Additional file 1: Figure S1. of Calreticulin-mutant proteins induce megakaryocytic signaling to transform hematopoietic cells and undergo accelerated degradation and Golgi-mediated secretion

Supplementary material & methods

IC50 determination ixazomib [n = 2] for TOM-1 and BV-173.

Ph+ cells BV-173 and TOM-1 were treated with DMSO and bortezomib at day 0, CCK8 assay was performed at day 1,2 and 3. The IC50 is depicted in a non-linear regression curve with the point of inflections representing the respective IC50. The calculated IC50 values are shown in the table and ranged around 57nM for TOM-1 at d2 and 41.2nM for BV-173 at day 2. Errobars = SD, n = 2 for each treatment. (TIF)</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 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

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

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

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

Quality control of next generation sequencing.

<p>(A) Quality of sequencing as assessed by Phred Quality Score (Q-Score) is shown for the second sequencing run, yielding 5.3 GB of data. 90.1% of all bases were called with a Q-Score of 30 or better (indicating that the probability of an incorrect base call was 1:1000 or less). (B) Coverage of wild-type and mutant reads from known mutations in HEL, HMC-1, and K562 cell lines is shown, as analyzed by SeqPilot Software. Percentages display the fraction of mutant reads per total reads in each case, with values close to 100% indicating homozygous and values close to 50% indicating heterozygous mutations.</p

Patient characteristics.

<p>Abbrevations: CML = chronic myeloid leukemia, CP = chronic phase, AP = accelerated phase, BC = blast crisis; ET = essential thrombocythemia; PV = polycythemia vera; HES = hypereosinophilic syndrome; MF = myelofibrosis, ALL = acute lymphoblastic leukemia, bm = bone marrow; pb = peripheral blood. If JAK2V617F positive, the allele burden of the c.1849G>T mutation (which leads to V617F) detected by NGS is shown in percent of total JAK2 sequences</p><p>Patient characteristics.</p