Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 1. Inhibition of neuroblastoma cell lines employing crizotinib and PF-06463922. Treatment of neuroblastoma cells with the ALK TKIs crizotinib and PF-06463922. (A,B) Proliferation was assessed over 5 days using the resazurin cell proliferation assay in the following neuroblastoma cell lines: CLB-BAR, CLB-GE, CLB-PE, SK-N-AS and IMR32. Neuroblastoma cell line characteristics: CLB-GE – MYCN amplified, 1p deletion, 17q gain, amplified ALK amplicon containing an ALKF1174V mutation; CLB-BAR – amplified MYCN/ALK, Δexon 4-11, 1p deletion, 17q gain; IMR32 – MYCN, wt sequence but exons 2-4 are amplified, 1p deletion, 17q gain; CLB-PE – 1p gain, amplified MYCN, 17q gain; SK-N-AS – IGF-1 overexpressing, 1p deletion, 1q gain, 17q gain, 17 deletion. Cells lines were treated with increasing concentrations of either PF-06463922 (A) or crizotinib (B) as indicated. Data are mean±s.d. of the fold-relative fluorescence from treated cells relative to untreated cells from three independent experiments. (C) CLB-BAR, CLB-GE neuroblastoma cells were treated for 6 h with either crizotinib or PF-04643922 as indicated. Cellswere harvested andpre-clearedcelllysates were analyzed onSDS PAGE followedbywesternblotting for ALK, phospho-ALK-Y1278,phospho-ERK5, pan-ERK5 phospho-ERK1/2 and pan-ERK. Actin was employed as a loading control. Protein band intensities were quantified by Image Studio Lite 3.1 and normalized to untreated samples.

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Inhibition, Proliferation Assay, Amplification, Mutagenesis, Sequencing, Fluorescence, Control

Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 3. Comparison of inhibition effects of crizotinib and PF-06463922 on WT and neuroblastoma gain-of-function mutant TKDs by in vitro kinase assay. (A,B) Different ALK TKD proteins were incubated with either PF-06463922 (A) or crizotinib (B) in the presence of ATP (0.1 mM) and substrate peptides (0.2 mM). The incorporation of labelled γ-32P was detected under different conditions. Background counts from no-enzyme controls were subtracted, and the data were normalized to the 0 nM inhibitor reactions. (C) IC50 values from A,B were calculated by fitting data to a log (inhibitor) versus normalized response (variable slope) equation in GraphPad Prism 6.0. All data are shown as mean±s.d. from at least two independent experiments. (D) Crystal structures of ALK kinase domain in complex with PF-06463922 (top) or crizotinib (bottom). Compounds indicated in black. Gain-of-function ALK mutations F1174, R1192P, F1245, G1269 and Y1278 are shown as red spheres. The ribbon diagram displays αC helix (1157-1173; orange), catalytic loop (1246-125; magenta), activation loop (1271-1288; cyan) with DFG motif marked in blue. Figures were generated with PyMol using published coordinates (Protein data bank code: 4CLI and 2XP2).

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Comparison, Inhibition, Mutagenesis, In Vitro, Kinase Assay, Incubation, Activation Assay, Generated

Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 5. Efficacy of PF-06463922 in an ALKF1174 CLB-GE xenograft neuroblastoma model. 4.5×106 CLB-GE cells were injected into left shoulder subcutaneously. (A) Tumor growth curves represent the average volume of vehicle group and PF-06463922-treated group, P≤0.05 (n=5 for each group). (B) Average tumor weights in vehicle group and PF-06463922 group are displayed, P=0.02. (C) Average body weight on the day of sacrifice is shown, P>0.05. (D) Immunoblotting analysis of indicated proteins from tumors collected after 8 days of treatment with vehicle or PF-0646399 and relapsed tumors. Tumor lysates were harvested, pre-cleared and analyzed by western blotting for ALK, phospho-ALK-Y1278, MYCN, phospho-ERK1/2 and phospho-AKT. Pan-ERK and pan-Akt were employed as loading controls. (E) Immunohistochemical staining of tumors for Ki-76 as a measure of proliferation rate as indicated. Ki67-positive cells were counted manually per field of vision and quantitative results (n=15). Statistical analysis shows significant difference between vehicle and PF-06463922- treated group, P<0.002 using unpaired t-test. Data in all graphs presented as mean±s.d.

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Injection, Western Blot, Immunohistochemical staining, Staining

Journal: Disease models & mechanisms

Article Title: The ALK inhibitor PF-06463922 is effective as a single agent in neuroblastoma driven by expression of ALK and MYCN.

doi: 10.1242/dmm.024448

Figure Lengend Snippet: Fig. 6. Preclinical efficacy of PF-06463922 in a murine model of Th-ALKF1174L/MYCN-driven neuroblastoma. (A) Waterfall plots of tumoral response in Th-ALKF1174L/MYCN mice treated with vehicle, crizotinib (100 mg/kg, once daily) or PF-06463922 (10 mg/kg, twice daily). Each bar indicates percent change in the volume of an individual tumor, as assessed by T2-weighted MRI, on day 7 compared with day 0 of treatment. (B) Representative MRI of each treatment group on day 0 and day 7 after treatment of indicated vehicle or drug. Hashed white line indicates tumor border. (C) Immunoblot analysis of phosphorylated ALK (phospho-ALK-Y1278), total ALK, and MYCN of tumors treated for 2 days with vehicle or PF-06463922. GAPDH was employed as loading control. Arrow indicates pALK-Y1278. (D) Quantification of band intensity of MYCN relative to GAPDH (left) and ratio of band intensity of pALK over total ALK (right) comparing vehicle versus treated samples. (E) Meso Scale Discovery assay depicted as electrochemiluminescence signal of treated samples relative to the respective vehicle controls for total ALK, phospho-ALK-Y1568, and the ratio of pALK to ALK. (F) Representative H&E (top) and immunohistochemical images for Ki67 (bottom) of vehicle and treated samples. Quantification of overall percentage of area positive for Ki67 (right). Error bars represent s.d. between individual animals (per group: n=4 in A, n=5 in C-F). P-values equal unpaired t-test comparison between vehicle and treatment groups.

Article Snippet: Primary antibodies were: phospho-ALK (Y1604 (1:2000; 3341) and Y1278 (1:2000; 12127), phospho-ERK1/2, actin (1:2000; 4967), phospho-Akt (1:2000; 92711), MYCN (1:2000; 9405), Ki67 (1:100; 9129), GADPH and phospho-ERK5 (1:1000; 3371) from Cell Signaling Technology (Danvers, MA).

Techniques: Western Blot, Control, Electrochemiluminescence, Immunohistochemical staining, Comparison

Journal: eLife

Article Title: FAM150A and FAM150B are activating ligands for anaplastic lymphoma kinase

doi: 10.7554/eLife.09811

Figure Lengend Snippet: ( A ) Schematic overview of anaplastic lymphoma kinase (ALK) and leukocyte tyrosine kinase (LTK) protein domain structure. ( B ) Alignment of the intracellular region of ALK and LTK, highlighting in yellow the Y1278 phosphoepitope in the activation loop which shares significant homology between ALK and LTK, and the Y1604 phosphoepitope of ALK which is not found in LTK. DOI: http://dx.doi.org/10.7554/eLife.09811.005

Article Snippet: The primary antibodies used were anti-pan-ERK (1:10,000; BD Transduction Laboratories), anti-ALK (for immunofluorescence 1:1000; ab4061, Abcam), anti-ALK (D5F3, 1:5000; Cell Signaling Technology), anti-ALK mAb135 (1:2000; [ ]), anti-pERK5 (1:1000; Cell Signaling Technology), anti-pALK-Y1278 (1:2000; Cell Signaling Technology), anti-pALK-Y1604 (1:2000; Cell Signaling Technology) and anti-pERK1/2-T202/Y204 (1:2000; Cell Signaling Technology), anti-FAM150A (1:4000, Atlas Antibodies), anti-HA (1:1000 for immunofluorescence, 1:6000 for immunoblotting; 16B12, Covance).

Techniques: Activation Assay

Journal: eLife

Article Title: FAM150A and FAM150B are activating ligands for anaplastic lymphoma kinase

doi: 10.7554/eLife.09811

Figure Lengend Snippet: ( A ) Neurite outgrowth in PC12 cells expressing either vector control or anaplastic lymphoma kinase (ALK) were cultured in medium from Human Embryonic Kidney (HEK) 293 cells transfected with either FAM150A or FAM150B, quantified below. Experiments were performed in triplicate and each sample within an experiment was performed in duplicate. Values represent mean ± SD from at least three independent experiments. ( B ) Whole cell lysates from PC12 cells expressing either vector control or ALK stimulated with medium from HEK293 cells transfected with vector control, FAM150A or FAM150B were analyzed by immunoblot. Analysis was carried out in the presence or absence of 250 nM crizotinib. Detection of ALK activation was visualized with pALK-Y1604 (arrowheads), ALK and pERK1/2 in whole cell lysates. The presence of FAM150A in supernatants was confirmed with anti-FAM150A antibodies, while the presence of FAM150B-HA was confirmed with anti-HA antibodies. Pan-ERK was employed for equal loading. ( C ) IMR32 cells harboring a wild-type ALK receptor were stimulated for 20 min with medium from HEK293 cells transfected with either vector control, FAM150A or FAM150B prior to analysis by immunoblot. Analysis was carried out in the presence or absence of 250 nM crizotinib. Stimulation with the ALK activating antibody mAb46 was employed as positive control. Detection of ALK activation was visualized with ALK, pALK-Y1604 (arrowheads) and pERK1/2. Pan-ERK was employed for equal loading. ( D ) IMR32 cells harboring a wild-type ALK receptor stimulated with increased amounts of recombinant His-tagged FAM150A purified from Sf21 cells. Detection of ALK activation was visualized with ALK, pALK-Y1278 (arrowheads) and pERK1/2. Pan-ERK was employed for equal loading. ( E ) Time course of IMR32 cells stimulated with FAM150A conditioned medium. Stimulation with ALK activating antibody mAb46 was employed as positive control. Detection of ALK activation was visualized with ALK, pALK-Y1604 (arrowheads), pERK5 and pERK1/2. Pan-ERK was employed for equal loading. ( F ) Time course of IMR32 cells harboring a wild type ALK receptor stimulated with FAM150B conditioned medium. Stimulation with ALK activating antibody mAb46 was employed as positive control. Detection of ALK activation was visualized with ALK, pALK-Y1604 (arrowheads), pERK5 and pERK1/2. Pan-ERK was employed for equal loading. DOI: http://dx.doi.org/10.7554/eLife.09811.006

Article Snippet: The primary antibodies used were anti-pan-ERK (1:10,000; BD Transduction Laboratories), anti-ALK (for immunofluorescence 1:1000; ab4061, Abcam), anti-ALK (D5F3, 1:5000; Cell Signaling Technology), anti-ALK mAb135 (1:2000; [ ]), anti-pERK5 (1:1000; Cell Signaling Technology), anti-pALK-Y1278 (1:2000; Cell Signaling Technology), anti-pALK-Y1604 (1:2000; Cell Signaling Technology) and anti-pERK1/2-T202/Y204 (1:2000; Cell Signaling Technology), anti-FAM150A (1:4000, Atlas Antibodies), anti-HA (1:1000 for immunofluorescence, 1:6000 for immunoblotting; 16B12, Covance).

Techniques: Expressing, Plasmid Preparation, Cell Culture, Transfection, Western Blot, Activation Assay, Positive Control, Recombinant, Purification