Axitinib and sorafenib are potent in tyrosine kinase inhibitor resistant chronic myeloid leukemia cells.

BACKGROUND: Chronic myeloid leukemia (CML) is driven by the fusion kinase Bcr-Abl. Bcr-Abl tyrosine kinase inhibitors (TKIs), such as imatinib mesylate (IM), revolutionized CML therapy. Nevertheless, about 20 % of CMLs display primary or acquired TKI resistance. TKI resistance can be either caused by mutations within the Bcr-Abl kinase domain or by aberrant signaling by its effectors, e.g. Lyn or Gab2. Bcr-Abl mutations are frequently observed in TKI resistance and can only in some cases be overcome by second line TKIs. In addition, we have previously shown that the formation of Gab2 complexes can be regulated by Bcr-Abl and that Gab2 signaling counteracts the efficacy of four distinct Bcr-Abl inhibitors. Therefore, TKI resistance still represents a challenge for disease management and alternative therapies are urgently needed. FINDINGS: Using different CML cell lines and models, we identified the clinically approved TKIs sorafenib (SF) and axitinib (AX) as drugs overcoming the resistance mediated by the Bcr Abl(T315I) mutant as well as the one mediated by Gab2 and Lyn(Y508F). In addition, we demonstrated that AX mainly affects the Bcr-Abl/Grb2/Gab2 axis, whereas SF seems to act independently of the fusion kinase and most likely by blocking signaling pathways up- and downstream of Gab2. CONCLUSION: We demonstrate that SF and AX show potency in various and mechanistically distinct scenarios of TKI resistance, including Bcr-Abl(T315I) as well as Lyn- and Gab2-mediated resistances. Our data invites for further evaluation und consideration of these inhibitors in the treatment of TKI resistant CML.

Alterations of Gab2 signalling complexes in imatinib and dasatinib treated chronic myeloid leukaemia cells.

BACKGROUND: The Gab2 docking protein acts as an important signal amplifier downstream of various growth factor receptors and Bcr-Abl, the driver of chronic myeloid leukaemia (CML). Despite the success of Bcr-Abl tyrosine kinase inhibitors (TKI) in the therapy of CML, TKI-resistance remains an unsolved problem in the clinic. We have recently shown that Gab2 signalling counteracts the efficacy of four distinct Bcr-Abl inhibitors. In the course of that project, we noticed that two clinically relevant drugs, imatinib and dasatinib, provoke distinct alterations in the electrophoretic mobility of Gab2, its signalling output and protein interactions. As the signalling potential of the docking protein is highly modulated by its phosphorylation status, we set out to obtain more insights into the impact of TKIs on Gab2 phosphorylation. FINDINGS: Using stable isotope labelling by amino acids in cell culture (SILAC)-based quantitative mass spectrometry (MS), we show now that imatinib and dasatinib provoke distinct effects on the phosphorylation status and interactome of Gab2. This study identifies several new phosphorylation sites on Gab2 and confirms many sites previously known from other experimental systems. At equimolar concentrations, dasatinib is more effective in preventing Gab2 tyrosine and serine/threonine phosphorylation than imatinib. It also affects the phosphorylation status of more residues than imatinib. In addition, we also identify novel components of the Gab2 signalling complex, such as casein kinases, stathmins and PIP1 as well as known interaction partners whose association with Gab2 is disrupted by imatinib and/or dasatinib. CONCLUSIONS: By using MS-based proteomics, we have identified new and confirmed known phosphorylation sites and interaction partners of Gab2, which may play an important role in the regulation of this docking protein. Given the growing importance of Gab2 in several tumour entities we expect that our results will help to understand the complex regulation of Gab2 and how this docking protein can contribute to malignancy.

A novel MCF-10A line allowing conditional oncogene expression in 3D culture.

INTRODUCTION: Non-transformed mammary epithelial cell lines such as MCF-10A recapitulate epithelial morphogenesis in three-dimensional (3D) tissue culture by forming acinar structures. They represent an important tool to characterize the biological properties of oncogenes and to model early carcinogenic events. So far, however, these approaches were restricted to cells with constitutive oncogene expression prior to the set-up of 3D cultures. Although very informative, this experimental setting has precluded the analysis of effects caused by sudden oncoprotein expression or withdrawal in established epithelial cultures. Here, we report the establishment and use of a stable MCF-10A cell line (MCF-10Atet) fitted with a novel and improved doxycycline (dox)-regulated expression system allowing the conditional expression of any transgene. METHODS: MCF-10Atet cells were generated by stable transfection with pWHE644, a vector expressing a second generation tetracycline-regulated transactivator and a novel transcriptional silencer. In order to test the properties of this new repressor/activator switch, MCF-10Atet cells were transfected with a second plasmid, pTET-HABRAF-IRES-GFP, which responds to dox treatment with the production of a bi-cistronic transcript encoding hemagglutinin-tagged B-Raf and green fluorescent protein (GFP). This improved conditional expression system was then characterized in detail in terms of its response to various dox concentrations and exposure times. The plasticity of the phenotype provoked by oncogenic B-RafV600E in MCF-10Atet cells was analyzed in 3D cultures by dox exposure and subsequent wash-out. RESULTS: MCF-10Atet cells represent a tightly controlled, conditional gene expression system. Using B-RafV600E as a model oncoprotein, we show that its sudden expression in established 3D cultures results in the loss of acinar organization, the induction of an invasive phenotype and hallmarks of epithelial-to-mesenchymal transition (EMT). Importantly, we show for the first time that this severe transformed phenotype can be reversed by dox wash-out and concomitant termination of oncogene expression. CONCLUSIONS: Taken together, we have generated a stable MCF-10A subline allowing tight dox-controlled and reversible expression of any transgene without the need to modify its product by introducing artificial dimerization or ligand-binding domains. This system will be very valuable to address phenomena such as EMT, oncogene addiction, oncogene-induced senescence and drug resistance.

The immunohistochemical staining pattern of Gab2 correlates with distinct stages of chronic myeloid leukemia.

Grb2-associated binder 2 protein (Gab2) is a member of scaffold proteins, playing crucial roles in (receptor-) tyrosine kinase and cytokine signaling. Chronic myeloid leukemia cells with t(9;22)(q34;q11) express the Bcr/Abl fusion protein, which interacts with Grb2 and Gab2 signaling, thereby triggering hematopoietic cell proliferation. The aim of this study was to examine in detail the total and subcellular Gab2 protein expression in myeloid cells in bone marrow biopsies of patients with chronic myeloid leukemia in different disease stages. The study included 50 fixed bone marrow biopsies of controls (unaffected hematopoiesis, n = 11) and Bcr/Abl-positive chronic myeloid leukemia cases (n = 39) of different stages (chronic phase, n = 13; accelerated phase, n = 4; blast crisis, n = 11; complete remission, n = 11). Immunohistochemistry and quantitative evaluation of Gab2 staining in 600 myeloid cells/bone marrow biopsy were performed before statistical analyses. Immunohistochemistry revealed Gab2 expression in hematopoietic cells. Gab2-positive myeloid cells occurred significantly more frequent in chronic myeloid leukemia cases than in controls (P < .001) and appeared to markedly increase from chronic phase to accelerated phase to blast crisis. Importantly, within the distinct stages of chronic myeloid leukemia, a significant switch of Gab2-positive myeloid cells with cytoplasmic or nuclear/perinuclear Gab2 staining occurred: Nuclear/perinuclear Gab2-positive myeloid cells significantly increased from chronic phase to accelerated phase (P = .001) and from chronic phase to blast crisis (P < .001). Still, an overlap and, hence, a wider range of Gab2 staining patterns were seen between and within chronic myeloid leukemia stages, most likely reflecting a high plasticity of Grb2-associated binder 2 functions in the progression of chronic myeloid leukemia. In summary, the present study, for the first time, analyzed Grb2-associated binder 2 protein expression in bone marrow biopsies of patients with chronic myeloid leukemia in detail, demonstrating a novel and distinct Grb2-associated binder 2 staining pattern in normal and chronic myeloid leukemia bone marrow biopsies as well as in distinct chronic myeloid leukemia stages. Grb2-associated binder 2 immunohistochemistry may provide a valuable supplementary tool to routine histopathology and standard immunohistochemistry for classification and staging of (borderline) chronic myeloid leukemia bone marrow biopsies and hence improved therapeutic disease management.

Function, regulation and pathological roles of the Gab/DOS docking proteins.

Since their discovery a little more than a decade ago, the docking proteins of the Gab/DOS family have emerged as important signalling elements in metazoans. Gab/DOS proteins integrate and amplify signals from a wide variety of sources including growth factor, cytokine and antigen receptors as well as cell adhesion molecules. They also contribute to signal diversification by channelling the information from activated receptors into signalling pathways with distinct biological functions. Recent approaches in protein biochemistry and systems biology have revealed that Gab proteins are subject to complex regulation by feed-forward and feedback phosphorylation events as well as protein-protein interactions. Thus, Gab/DOS docking proteins are at the centre of entire signalling subsystems and fulfil an important if not essential role in many physiological processes. Furthermore, aberrant signalling by Gab proteins has been increasingly linked to human diseases from various forms of neoplasia to Alzheimer's disease.In this review, we provide a detailed overview of the structure, effector functions, regulation and evolution of the Gab/DOS family. We also summarize recent findings implicating Gab proteins, in particular the Gab2 isoform, in leukaemia, solid tumours and other human diseases.

How to Grb2 a Gab.

In this issue of Structure, Harkiolaki et al. use crystallography, peptide arrays and isothermal calorimetry to provide a detailed insight into the interaction between the C-terminal SH3 domain of adaptor protein Grb2 bound to the docking protein Gab2.