Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Activating mutations in ALK kinase domain confer resistance to structurally unrelated ALK inhibitors in NPM-ALK-positive anaplastic large-cell lymphoma

doi: 10.1007/s00432-014-1589-3

Figure Lengend Snippet: Identification of the resistance mechanism in Karpas299CR and Karpas299CHR cells. a Analysis of phosphorylation status of key signaling molecules assessed by the PathScan ® RTK Signaling Antibody Array. b Immunoblot analysis of pALK and downstream signaling proteins following 24 h of incubation in the inhibitor-free medium. c Sequencing of ALK KD coding fragment in Karpas299CR and Karpas299CHR cells

Article Snippet: Primary antibodies against pALK (Y1604), total ALK, pSTAT3 (Y705), total STAT3, pERK1/2 (T202/Y204), total ERK1/2 and pS6 (S240/244) were purchased from Cell Signaling Technology.

Techniques: Phospho-proteomics, Ab Array, Western Blot, Incubation, Sequencing

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Activating mutations in ALK kinase domain confer resistance to structurally unrelated ALK inhibitors in NPM-ALK-positive anaplastic large-cell lymphoma

doi: 10.1007/s00432-014-1589-3

Figure Lengend Snippet: Sensitivity of Karpas299wt and resistant cells to ALK inhibitors. a ATPlite cell viability assay following 72 h of incubation with ALK inhibitors. Data expressed as percentage viability relative to untreated controls. Corresponding IC 50 values are summarized in Table . b BrdU cell proliferation assay and flow cytometry analysis following 48-h incubation with inhibitors. Data expressed as percentage of cells in S phase relative to untreated control. c Immunoblot analysis of pALK and downstream signaling following incubation with inhibitors for 2 h

Article Snippet: Primary antibodies against pALK (Y1604), total ALK, pSTAT3 (Y705), total STAT3, pERK1/2 (T202/Y204), total ERK1/2 and pS6 (S240/244) were purchased from Cell Signaling Technology.

Techniques: Viability Assay, Incubation, BrdU Cell Proliferation Assay, Flow Cytometry, Control, Western Blot

Journal: Oncotarget

Article Title: Targeting stemness is an effective strategy to control EML4-ALK + non-small cell lung cancer cells

doi:

Figure Lengend Snippet: A. Sphere-forming capacity of BEAS-2B, A549, H3122 and H2228 cells in a low-density suspension culture. Original magnification, x40. B. ALK, pALK, NANOG, OCT4, SOX2, KLF4, c-MYC and β-ACTIN expression in BEAS-2B, A549, H3122 and H2228 cells in a low-density suspension culture. Original magnification, x40. (B) ALK, pALK, NANOG, OCT4, SOX2, KLF4, c-MYC and β-ACTIN expression in BEAS-2B, A549, H3122 and H2228 cells was visualized by western blot analysis with lysates from the monolayer cultured cells. (C) H3122 and H2228 cells were treated with siGFP (control) or siALK and the levels of ALK, NANOG, OCT4, SOX2, KLF4, and c-MYC proteins were analyzed. β-ACTIN was used as an internal loading control. Numbers below blots indicate expression as measured by fold change. D. Flow cytometry analysis of the frequency of ALDH1 + cells in H3122 and H2228 cells treated with siALK or siGFP (control). E. Sphere-forming capacity of H3122 and H2228 cells treated with siGFP or siALK in a low-density suspension culture. Original magnification, × 40. F. Tumorigenicity of siGFP -versus siALK -treated H3122 cells inoculated at indicated doses into 5 NOD/SCID mice per group. G. Tumors were extracted at 20 days after injection of 10 5 siGFP - or siALK -treated H3122 cells. Error bars represent mean ± SD. Individual data analysis was performed using two-tailed Student's t -test.

Article Snippet: Primary antibodies against ALK, pALK (Y1604), c-MYC, EGFR, pEGFR (Y1068), AKT, pAKT (T308), pAKT (S473), mTORC1, pmTORC1 (S2448), S6K, pS6K (S240/244) (Cell Signaling Technology, MA, USA); SOX2, OCT4, and KLF4 (Santa Cruz Biotechnology, Dallas, TX, USA); NANOG (Abnova, CA, USA) were used at 1:1000–1:3000 dilution.

Techniques: Suspension, Expressing, Western Blot, Cell Culture, Control, Flow Cytometry, Injection, Two Tailed Test

Journal: Oncotarget

Article Title: Targeting stemness is an effective strategy to control EML4-ALK + non-small cell lung cancer cells

doi:

Figure Lengend Snippet: A. A549 cells were transfected with an empty vector or EML4-ALK variant 1 ( EAV1 ) cDNA and the levels of ALK, pALK, NANOG, and OCT4 were analyzed. β-ACTIN was used as an internal loading control. Numbers below blots indicate expression as measured by fold change. B. Flow cytometry analysis of the frequency of ALDH1 + cells in A549 cells transfected with EAV1 or empty vector. C. Sphere-forming capacity of EAV1 versus empty vector-transfected A549 cells in a low-density suspension culture. Original magnification, × 40. Error bars represent mean ± SD. Individual data analysis was performed using two-tailed Student's t -test.

Article Snippet: Primary antibodies against ALK, pALK (Y1604), c-MYC, EGFR, pEGFR (Y1068), AKT, pAKT (T308), pAKT (S473), mTORC1, pmTORC1 (S2448), S6K, pS6K (S240/244) (Cell Signaling Technology, MA, USA); SOX2, OCT4, and KLF4 (Santa Cruz Biotechnology, Dallas, TX, USA); NANOG (Abnova, CA, USA) were used at 1:1000–1:3000 dilution.

Techniques: Transfection, Plasmid Preparation, Variant Assay, Control, Expressing, Flow Cytometry, Suspension, Two Tailed Test

Journal: Oncotarget

Article Title: Targeting stemness is an effective strategy to control EML4-ALK + non-small cell lung cancer cells

doi:

Figure Lengend Snippet: H3122 cells were treated with crizotinib at the indicated concentration. A. ALK, pALK, NANOG, OCT4, and β-ACTIN expression in H3122 cells treated with crizotinib or DMSO (control) for 24 hr was visualized by western blot analysis. Numbers below blots indicate expression as measured by fold change. B. Flow cytometry analysis of the frequency of ALDH + cells in H3122 cells treated with crizotinib or DMSO (control) for 24 hr. C. Sphere-forming capacity of H3122 cells treated with crizotinib or DMSO (control) in a low-density suspension culture. Original magnification, × 40. Error bars represent mean ± SD. Individual data analysis was performed using two-tailed Student's t -test.

Article Snippet: Primary antibodies against ALK, pALK (Y1604), c-MYC, EGFR, pEGFR (Y1068), AKT, pAKT (T308), pAKT (S473), mTORC1, pmTORC1 (S2448), S6K, pS6K (S240/244) (Cell Signaling Technology, MA, USA); SOX2, OCT4, and KLF4 (Santa Cruz Biotechnology, Dallas, TX, USA); NANOG (Abnova, CA, USA) were used at 1:1000–1:3000 dilution.

Techniques: Concentration Assay, Expressing, Control, Western Blot, Flow Cytometry, Suspension, Two Tailed Test

Journal: Molecular cancer research : MCR

Article Title: ALK Fusion Partners Impact Response to ALK Inhibition: Differential Effects on Sensitivity, Cellular Phenotypes, and Biochemical Properties

doi: 10.1158/1541-7786.MCR-18-0171

Figure Lengend Snippet: (A) Lysates from the ALK Variant 3T3 cell line models were run out by SDS-PAGE and blotted for pALK (Y1604), total ALK, and actin. Membranes were visualized using the LiCor Odyssey and quantified with iMageStudio Lite. The pALK and ALK signals were normalized to the actin in the corresponding lane. The respective pALK/ALK ratios are shown below each lane. (B) Kinase assays were performed in triplicate for each ALK variant. ALK variants were immunoprecipitated from the 3T3 cell line model lysates using a polyclonal ALK antibody (Cell Signaling) and Sepharose A beads. An additional aliquot of the immunoprecipitated product was run on SDS-PAGE for quantification of total ALK per sample. Kinase activity was determined using a standard curve provided with the assay. Activity was normalized to the total ALK immunoprecipitated. Represented is the expression-normalized activity relative to the WT ALK control. Experiments were performed three times. One representative experiment is shown here. (C) ALK variant expressing 3T3 cell line models were treated with 50ug/mL of cycloheximide for 2, 8, 16, or 24 hours. Lysates were run on SDS-PAGE and probed for total ALK (Cell Signaling). Percent ALK remaining in each sample compared to the vehicle control treated sample is depicted below each respective lane. NOTE: Quantifications for FN1-ALK and ALK F1174L include both the high and low molecular weight bands. Experiments were repeated three times. Once representative experiment is shown here.

Article Snippet: The following primary antibodies were obtained from Cell Signaling Technology (Danvers, MA, USA): ALK mAb Rabbit (#3333), ALK mAb Mouse (3791S), ALK (D5F3) mAb Rabbit (#3633) and pALK Y1604 (#3341S).

Techniques: Variant Assay, SDS Page, Immunoprecipitation, Activity Assay, Expressing, Control, Molecular Weight

Journal: Cellular Oncology (Dordrecht)

Article Title: Anaplastic lymphoma kinase (ALK) inhibitor response in neuroblastoma is highly correlated with ALK mutation status, ALK mRNA and protein levels

doi: 10.1007/s13402-011-0048-2

Figure Lengend Snippet: ALK levels are higher in ALK mutant than wild type NBL cell lines. Higher a ALK mRNA ( p < 0.01) b ALK 220 kDa protein ( p < 0.01) and c ALK 140 kDa protein ( p < 0.01) levels in ALK mutant than WT NBL cell lines. d No significant difference of Y1604 pALK levels between mutant ( squares ) and wild type ( dots ) cell lines ( p = 0.70). MUT, ALK point mutation; WT, ALK wild type gene, including copy number gains (amplified lines are indicated as dots ). Western blots displayed in Supplementary Figure and

Article Snippet: The primary antibodies used are α-pALK Y1604 (Epitomics, Burlingame, USA), α-ALK (Thermo Fisher Scientific,Fremont, USA), α-AKT, α-pAKT Ser473, α-p44/42 MAPK, α-phospho-p44/42 MAPK, α-GAPDH and the secondary antibodies used were α-mouse and α-rabbit HRP linked.

Techniques: Mutagenesis, Amplification, Western Blot

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Activating mutations in ALK kinase domain confer resistance to structurally unrelated ALK inhibitors in NPM-ALK-positive anaplastic large-cell lymphoma

doi: 10.1007/s00432-014-1589-3

Figure Lengend Snippet: Identification of the resistance mechanism in Karpas299CR and Karpas299CHR cells. a Analysis of phosphorylation status of key signaling molecules assessed by the PathScan ® RTK Signaling Antibody Array. b Immunoblot analysis of pALK and downstream signaling proteins following 24 h of incubation in the inhibitor-free medium. c Sequencing of ALK KD coding fragment in Karpas299CR and Karpas299CHR cells

Article Snippet: Primary antibodies against pALK (Y1604), total ALK, pSTAT3 (Y705), total STAT3, pERK1/2 (T202/Y204), total ERK1/2 and pS6 (S240/244) were purchased from Cell Signaling Technology.

Techniques: Ab Array, Western Blot, Incubation, Sequencing

Journal: Journal of Cancer Research and Clinical Oncology

Article Title: Activating mutations in ALK kinase domain confer resistance to structurally unrelated ALK inhibitors in NPM-ALK-positive anaplastic large-cell lymphoma

doi: 10.1007/s00432-014-1589-3

Figure Lengend Snippet: Sensitivity of Karpas299wt and resistant cells to ALK inhibitors. a ATPlite cell viability assay following 72 h of incubation with ALK inhibitors. Data expressed as percentage viability relative to untreated controls. Corresponding IC 50 values are summarized in Table . b BrdU cell proliferation assay and flow cytometry analysis following 48-h incubation with inhibitors. Data expressed as percentage of cells in S phase relative to untreated control. c Immunoblot analysis of pALK and downstream signaling following incubation with inhibitors for 2 h

Article Snippet: Primary antibodies against pALK (Y1604), total ALK, pSTAT3 (Y705), total STAT3, pERK1/2 (T202/Y204), total ERK1/2 and pS6 (S240/244) were purchased from Cell Signaling Technology.

Techniques: Viability Assay, Incubation, BrdU Cell Proliferation Assay, Flow Cytometry, Western Blot

Journal: Cold Spring Harbor Molecular Case Studies

Article Title: Clinical response of the novel activating ALK-I1171T mutation in neuroblastoma to the ALK inhibitor ceritinib

doi: 10.1101/mcs.a002550

Figure Lengend Snippet: Characterization of ALK-I1171T with cell culture systems. ( A ) Model of the ALK kinase domain (PDB #3LCS) using PyMol, which can be divided into the upper amino-terminal lobe and the lower carboxy-terminal lobe by the hinge region and ceritinib binding/ATP (red) binding pocket. Regulatory spine (R-spine) residues are shown in magenta. The spine is anchored in the αF-helix (D1311) and includes the DFG (F1271) and the HRD motif (H1247) of the C-lobe. R-spine residues in the amino-terminal lobe include the β4 strand (C1182) and the residue mutated in this patient in the αC-helix (I1171). Mutation of residue I1171 to threonine in PyMol, results in a small shift, which potentially drives a dynamic allostery that results in a gain-of-function ALK activity ( upper insert: wild-type ALK kinase domain; lower insert: ALK-I1171T kinase domain). ( B ) ALK kinase activity and activation of downstream signaling pathways were visualized by western blot with antibodies against pALK (Y1604) and pERK1/2. Total ALK and pan-ERK were used as loading controls. Blots are representative of three independent experiments. ( C ) Neurite outgrowth of PC12 cells as a readout for ALK kinase activity was performed with wild-type ALK and ALK variants in the absence or presence of ALKAL1 ligand. Bars represent mean percentage ± STD of neurite-carrying cells among GFP-positive cells from three independent experiments. ( D ) Representative focus formation assays for NIH 3T3 cells transfected with wild-type ALK, ALK variants (ALK-I1171T and ALK-F1174L), or empty vector. ( E ) Inhibition profiling of ALK TKIs on ALK-I1171T. PC12 cells expressing ALK-I1171T were treated with serial dilution of ALK inhibitors as indicated. Phosphorylation of ALK was detected with pALK (Y1604) antibody and total ALK was used as loading control. ( F ) IC 50 values of different ALK inhibitors were calculated with GraphPad Prism 6.0 by fitting data to a log (inhibitor concentration) versus normalized response (variable slope) equation and shown in the accompanying table. Values represent average ± STD from three independent experiments.

Article Snippet: Primary antibodies used for immunoblotting were: anti-pan-ERK (1:10,000), purchased from BD Transduction Laboratories, anti-pALK (Y1604) and anti-pERK1/2 (T202/Y204) from Cell Signaling Technology.

Techniques: Cell Culture, Binding Assay, Mutagenesis, Activity Assay, Activation Assay, Western Blot, Transfection, Plasmid Preparation, Inhibition, Expressing, Serial Dilution, Concentration Assay