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Objectives: Mature dendritic cells (DCs) may be derived from the BCR/ABL1 expressing monocytes in chronic myeloid leukaemia. These cells have potential therapeutic applications, but are recognised to have defective function. In normal DCs, activation and maturation depend on ABL1 dependent signals. We therefore tested the hypothesis that in the DCs of chronic myeloid leukaemia, the presence of the BCR/ABL1 molecule disrupts normal ABL1 signal pathways, and contributes to the observed functional defects of the cells., Methods: We employed in vitro culture of clinical samples, combining microscopic and biochemical techniques with a phosphoproteomic approach to compare and characterise DCs from normal individuals and chronic myeloid leukaemia patients., Results and Conclusions: We identified an altered intracellular localisation for ABL1 within DCs derived from the monocytes of chronic myeloid leukaemia. The protein was found in the perinuclear region co-distributed with the adapter-protein CRKL and the BCR/ABL1 protein. This altered distribution was associated with defective generation of ABL1-dependent maturation signals, and a dislocation of ABL1 from the F-actin cytoskeleton. We suggest that abnormal ABL1-dependent signals contribute to the recognised functional defects affecting chronic myeloid leukaemia DCs., (© 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Signal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors linking extracellular signals to target gene transcription. Hematopoietic cells express two highly conserved STAT5-isoforms (STAT5A/STAT5B), and STAT5 is directly activated by JAK2 downstream of several cytokine receptors and the oncogenic BCR-ABL tyrosine kinase. Using an IL-3-dependent cell line with inducible BCR-ABL-expression we compared STAT5-activation by IL-3 and BCR-ABL in a STAT5-isoform specific manner. RNAi targeting of STAT5B strongly inhibits BCR-ABL-dependent cell proliferation, and STAT5B but not STAT5A is essential for BCL-XL-expression in the presence of BCR-ABL. Although BCR-ABL induces STAT5-tyrosine phosphorylation independent of JAK2-kinase activity, BCR-ABL is less efficient in inducing active STAT5A:STAT5B-heterodimerization than IL-3, leaving constitutive STAT5A and STAT5B-homodimerization unaffected. In comparison to IL-3, nuclear accumulation of a STAT5A-eGFP fusion protein is reduced by BCR-ABL, and BCR-ABL tyrosine kinase activity induces STAT5A-eGFP translocation to the cell membrane and co-localization with the IL-3 receptor. Furthermore, BCR-ABL-dependent phosphorylation of Y682 in STAT5A was detected by mass-spectrometry. Finally, RNAi targeting STAT5B but not STAT5A sensitizes human BCR-ABL-positive cell lines to imatinib-treatment. These data demonstrate differences between IL-3 and BCR-ABL-mediated STAT5-activation and isoform-specific effects, indicating therapeutic options for isoform-specific STAT5-inhibition in BCR-ABL-positive leukemia.

BCR/ABL expression is the key characteristic of chronic myeloid leukaemia (CML). The progression of CML is associated with genomic instability. Systematic analysis of DNA damage signalling in the presence of BCR/ABL thus offers opportunities to identify mechanisms of leukaemic progression. We therefore undertook a quantitative phosphoproteomics study to test whether BCR/ABL expression could globally affect the response to genotoxic stress signalling. Etoposide-induced DNA damage was chosen and the concentration and time of exposure determined such that apoptosis was not associated with the study. More than 1100 phosphoentities were identified. BCR/ABL was shown to significantly alter the response to etoposide in many cases. These include sites on MDC1, a key component of DNA repair, and Hemogen. Hemogen is a transcriptional target of HOXB4 and GATA1, two transcription factors involved in haemopoietic development, and is overexpressed in acute myeloid leukaemia. To validate Hemogen phosphorylation, absolute quantification using an isotopomeric standard and selected reaction monitoring was performed. This revealed a strong correlation with isobaric tagging data and offering quantification at about 10 fmol per million cells. Furthermore we found that multiple protein phosphorylation changes mediated by BCR/ABL were connected to the increased activation of NFκB, a key survival transcription factor, after etoposide exposure., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Leukaemic transformation is frequently associated with the aberrant activity of a protein tyrosine kinase (PTK). As such it is of clinical relevance to be able to map the effects of these leukaemogenic PTKs on haemopoietic cells at the level of phosphorylation modulation. In this paradigm study we have employed a range of proteomic approaches to analyse the effects of one such PTK, BCR/ABL. We have employed phosphoproteome enrichment techniques allied to peptide and protein quantification to identify proteins and pathways involved in cellular transformation. Amongst the proteins shown to be regulated at the post-translational level were cofilin, an actin-severing protein thus linked to altered motility and Cbl an E3 ubiquitin ligase integrally linked to the control of tyrosine kinase signalling (regulated by 5 and 6 PTKs respectively). The major class of proteins identified however were molecular chaperones. We also showed that HSP90 phosphorylation is altered by BCR/ABL action and that HSP90 plays a crucial role in oncogene stability. Further investigation with another six leukaemogenic PTKs demonstrates that this HSP90 role in oncogene stability appears to be a common phenomenon in a range of leukaemias. This opens up the potential opportunity to treat different leukaemias with HSP90 inhibitors.

Chronic myeloid leukemia (CML) is characterized by the presence of the constitutively active BCR-ABL protein tyrosine kinase. Using a multipotent hemopoietic cell line, FDCP-Mix, expressing BCR-ABL tyrosine kinase, we investigated the initial effects of this kinase in primitive hematopoietic stem cells. We identified down-regulation of a novel gene, CCN3, as a direct consequence of BCR-ABL kinase activity. CCN3 has been reported to function as a tumor suppressor gene in solid tumors. Northern and Western blotting plus immunocytochemical analysis confirmed CCN3 expression is decreased and is tyrosine-phosphorylated in BCR-ABL kinase active FDCP-Mix cells. Decreased cellular CCN3 correlated with increased CCN3 secretion in BCR-ABL kinase active cells. In vitro treatment of human CML cell lines with imatinib or siRNA directed against BCR-ABL significantly reduced BCR-ABL while increasing CCN3 expression. Cells from patients responding to imatinib showed a similar decrease in BCR-ABL and increase in CCN3. CML CD34+ cells treated with imatinib in vitro demonstrated increased CCN3 protein. Transfecting CCN3 into BCR-ABL+ cells inhibited proliferation and decreased clonogenic potential. CCN3 plays an important role in internal and external cell-signaling pathways. Thus, BCR-ABL can regulate protein levels by governing secretion, a novel mechanism for this tyrosine kinase.

The chemokine, stromal cell-derived factor-1 (SDF-1) is a crucial regulator of stem cell homing and tethering, and potentiation of this pathway in leukaemias may contribute to the pathogenesis of the disease. A key second messenger in SDF-1 signal/response coupling is phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3]. SDF-1 elevated PtdIns(3,4,5)P3 levels markedly in the multipotent FDCP-mix stem cell line. Similarly, transfection with BCR/ABL or TEL/PDGFRbeta leukaemogenic tyrosine kinases chronically elevated PtdIns(3,4,5)P3 levels. However, whilst an SDF-1 chemotactic response was observed in TEL/PDGFRbeta-transfected cells, in BCR/ABL cells this was markedly decreased, which was not due to Ras-pathway activation. Thus, multipotent cells can respond to SDF-1, despite chronic increases in this second messenger indicating that a discrete pool of SDF-1-stimulated PtdIns(3,4,5)P3 production drives the chemotactic response. To discern the mechanism for the differential effects of these oncogenes we considered subcellular localisation. As TEL/PDGFRbeta has a cytosolic location whilst BCR/ABL associates with actin, we removed the actin-binding domain from BCR/ABL. We observed relocation of BCR/ABL to the cytosol and increased SDF-1 responses. We conclude that the localisation of BCR/ABL to the cytoskeleton is essential for effects on motility and moderating SDF-1 responses is not essential in tyrosine kinase-mediated leukaemic transformation.

Philadelphia chromosome-positive, chronic myeloid leukaemia (CML) stem and progenitor cells have a survival and growth advantage compared with their normal counterparts. The mechanisms through which the BCR/ABL protein tyrosine kinase (PTK) induces these effects and the important domains within this protein are not fully defined. The F- and G-actin binding region of the BCR/ABL C-terminus may be important in BCR/ABL-mediated events, and we have investigated this by expressing a C-terminus deletion mutant of the temperature-sensitive BCR/ABL PTK, in a haemopoietic progenitor cell line, which models the chronic phase of CML. The truncated BCR/ABL PTK displayed similar levels of PTK activity when compared with wild type and activation of second messenger formation (in the form of sn-1,2-diacylglycerol) remains intact. On fibronectin substrata, localisation of the protein to the periphery of the cell was, however, dependent on the C-terminus of BCR/ABL PTK. Deletion of the C-terminus reversed both BCR/ABL-mediated apoptotic suppression and drug resistance although the progenitor cells did retain a proliferative advantage at low concentrations of growth factor. These results demonstrated that the C-terminal actin-binding domain of BCR/ABL is important for some of BCR/ABL PTK-mediated leukaemogenic effects.

In chronic myeloid leukaemia (CML) expression of the chimeric tyrosine kinase, Bcr-Abl, promotes the inappropriate survival of haemopoietic stem cells by a nonautocrine mechanism in the absence of IL-3. Stimulation of glucose uptake appears to play an important role in the suppression of apoptosis by this cytokine in normal haemopoietic cells. To investigate whether the cell survival mechanisms mediated by the oncoprotein and cytokine showed any similarities, we employed a haemopoietic cell line, TonB210, engineered for inducible expression of Bcr-Abl. Tyrosine kinase expression in cytokine-deprived cells was found to mimic the effect of IL-3 in maintaining a higher V(max) for hexose uptake. In both IL-3- treated cells and those expressing Bcr-Abl, high rates of hexose uptake were associated with the retention at the cell surface of approximately 80% of the total cellular content of the GLUT1 glucose transporter. In contrast, treatment of Bcr-Abl-expressing cells for 6 h with the Bcr-Abl kinase inhibitor Glivec (10 muM), in the absence of IL-3, led to internalization of approximately 90% of the cell-surface transporters and drastically decreased (4.4+/-0.9 (mean+/-s.e.m., 4)-fold) the V(max) for hexose uptake, without significant effect on the K(m) for this process or on the total cellular transporter content. These effects were not the result of any significant loss in cell viability, and preceded the onset of apoptosis caused by inhibition of Bcr-Abl. Both IL-3 treatment and expression of Bcr-Abl led to enhanced phosphorylation of Akt (protein kinase B). The stimulation of transport by IL-3 and Bcr-Abl in TonB210 cells was inhibitable by phosphatidylinositol 3-kinase inhibitors, indicating the involvement of this kinase in the signal transduction pathway. These findings suggest that inhibition of glucose transport plays an important role in the therapeutic action of Glivec, and that the signal transduction pathways involved in transport stimulation by Bcr-Abl may offer novel therapeutic targets for CML.

Many leukemic oncogenes form as a consequence of gene fusions or mutation that result in the activation or overexpression of a tyrosine kinase. To identify commonalities and differences in the action of two such kinases, breakpoint cluster region (BCR)/ABL and TEL/PDGFRbeta, two-dimensional gel electrophoresis was employed to characterize their effects on the proteome. While both oncogenes affected expression of specific proteins, few common effects were observed. A number of proteins whose expression is altered by BCR/ABL, including gelsolin and stathmin, are related to cytoskeletal function whereas no such changes were seen in TEL/PDGFRbeta-transfected cells. Treatment of cells with the kinase inhibitor STI571 for 4-h reversed changes in expression of some of these cytoskeletal proteins. Correspondingly, BCR/ABL-transfected cells were less responsive to chemotactic and chemokinetic stimuli than non-transfected cells and TEL/PDGFRbeta-transfected Ba/F3 cells. Decreased motile response was reversed by a 16-h treatment with STI571. A phosphoprotein-specific gel stain was used to identify TEL/PDGFRbeta and BCR/ABL-mediated changes in the phosphoproteome. These included changes on Crkl, Ras-GAP-binding protein 1, and for BCR/ABL, cytoskeletal proteins such as tubulin, and Nedd5. Decreased phosphorylation of Rho-GTPase dissociation inhibitor (Rho GDI) was also observed in BCR/ABL-transfected cells. This results in the activation of the Rho pathway, and treatment of cells with Y27632, an inhibitor of Rho kinase, inhibited DNA synthesis in BCR/ABL-transfected Ba/F3 cells but not TEL/PDGFRbeta-expressing cells. Expression of a dominant-negative RhoA inhibited both DNA synthesis and transwell migration, demonstrating the significance of this pathway in BCR/ABL-mediated transformation.

Chronic myeloid leukaemia (CML), a hematopoietic stem cell disorder is characterized by the expression of BCR-ABL. To investigate the effects of BCR-ABL on multipotent hematopoietic cells, a temperature sensitive BCR-ABL tyrosine kinase was expressed in the cell line, FDCP-Mix. BCR-ABL mediated an increase in c-kit expression that correlated with an enhanced mitogenic response to SCF. This was not observed in the absence of Bcr-Abl kinase activity or presence of the BCR-ABL inhibitor STI571, which also inhibits c-kit. When cultured in a combination of SCF plus G-CSF the FDCP-Mix cells undergo neutrophilic differentiation over a 7-10 day period. When BCR-ABL was active there was a marked inhibition of cell maturation compared to control cells in which BCR-ABL was either inactive or not present. However, BCR-ABL did not block differentiation as the cells eventually undergo terminal maturation. These data argue that BCR-ABL is directly responsible for the enhanced response to SCF reported in CML progenitor cells. Furthermore, although the primary effect of STI571 is via direct inhibition of BCR-ABL, STI571 additionally reduces the enhanced response to SCF. Thus there are two sites of STI571 action of potential importance in Bcr-Abl expressing cells.

The regulation of nutrient transport by both cytokines and oncogenes has been linked to haemopoietic cell survival. In this study, we found that activation of Bcr--Abl protein tyrosine kinase was associated with the stimulation of glucose transport in the multipotent haemopoietic cell line FDCP-mix, a cell model for chronic-phase chronic myeloid leukaemia (CML). Bcr--Abl upregulation of glucose transport was mediated by phosphatidylinositol-3-kinase. The observation that Bcr--Abl can regulate glucose transport in a CML cell model raises the possibility that glucose transport regulation may have a role to play in the aberrant survival of stem cells in the chronic phase of CML.

Introduction: The causative oncogene in CML is the BCR/ABL protein tyrosine kinase. This stem cell disease is often treated with interferon alpha (IFN-alpha) which can initiate haematological and cytological remission which is associated with increased survival. There is however no clear indication of why CML cells are more responsive to IFN-alpha., Materials and Methods: To establish if BCR/ABL increases the sensitivity of multipotent cells to IFN-alpha a temperature sensitive mutant of BCR/ABL was expressed in the multipotent haemopoietic stem cell line FDCP-Mix. The effect of IFN-alpha in terms of proliferation, induction of apoptosis, changes in cell cycle inhibitor proteins, and differentiation was assessed by [3H]thymidine incorporation, Annexin V and Western blot analysis., Results: When the BCR/ABL tyrosine kinase was activated, the IFN-alpha-induced inhibition on the growth rate of the FDCP-Mix cell population was more marked than in control populations. The BCR/ABL-mediated effect was due to decreased rates of DNA synthesis. There was no IFN-alpha-mediated induction of apoptosis. This enhanced BCR/ABL mediated growth inhibition occurred over a range of growth factor concentrations and was independent of changes in p21(Cip1) and p27(Kip) levels. When FDCP-Mix cells were induced to differentiate into mature macrophages and neutrophils in the presence of IFN-alpha, there was increased sensitivity to IFN-alpha that was independent of BCR/ABL activity., Conclusion: BCR/ABL PTK expression in this primitive multipotent haematopoietic cell line results in an enhanced response to IFN-alpha. In contrast, the more mature myeloid progenitor cells are equally responsive to this growth inhibitor. This data may explain some of the clinical effects of IFN-alpha.

Chronic myeloid leukaemia is a haemopoietic stem cell disorder, the hallmark of which is the expression of the Bcr-Abl Protein Tyrosine Kinase (PTK). We have previously reported that activation of a temperature sensitive Bcr-Abl PTK in the multipotent haemopoietic cell line FDCP-Mix for short periods resulted in subtle changes including, a transient suppression of apoptosis and no inhibition of differentiation. In contrast, activation of the Bcr-Abl PTK for 12 weeks results in cells that display a delay in differentiation at the early granulocyte stage. Flow cytometric analysis also indicates that the expression of cell surface differentiation markers and nuclear morphology are uncoupled. Furthermore, a significant number of the mature neutrophils display abnormal morphological features. Prolonged exposure to Bcr-Abl PTK results in interleukin-3 independent growth and decreased p53 protein levels. FDCP-Mix cells expressing a dominant negative p53 and p53null FDCP-Mix cells demonstrate that the reduction in p53 is causally related to the delay in development. Returning the cells to the restrictive temperature restores the p53 protein levels, the growth factor dependence and largely relieves the effects on development. We conclude that prolonged Bcr-Abl PTK activity within multipotent cells results in a reduction of p53 that drives a delayed and abnormal differentiation.

Background: Chronic Myeloid Leukaemia (CML) is characterised by the chromosomal translocation resulting in expression of the Bcr-Abl protein tyrosine kinase (PTK) in early stem cells and their progeny. However the precise nature of Bcr-Abl effects in primitive CML stem cells remains a matter of active debate., Materials and Methods: Extremely primitive Bcr-Abl fusion positive cells were purified from patients with CML using multiparameter flow cytometric analysis of CD34, Thy, and lineage marker (Lin) expression, plus rhodamine-123 (Rh-123) brightness. Progenitor cells of increasing maturity were examined for cycling status by flow cytometry and their proliferative status directly correlated with cell phenotype. The activation status of a key transcription factor, signal transducers and activators of transcription (STAT-5), was also analyzed by immunocytochemistry., Results: The most primitive stem cells currently defined (CD34+Lin-Thy+ Rh-1231o) were present as a lower proportion of the stem cell compartment (CD34+Lin-) of CML patients at presentation than of normal individuals (2.3% +/- 0.4 compared with 5.1% +/- 0.6 respectively). Conversely there was a significantly higher proportion of the more mature cells (CD34+Lin-Thy-Rh-123 hi) in CML patients than in normal individuals (79.3 +/- 1.8 compared with 70.9 +/- 3.3). No primitive subpopulation of CML CD34+Lin- cells was cycling to a significantly greater degree than cells from normal donors, in fact, late progenitor cells (CD34+Lin+) were cycling significantly less in CML samples than normal samples. STAT5, however, was observed to be activated in CML cells., Conclusions: We conclude that no subpopulation of CML stem cells displays significantly increased cell cycling. Thus, increased cycling cannot be a direct consequence of Bcr-Abl PTK acquisition in highly enriched stem cells from patients with CML. In vivo CML need not be considered a disease of unbridled stem cell proliferation, but a subtle defect in the balance between self renewal and maturation.

Chronic myeloid leukaemia (CML) is a clonal disorder of the pluripotent haemopoietic stem cell, the hallmark of which is the constitutively activated Bcr-Abl protein tyrosine kinase. During the initial chronic phase of CML the primitive multipotent leukaemic progenitor cells remain growth factor dependent and are capable of producing terminally differentiated cells. Although the available evidence suggests that Bcr-Abl directly affects signalling pathways involved in controlling the development of primitive haemopoietic progenitors the identification of the specific biological consequences of Bcr-Abl activity in these progenitors has been hampered by the lack of suitable systems modelling CML. By transfecting the multipotent haemopoietic cell line FDCP-Mix with a temperature sensitive mutant of Bcr-Abl we have developed the first working model that mirrors the chronic phase of CML. FDCP-Mix cells expressing Bcr-Abl tyrosine kinase activity remain growth factor dependent and retain their ability to differentiate. Normal neutrophilic cells are formed in response to G-CSF and GM-CSF. In addition, the transfected FDCP-Mix cells grown at the permissive temperature for Bcr-Abl tyrosine kinase activity display enhanced survival and proliferation in low concentrations of growth factor. These findings are consistent with the initial subtle changes seen in CML progenitor cells during the chronic phase and confirm that Bcr-Abl effects are context specific, i.e. they depend on the origin and developmental potential of the transfected cells. This questions the significance of studies in non-haemopoietic and differentiation blocked haemopoietic cells.