Kinetics and dynamics of BCR-ABL signaling upon tyrosine-kinase inhibition.

<p>K562 cells were treated with increasing concentrations of tyrosine kinase inhibitors. Untreated cells served as a control. For analysis of signaling pathway inhibition cells were either lysed for Western Blot analysis or fixed for FACS. (<b>A</b>): <i>Left panel</i>: Western Blot analysis of signaling pathway inhibition after exposure of Ba/F3-BCR-ABL cells to imatinib for 2 h. <i>Right panel</i>: FACS analysis of STAT5 and CRKL inhibition using phosphorylation-specific antibodies. To assess signaling dynamics, this analysis was performed at three different time-points (0.3, 2, and 12 h). (<b>B</b>) The same analysis was performed using the second-generation BCR-ABL tyrosine kinase inhibitor dasatinib. Experiments were performed at least in triplicate and one representative experiment is shown.</p

Experimental set-up for analysis of induction of apoptosis upon HD-TKI exposure.

<p>(<b>A</b>) Cells were seeded at a density of 5×10⁴ cells/ml in a total volume of 2 ml in RPMI 1640 supplemented with 10% FCS. Cells were treated for 2 h with TKI as indicated. Then, cells were washed twice with 2 ml PBS at room temperature and replated in fresh cell culture media (2 ml final volume). Cells exposed to 0.35% DMSO served as controls. 24 h after start of TKI exposure percentage of cells in subG1 phase was measured by flow cytometry after propidium iodide staining. (<b>B</b>) To analyze for residual TKI activity upon HD-TKI pulse exposure, a second and third drug wash-out procedure (each consisting of 2×2 ml PBS washing) was performed: Cells were treated with TKI for 2 h. Cells initially pulse-exposed to HD-TKI were washed twice with 2 ml PBS at room temperature and replated in 2 ml fresh media (density: 5×10⁴ cells/ml) as described in (a) (“<i>1x”</i>). To test for residual TKI-activity, the cell culture supernatant was transferred to previously untreated cells <i>(“S1”)</i>, which were subsequently incubated for 24 h. Two hours after replating, a second drug wash-out was performed (2×2 ml PBS). Cells were again replated in 2 ml fresh media (“<i>2x”</i>). Again supernatants were transferred to previously untreated cells (“<i>S2”</i>). This procedure was repeated for a third time (“<i>3x”, “S3”</i>).</p

Repetitive washing prevents apoptosis after high-dose TKI pulse-exposure.

<p>To analyze for residual TKI activity upon HD-TKI pulse-exposure, we employed repetitive wash-out procedures and exposure of previously untreated cells to cell culture supernatants. Therefore, 5×10⁴ cells/ml were seeded in a total volume of 2 ml in RPMI 1640 supplemented with 10% FCS and treated with TKI as indicated for 2 hours. Cells exposed to 0.35% DMSO served as controls (“M”). Then, cells were washed and replated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040853#pone-0040853-g001" target="_blank"><b>Figure 1B</b></a> (“1x”, “2x”, “3x”; upper panel). To test for residual TKI-activity, the cell culture supernatants were transferred to previously untreated cells (“S1”, “S2”, “S3”; lower panel). For further details of the experimental procedure please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040853#pone-0040853-g001" target="_blank"><b>Figure 1B</b></a>. Percentage of cells in subG1 phase was measured by flow cytometry after propidium iodide staining 24 hours after start of TKI treatment and is depicted for Ba/F3-BCR-ABL cells (<b>A</b>) and K562 cells (<b>B</b>), respectively. Experiments were performed in triplicate. Data are presented as mean percentage of cells in subG1 phase + SEM.</p

AnnexinV- and Cleaved Caspase3-staining after high-dose TKI pulse-exposure.

<p>To confirm results obtained with propidium iodide staining, AnnexinV-PE staining (<b>A</b>) and Cleaved Caspase3-Alexa488 staining (<b>B</b>) of Ba/F3-BCR-ABL cells was performed at 24 hours after HD-TKI pulse-exposure using either imatinib or dasatinib as indicated followed by repetitive wash-out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040853#pone-0040853-g002" target="_blank"><b>Figure 2</b></a>. Experiments were performed in triplicate and one representative experiment is shown.</p

Measurement of cellular imatinib uptake and release upon HD-TKI pulse-exposure.

<p>5×10⁴ K562 cells were treated for 2 h with 25 µM ¹⁴C-labeled imatinib in a total volume of 1 ml cell culture media followed by drug wash-out as described for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040853#pone-0040853-g001" target="_blank"><b>Figure 1B</b></a>. Specific activity of cell culture supernatants and cell pellets was measured using a scintillation counter. Imatinib concentrations were calculated by fitting the dpm (disintegrations per minute) values to a standard curve. All measurements were performed in triplicate. Depicted are mean values + SEM of 4 independent experiments. (<b>A</b>): Dynamics of cellular imatinib content was measured. Cells were pelleted and washed according to our drug wash-out protocol immediately at the end of the incubation period (end of exposure, “EOE”) and then subjected to scintillation counter assessment of cellular imatinib uptake. In parallel, for analysis of imatinib release, cells were replated in 1 ml fresh cell culture media and 0, 15, 30, and 120 min after replating cells were again pelleted, washed and then subjected to measurement in a scintillation counter. (<b>B</b>): Corresponding dynamics of imatinib concentration in cell culture supernatants upon HD-TKI pulse-exposure followed by drug wash-out. Measurement of cell culture supernatants immediately after 2 h incubation with 25 µM ¹⁴C-labeled imatinib represents the amount of imatinib present in the incubation medium (“EOE”). Intervals 0, 15, 30, and 120 min represent the time elapsed after initial drug wash-out and replating in fresh cell culture media.</p

Measurement of TKI concentrations in cell culture supernatants using chromatographic methods.

<p>Imatinib and dasatinib concentrations were measured in cell culture supernatants using HPLC and LC/MS/MS, respectively. The sampling procedure and time-scale were identical to that described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040853#pone-0040853-g005" target="_blank"><b>Figure 5</b></a>. (<b>A</b>) Ba/F3-BCR-ABL cells <i>(I)</i>, K562 cells <i>(II)</i>, primary human CML patient MNCs (<i>III</i>) and normal CD34⁺ enriched cells (<i>IV</i>) were pulse exposed to 25 µM imatinib followed by drug wash-out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040853#pone-0040853-g001" target="_blank"><b>Figure 1B</b></a>. (<b>B</b>) Ba/F3-BCR-ABL cells (<i>I</i>) and normal enriched CD34⁺ cells (<i>II</i>) were pulse exposed to 100 nM dasatinib followed by drug wash-out as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0040853#pone-0040853-g001" target="_blank"><b>Figure 1B</b></a>. The red lines represent the <i>in vitro</i> IC50 for the respective TKI used. Supernatant 1 (“S1”) was measured and served as a positive control. Measurement of cell culture media taken immediately upon replating served as a control for efficient washing (“0 min”). Depicted are mean values + SEM; at least 3 independent experiments were performed. For primary material one representative experiment is shown.</p

Differential drug effects on cellular tyrosine phosphorylation.

<p>Cells were treated for 30 min with bafetinib (800 nM), bosutinib (400 nM), dasatinib (100 nM) and nilotinib (4 µM), which are concentrations equivalent to reported maximum patient plasma concentrations, and DMSO control. Effects of individual drugs were determined by immunoblot analysis for BCR-ABL (α-ABL) and total phosphotyrosine (α-pY). Actin served as loading control. <b>A</b>. Dasatinib had the strongest impact on cellular tyrosine phosphorylation in BV-173 cells while the effects of bafetinib, nilotinib and particularly bosutinib were less pronounced. <b>B</b>. Dasatinib completely abolished cellular tyrosine phosphorylation in Z-119 cells. BCR-ABL levels were not appreciably affected, but it’s phosphorylation (marked by arrow) was inhibited by the drugs in either cell line.</p

Graphical representation of binding specificity assessment.

<p>Using the example of dasatinib and BV-173 cells, the average spectral counts obtained from chemical proteomics were compared with the respective competition experiments in the presence of 20 µM free drug in a double-logarithmic plot. Specific ( ♦) and non-specific (◊) binders were identified by definition of a specificity gate (grey area) with a ratio threshold of 2 and a minimum average spectral count of 10. For proteins that were not identified in the competition experiment, the minimum average spectral count was lowered to 1. </p

Schematic outline of the integrated chemical proteomics and computational biology strategy.

<p><b>A</b>. Drug-protein interaction networks are generated by chemical proteomics while the protein-protein interaction (PPI) network is derived from public databases and modified to represent the specific disease. The interaction networks are correlated through a random walk approach across the PPI network using proteins from the drug-protein network as entry points. The resulting correlation scores are subsequently validated by cell proliferation assays. <b>B</b>. Chemical structures of the four second-generation BCR-ABL tyrosine kinase inhibitors dasatinib (Sprycel, BMS-354825), nilotinib (<i>Tasigna</i>, AMN107), bosutinib (SKI-606) and bafetinib (INNO-406, NS-187) as well as their coupleable analogues c-dasatinib, c-nilotinib, c-bosutinib and c-bafetinib that were used for immobilization and subsequent chemical proteomics experiments.</p

Hybrid drug-protein/protein-protein interaction networks of specific drug binding proteins.

<p>Individual cellular target profiles of nilotinib (green), dasatinib (red), bosutinib (yellow) and bafetinib (blue) were intersected with each other and overlaid with PPI data from public databases. Protein kinases and the oxidoreductase NQO2, as a validated target of nilotinib and to lesser extent of bafetinib, were considered to be direct drug binders (solid lines) and color-coded according to the drug they were interacting with. Shared kinase targets display a split color code. All other non-kinase proteins were assumed to be indirect binders (dashed lines) and displayed in grey. The analysis reveals distinct protein complexes, which are enriched by particular drugs and which are highlighted with the respectively colored background. <b>A</b>. Z-119 drug-protein interaction network. <b>B</b>. BV-173 drug-protein interaction network.</p