of hemin on cellular sensitivity to imatinib and other anti-leukemia reagents. Hemin treatment of human BCR/ABL-positive KCL22 leukemia cells increased IC(50) values of imatinib, that is, the drug resistance, in a dose-dependent manner without any change in the BCR/ABL kinase activity. Imatinib-induced apoptosis was also suppressed by hemin treatment in KCL22 cells. Hemin treatment increased the activity of gamma-glutamylcysteine synthetase (gamma-GCS) light subunit gene promoter, which contains a Maf recognition element (MARE). Protein levels of gamma-GCS and heme oxygenase-1 (HO-1), two MARE-containing genes, were also increased after hemin treatment. Knockdown of Nrf2 expression by RNA interference largely abolished the effect of hemin on imatinib-treated cells, suggesting that Nrf2 recognition of MARE is essential for the hemin-mediated protective effect. Similar to hemin, treatment of cells with delta-aminolevulinic acid (delta-ALA), the obligatory heme precursor, also increased IC(50) values of imatinib. In contrast, inhibition of cellular heme synthesis by succinylacetone increased the sensitivity of cells to imatinib in two imatinib-resistant cell lines, KCL22/SR and KU812/SR. Hemin treatment also decreased the sensitivity of cells to four anthracyclins, daunorubicin, idarubicin, doxorubicin, and mitoxantrone, in BCR/ABL-negative leukemia U937 and THP-1 cells, as well as in KCL22 cells. These findings thus indicate that cellular heme level plays an important role in determining the sensitivity of cells to imatinib and certain other anti-leukemia drugs and that the effect of heme may be mediated via its ability to upregulate Nrf2 activity.     

Comparative proteomic analysis of chronic myelogenous leukemia cells: inside the mechanism of imatinib resistance

Imatinib is the first molecular targeted therapy that has shown clinical success, but imatinib acquired resistance, although a rare event, is critical during the therapy of chronic myelogenous leukaemia (CML). With the aim of better understanding the molecular mechanisms accompanying acquisition of resistance to this drug, a comparative proteomic approach was undertaken on CML cell lines LAMA 84 S (imatinib sensitive) and LAMA 84 R (imatinib resistant). Forty-four differentially expressed proteins were identified and categorized into five main functional classes: (I) heat shock proteins and chaperones; (II) nucleic acid interacting proteins (binding/synthesis/stability); (III) structural proteins, (IV) cell signaling, and (V) metabolic enzymes. Several heat shock proteins known to complex Bcr-Abl were overexpressed in imatinib resistant cells, showing a possible involvement of these proteins in the mechanism of resistance. HnRNPs also resulted in being up-regulated in imatinib resistant cells. These proteins have been shown to be strongly and directly related to Bcr-Abl activity. To our knowledge, this is the first direct proteomic comparison of imatinib sensitive/resistant CML cell lines.     

Comparative proteome profiling and functional analysis of chronic myelogenous leukemia cell lines

The aim of the present study was the molecular profiling of different Ph+ chronic myelogenous leukemia (CML) cell lines (LAMA84, K562, and KCL22) by a proteomic approach. By employing two-dimensional gel electrophoresis combined with mass spectrometry analysis, we have identified 191 protein spots corresponding to 142 different proteins. Among these, 63% were cancer-related proteins and 74% were described for the first time in leukemia cells. Multivariate analysis highlighted significant differences in the global proteomic profile of the three CML cell lines. In particular, the detailed analysis of 35 differentially expressed proteins revealed that LAMA84 cells preferentially expressed proteins associated with an invasive behavior, while K562 and KCL22 cells preferentially expressed proteins involved in drug resistance. These data demonstrate that these CML cell lines, although representing the same pathological phenotype, show characteristics in their protein expression profile that suggest different phenotypic leukemia subclasses. These data contribute a new potential characterization of the CML phenotype and may help to understand interpatient variability in the progression of disease and in the efficacy of a treatment.     

Functional phosphoproteomic analysis reveals cold-shock domain protein A to be a Bcr-Abl effector-regulating proliferation and transformation in chronic myeloid leukemia

One proposed strategy to suppress the proliferation of imatinib-resistant cells in chronic myeloid leukemia (CML) is to inhibit key proteins downstream of Bcr-Abl. The PI3K/Akt pathway is activated by Bcr-Abl and is specifically required for the growth of CML cells. To identify targets of this pathway, we undertook a proteomic screen and identified several proteins that differentially bind 14-3-3, dependent on Bcr-Abl kinase activity. An siRNA screen of candidates selected by bioinformatics analysis reveals cold-shock domain protein A (CSDA), shown previously to regulate cell cycle progression in epithelial cells, to be a positive regulator of proliferation in a CML cell line. We show that Akt can phosphorylate the serine 134 residue of CSDA but, downstream of Bcr-Abl activity, this modification is mediated through the activation of MEK/p90 ribosomal S6 kinase (RSK) signaling. Inhibition of RSK, similarly to treatment with imatinib, blocked proliferation specifically in Bcr-Abl-positive leukemia cell lines, as well as cells from CML patients. Furthermore, these primary CML cells showed an increase in CSDA phosphorylation. Expression of a CSDA phospho-deficient mutant resulted in the decrease of Bcr-Abl-dependent transformation in Rat1 cells. Our results support a model whereby phosphorylation of CSDA downstream of Bcr-Abl enhances proliferation in CML cells to drive leukemogenesis.     

Translational Regulation of GPx-1 and GPx-4 by the mTOR Pathway

Glutathione peroxidase activity was previously determined to be elevated in lymphocytes obtained from patients treated with the Bcr-Abl kinase inhibitor imatinib mesylate. In order to expand upon this observation, the established chronic myelogenous leukemia cell lines KU812 and MEG-01 were treated with imatinib and the effect on several anti-oxidant proteins was determined. The levels of GPx-1 were significantly increased following treatment with imatinib. This increase was not due to altered steady-state mRNA levels, and appeared to be dependent on the expression of Bcr-Abl, as no increases were observed following imatinib treatment of cells that did not express the fusion protein. The nutrient-sensing signaling protein, mammalian target of rapamycin (mTOR), can be activated by Bcr-Abl and its activity regulates the translation of many different proteins. Treatment of those same cells used in the imatinib studies with rapamycin, an inhibitor of mTOR, resulted in elevated GPx-1 and GPx-4 protein levels independent of Bcr-Abl expression. These proteins all belong to the selenoprotein family of peptides that contain the UGA-encoded amino acid selenocysteine. Collectively, these data provide evidence of a novel means of regulating anti-oxidants of the selenoprotein family via the mTOR pathway.     

Imatinib interferes with survival of multi drug resistant Kaposi's sarcoma cells

Multi drug resistance (MDR) is defined as the ability of tumor cells to become resistant to unrelated drugs. Tyrosine kinase inhibitor imatinib has been demonstrated to be effective in the treatment of certain tumors. In particular, imatinib inhibits Bcr-Abl kinase activity, c-kit and the phosphorylation of platelet-derived growth factor (PDGF) receptors. In this work, we show that imatinib inhibits PDGF phosphorylation not only in wt Kaposi sarcoma (KS) but also in multi drug resistant KS cells. This was associated with an increased apoptosis in wt cells and an increased autophagy in MDR-KS cells. These data add new insights to the possible use of imatinib in the overcoming of MDR in KS cells.     

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.     

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.     

Inhibition of protein-tyrosine phosphatase 1B (PTP1B) mediates ubiquitination and degradation of Bcr-Abl protein

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder characterized at the molecular level by the expression of Bcr-Abl, a chimeric protein with deregulated tyrosine kinase activity. The protein-tyrosine phosphatase 1B (PTP1B) is up-regulated in Bcr-Abl-expressing cells, suggesting a regulatory link between the two proteins. To investigate the interplay between these two proteins, we inhibited the activity of PTP1B in Bcr-Abl-expressing TonB.210 cells by either pharmacological or siRNA means and examined the effects of such inhibition on Bcr-Abl expression and function. Herein we describe a novel mechanism by which the phosphatase activity of PTP1B is required for Bcr-Abl protein stability. Inhibition of PTP1B elicits tyrosine phosphorylation of Bcr-Abl that triggers the degradation of Bcr-Abl through ubiquitination via the lysosomal pathway. The degradation of Bcr-Abl consequently inhibits tyrosine phosphorylation of Bcr-Abl substrates and the downstream production of intracellular reactive oxygen species. Furthermore, PTP1B inhibition reduces cell viability and the IC(50) of the Bcr-Abl inhibitor imatinib mesylate. Degradation of Bcr-Abl via PTP1B inhibition is also observed in human CML cell lines K562 and LAMA-84. These results suggest that inhibition of PTP1B may be a useful strategy to explore in the development of novel therapeutic agents for the treatment of CML, particularly because host drugs currently used in CML such as imatinib focus on inhibiting the kinase activity of Bcr-Abl.     

Erythropoietin promotes resistance against the Abl tyrosine kinase inhibitor imatinib (STI571) in K562 human leukemia cells

Chronic myeloid leukemia is characterized by the Philadelphia chromosome translocation that causes expression of Bcr-Abl, a deregulated tyrosine kinase. Imatinib mesylate (STI571, Gleevec), a therapeutically used inhibitor of Bcr-Abl, causes apoptosis of Bcr-Abl-positive cells. In the leukemia cell line K562, we observed spontaneous resistance to imatinib at very low frequencies when cells were exposed to the drug (1 micro M) for more than 4 weeks. Surprisingly, in the presence of erythropoietin (Epo), K562 cells were temporarily able to sustain proliferation in the presence of imatinib, and imatinib-resistant clones could be isolated with high frequencies. From such imatinib-resistant, Epo-dependent clones, sublines could be established that were resistant to imatinib in the absence of Epo. Mitogen-activated protein (MAP) kinase activity was inhibited by imatinib treatment but could be partially restored by Epo. Inhibition of MAP kinase or phosphatidylinositol 3-kinase blocked the protective effect of Epo. The data suggest that K562 cells acquire factor dependency under imatinib/Epo treatment, allowing them to escape from imatinib-induced, immediate cell death. This pool of cells provides the basis for the outgrowth of imatinib-resistant clones of unlimited proliferative capacity. Thus, Epo, an endogenous regulator of hematopoiesis, promotes the development of resistance to imatinib.     

Oncogenic stress induced by acute hyper-activation of Bcr-Abl leads to cell death upon induction of excessive aerobic glycolysis

In response to deregulated oncogene activation, mammalian cells activate disposal programs such as programmed cell death. To investigate the mechanisms behind this oncogenic stress response we used Bcr-Abl over-expressing cells cultivated in presence of imatinib. Imatinib deprivation led to rapid induction of Bcr-Abl activity and over-stimulation of PI3K/Akt-, Ras/MAPK-, and JAK/STAT pathways. This resulted in a delayed necrosis-like cell death starting not before 48 hours after imatinib withdrawal. Cell death was preceded by enhanced glycolysis, glutaminolysis, and amino acid metabolism leading to elevated ATP and protein levels. This enhanced metabolism could be linked to induction of cell death as inhibition of glycolysis or glutaminolysis was sufficient to sustain cell viability. Therefore, these data provide first evidence that metabolic changes induced by Bcr-Abl hyper-activation are important mediators of oncogenic stress-induced cell death.During the first 30 hours after imatinib deprivation, Bcr-Abl hyper-activation did not affect proliferation but resulted in cellular swelling, vacuolization, and induction of eIF2α phosphorylation, CHOP expression, as well as alternative splicing of XPB, indicating endoplasmic reticulum stress response. Cell death was dependent on p38 and RIP1 signaling, whereas classical death effectors of ER stress, namely CHOP-BIM were antagonized by concomitant up-regulation of Bcl-xL.Screening of 1,120 compounds for their potential effects on oncogenic stress-induced cell death uncovered that corticosteroids antagonize cell death upon Bcr-Abl hyper-activation by normalizing cellular metabolism. This protective effect is further demonstrated by the finding that corticosteroids rendered lymphocytes permissive to the transforming activity of Bcr-Abl. As corticosteroids are used together with imatinib for treatment of Bcr-Abl positive acute lymphoblastic leukemia these data could have important implications for the design of combination therapy protocols.In conclusion, excessive induction of Warburg type metabolic alterations can cause cell death. Our data indicate that these metabolic changes are major mediators of oncogenic stress induced by Bcr-Abl.     

A Multiple Reaction Monitoring (MRM) Method to Detect Bcr-Abl Kinase Activity in CML Using a Peptide Biosensor

The protein kinase Bcr-Abl plays a major role in the pathogenesis of chronic myelogenous leukemia (CML), and is the target of the breakthrough drug imatinib (Gleevec™). While most patients respond well to imatinib, approximately 30% never achieve remission or develop resistance within 1–5 years of starting imatinib treatment. Evidence from clinical studies suggests that achieving at least 50% inhibition of a patient’s Bcr-Abl kinase activity (relative to their level at diagnosis) is associated with improved patient outcomes, including reduced occurrence of resistance and longer maintenance of remission. Accordingly, sensitive assays for detecting Bcr-Abl kinase activity compatible with small amounts of patient material are desirable as potential companion diagnostics for imatinib. Here we report the detection of Bcr-Abl activity and inhibition by imatinib in the human CML cell line K562 using a cell-penetrating peptide biosensor and multiple reaction monitoring (MRM) on a triple quadrupole mass spectrometer. MRM enabled reproducible, selective detection of the peptide biosensor at fmol levels from aliquots of cell lysate equivalent to ∼15,000 cells. This degree of sensitivity will facilitate the miniaturization of the entire assay procedure down to cell numbers approaching 15,000, making it practical for translational applications in patient cells in which the limited amount of available patient material often presents a major challenge.     

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.     

Aclacinomycin A Sensitizes K562 Chronic Myeloid Leukemia Cells to Imatinib through p38MAPK-Mediated Erythroid Differentiation

Expression of oncogenic Bcr-Abl inhibits cell differentiation of hematopoietic stem/progenitor cells in chronic myeloid leukemia (CML). Differentiation therapy is considered to be a new strategy for treating this type of leukemia. Aclacinomycin A (ACM) is an antitumor antibiotic. Previous studies have shown that ACM induced erythroid differentiation of CML cells. In this study, we investigate the effect of ACM on the sensitivity of human CML cell line K562 to Bcr-Abl specific inhibitor imatinib (STI571, Gleevec). We first determined the optimal concentration of ACM for erythroid differentiation but not growth inhibition and apoptosis in K562 cells. Then, pretreatment with this optimal concentration of ACM followed by a minimally toxic concentration of imatinib strongly induced growth inhibition and apoptosis compared to that with simultaneous co-treatment, indicating that ACM-induced erythroid differentiation sensitizes K562 cells to imatinib. Sequential treatment with ACM and imatinib induced Bcr-Abl down-regulation, cytochrome c release into the cytosol, and caspase-3 activation, as well as decreased Mcl-1 and Bcl-xL expressions, but did not affect Fas ligand/Fas death receptor and procaspase-8 expressions. ACM/imatinib sequential treatment-induced apoptosis was suppressed by a caspase-9 inhibitor and a caspase-3 inhibitor, indicating that the caspase cascade is involved in this apoptosis. Furthermore, we demonstrated that ACM induced erythroid differentiation through the p38 mitogen-activated protein kinase (MAPK) pathway. The inhibition of erythroid differentiation by p38MAPK inhibitor SB202190, p38MAPK dominant negative mutant or p38MAPK shRNA knockdown, reduced the ACM/imatinib sequential treatment-mediated growth inhibition and apoptosis. These results suggest that differentiated K562 cells induced by ACM-mediated p38MAPK pathway become more sensitive to imatinib and result in down-regulations of Bcr-Abl and anti-apoptotic proteins, growth inhibition and apoptosis. These results provided a potential management by which ACM might have a crucial impact on increasing sensitivity of CML cells to imatinib in the differentiation therapeutic approaches.     

Apoptosis-based dual molecular targeting by INNO-406, a second-generation Bcr-Abl inhibitor, and ABT-737, an inhibitor of antiapoptotic Bcl-2 proteins, against Bcr-Abl-positive leukemia

Bcr-Abl is the cause of Philadelphia-positive (Ph(+)) leukemias and also constitutes their principal therapeutic target, as exemplified by dramatic effects of imatinib mesylate. However, mono-targeting of Bcr-Abl does not always achieve complete leukemia eradication, and additional strategies those enable complete elimination of leukemic cells are desired to develop. Here we demonstrate that INNO-406, a much more active Bcr-Abl tyrosine kinase inhibitor than imatinib, augments the activities of several proapoptotic Bcl-2 homology (BH)3-only proteins (Bim, Bad, Bmf and Bik) and induces apoptosis in Ph(+) leukemia cells via Bcl-2 family-regulated intrinsic apoptosis pathway. ABT-737, an inhibitor of antiapoptotic Bcl-2 and Bcl-X(L), greatly enhanced the apoptosis by INNO-406, even in INNO-406-less sensitive cells with Bcr-Abl point mutations except T315I mutation. In contrast, co-treatment with INNO-406 and other pharmacologic inducers of those BH3-only proteins, such as 17-allylaminogeldanamycin, an heat shock protein-90 inhibitor, or PS-341, a proteasome inhibitor, did not further increase the BH3-only protein levels or sensitize leukemic cells to INNO-406-induced apoptosis, suggesting a limit to how much expression levels of BH3-only proteins can be increased by anticancer agents. Thus, double-barrelled molecular targeting for Bcr-Abl-driven oncogenic signaling and the cell protection by antiapoptotic Bcl-2 family proteins may be the rational therapeutic approach for eradicating Ph(+) leukemic cells.     

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.