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Phosphoproteome and gene expression profiling of ALK inhibition in neuroblastoma cell lines reveals conserved oncogenic pathways

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Science Signaling  20 Nov 2018:
Vol. 11, Issue 557, eaar5680
DOI: 10.1126/scisignal.aar5680
  • Fig. 1 ALK inhibition experimental setup.

    (A) Three different neuroblastoma cell lines (CLB-BAR, CLB-GE, and SK-N-AS) were cultured in control conditions or in the presence of the ALK inhibitors crizotinib or lorlatinib. Cells were harvested for tyrosine (pTyr) and serine/threonine (pSer and pThr) phosphoproteomic analysis or RNA-seq analysis after 1 and 24 hours, respectively. Pie charts show the number of targeted sites for each drug. n = 1 biological replicate with 2 technical replicates for each cell line and treatment condition. (B) Boxplots show raw ALK phosphorylation signal intensities of all targeted ALK sites in untreated cells or in cells treated with the indicated ALK inhibitor. Phosphorylation signal was not detected in SK-N-AS cells. P values were calculated using Wilcoxon rank sum test. (C) Bar plots show the ALK mRNA normalized counts for all three cell lines in control or ALK inhibitor conditions as indicated, as determined by RNA-seq analysis. n = 1 biological replicate for each cell line and treatment condition.

  • Fig. 2 Phosphoproteomic analysis after ALK inhibition in CLB-BAR cells.

    (A) Correlation between the effects of the ALK inhibitors crizotinib and lorlatinib on protein phosphorylation states. Each dot represents a phosphorylation site. Targeted ALK sites are indicated in blue. Proteins with the largest response to both drugs are indicated with the targeted site in parentheses. Thresholds used to determine differential phosphorylation (−1.5/1.5) are indicated by dashed lines. (B) Reactome GSEA on all 56 proteins that were identified with decreased phosphorylation levels upon ALK inhibition. The 10 most significantly enriched pathways are shown and ranked on the basis of FDR values. GSEA was performed using Fisher’s exact test. Results from the complete analysis are available in data file S2. (C) Schematic overview of ALK showing the tyrosine residues in the ALK intracellular domain that are modulated in response to ALK inhibitor treatment. (D) Effect of ALK inhibition on other receptor tyrosine kinases (RTKs). Median values are indicated by horizontal dashed lines. n = 1 biological replicate with 2 technical replicates for each cell line and treatment condition.

  • Fig. 3 Gene expression analysis 24 hours after ALK inhibition in CLB-BAR cells.

    (A) Correlation between the effects of both ALK inhibitors on gene expression. ALK (blue), genes encoding factors involved in ALK signaling (green), and genes with the largest expression changes (black) after treatment with both drugs are labeled. Thresholds used to determine differential expression (−1.5/1.5) are indicated by dashed lines. Pearson correlation coefficient is indicated on the top left of the plot. (B) Overlap of differentially expressed genes with the 77-gene ALK signature as described for TAE684 by Lambertz et al. (43). The Venn diagram shows the overlap with genes differentially expressed upon crizotinib and lorlatinib treatment. The bar plot shows the proportions of this gene signature that are differentially expressed after ALK TKI treatment as indicated. Note that only 75 coding genes from the 77-gene signature dataset were used for analysis (the long noncoding RNAs MALAT1 and NEAT1 were excluded). (C) GSEA on all 764 genes that were differentially expressed upon ALK inhibition using four different transcription factor target databases. The five most significantly enriched transcription factors are shown and ranked on FDR values. Results from the complete analysis are available in data file S4. TRANSFAC, TRANScription FACtor Database; TRRUST, Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining. (D) Differential expression of MYCN targets as determined by GSEA. Bar plots indicate the proportion of MYCN targets (+) and nontargets (−), derived from different databases, as indicated on top, that were found to be differentially expressed. P values were calculated using Fisher’s exact test. n = 1 biological replicate for each cell line and treatment condition.

  • Fig. 4 Interaction between the signaling proteins with decreased phosphorylation and transcription factors in the HPRD PPI network.

    Seventy-four proteins with decreased phosphorylation (red) and 53 transcription factors (blue) were mapped to the HPRD protein interaction network. For visualization purposes, only direct interactions between the identified proteins are shown, with the exception of MYCN. Genes with increased abundance (red borders) and decreased abundance (blue borders) are also indicated. A protein was considered to show decreased phosphorylation when it contained sites with decreased phosphorylation signals in both crizotinib- and lorlatinib-treated cells (log2 FC threshold of −1.5; 56 proteins) or when it contained nonphosphorylated sites in cells treated with either drug when the corresponding site was not measured for the other drug (in this case, log2 FC threshold of −4; 18 additional proteins).

  • Fig. 5 Oncogenic ALK regulates ETV3 and ETV4 in neuroblastoma.

    (A and B) Kaplan-Meier event-free survival curves of 498 patients with neuroblastoma from the SEQC cohort stratified according to ETV3 and ETV4 expression. Patients with higher expression are highlighted in blue, whereas patients with lower expression are highlighted in red. The log-rank test P values are indicated. (C) Immunoblotting of lysates from IMR-32 cells pretreated with crizotinib and stimulated with ALKAL1 for 30 min and 6 hours. Actin served as the loading control. n = 3 biological replicates. (D) Western blotting for the indicated proteins in lysates from ALK-positive neuroblastoma cell lines CLB-BAR and CLB-GE treated with crizotinib or lorlatinib for the indicated times. n = 3 biological replicates. (E) CLB-BAR cells were transfected with siRNAs targeting ETV3. Proliferation relative to day 0 and relative to scrambled control transfected cells (SiC) was analyzed after 3 and 6 days using resazurin assay. Data are means ± SD from three independent experiments. RFU, relative fluorescence units. (F) CLB-BAR cells were transfected with either scrambled control or three independent siRNAs targeting ETV3. Lysates were immunoblotted for ALK, pALK, ETV3, and MYCN, and actin was used as a loading control. n = 3 biological replicates. (G) CLB-BAR cells were transfected with siRNAs targeting ETV4. Proliferation relative to day 0 and relative to scrambled control transfected cells was analyzed after 2, 4, and 6 days using resazurin assay. Data are means ± SD from three independent experiments. (H) CLB-BAR cells were transfected with either scrambled control or three independent siRNAs targeting ETV4. Lysates were immunoblotted for ALK, pALK, ETV4, and MYCN, and actin was used as a loading control. n = 3 biological replicates. Immunoblot quantification bar plots show means ± SD. **P < 0.01, ***P < 0.005, paired two-sided Student’s t test.

  • Fig. 6 Lorlatinib reduces DUSP4 protein levels in an ALK-dependent manner.

    (A) Kaplan-Meier event-free survival curves of 498 patients with neuroblastoma from the SEQC cohort stratified according to DUSP4 expression. Patients with higher expression are highlighted in blue, whereas patients with lower expression are highlighted in red. The log-rank test P value is indicated. (B) Immunoblotting of lysates from CLB-BAR, CLB-GE, and SK-N-AS cells treated with lorlatinib for 30 min and 6 hours. n = 3 biological replicates. (C) Immunoblots from IMR-32 cells stimulated with ALKAL1 ligand for the specified times. n = 3 biological replicates. Immunoblot quantification bar plots show means ± SD. *P < 0.05, ***P < 0.005, paired two-sided Student’s t test.

  • Fig. 7 ALK regulates subcellular localization of FOXO3.

    (A) Schematic representation of FOXO relocalization. (B) Kaplan-Meier event-free survival curves of 498 patients with neuroblastoma from the SEQC cohort stratified according to FOXO3 with event-free survival probability. Patients with higher expression are highlighted in blue, whereas patients with lower expression are highlighted in red. The log-rank test P value is indicated. (C and D) Immunofluorescence staining for both CLB-BAR and CLB-GE neuroblastoma cell lines for FOXO3a with or without lorlatinib and quantification. n = 3 biological replicates. Immunofluorescence quantification bar plots show means ± SD. *P < 0.05, ***P < 0.005, paired two-sided Student’s t test. Scale bars, 10 μm. DAPI, 4′,6-diamidino-2-phenylindole.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/557/eaar5680/DC1

    Fig. S1. Immunoblot validation of response to ALK inhibition in neuroblastoma cells.

    Fig. S2. RTK inhibition effects on three neuroblastoma cell lines.

    Fig. S3. Cell cycle analysis and apoptosis analyses upon treatment with ALK inhibitors.

    Fig. S4. Phosphoproteomic analysis after ALK inhibition in different neuroblastoma cell lines.

    Fig. S5. Identification of 74 proteins with decreased phosphorylation in the CLB-BAR neuroblastoma cell line.

    Fig. S6. ALK RNA-seq expression analysis early (1 hour) after ALK inhibition.

    Fig. S7. RNA-seq gene expression analysis 24 hours after ALK inhibition in different neuroblastoma cell lines.

    Fig. S8. Changes in expression of genes encoding TRKs upon treatment with ALK inhibitors in different neuroblastoma cell lines.

    Fig. S9. Effect of ALK inhibition on RET phosphorylation and expression.

    Fig. S10. Integrative analysis of ALK-induced phosphorylation signaling and transcriptional response.

    Fig. S11. Kaplan-Meier event-free survival curves of 498 patients with neuroblastoma from the SEQC cohort.

    Fig. S12. Phosphatase treatment of ETV3 and DUSP4.

    Data File S1. Phosphoproteomic responses 1 hour after ALK TKI treatment.

    Data File S2. Reactome GSEA on genes encoding differentially phosphorylated proteins.

    Data File S3. Expression responses 24 hours after ALK TKI treatment.

    Data File S4. Transcription factor GSEA results on 764 differentially expressed genes in the CLB-BAR cell line.

    Data File S5. Transcription factor nearest-neighborhood analysis results in CLB-BAR.

    Data File S6. Cell Signaling Technology Phosphoscan raw data.

  • The PDF file includes:

    • Fig. S1. Immunoblot validation of response to ALK inhibition in neuroblastoma cells.
    • Fig. S2. RTK inhibition effects on three neuroblastoma cell lines.
    • Fig. S3. Cell cycle analysis and apoptosis analyses upon treatment with ALK inhibitors.
    • Fig. S4. Phosphoproteomic analysis after ALK inhibition in different neuroblastoma cell lines.
    • Fig. S5. Identification of 74 proteins with decreased phosphorylation in the CLB-BAR neuroblastoma cell line.
    • Fig. S6. ALK RNA-seq expression analysis early (1 hour) after ALK inhibition.
    • Fig. S7. RNA-seq gene expression analysis 24 hours after ALK inhibition in different neuroblastoma cell lines.
    • Fig. S8. Changes in expression of genes encoding TRKs upon treatment with ALK inhibitors in different neuroblastoma cell lines.
    • Fig. S9. Effect of ALK inhibition on RET phosphorylation and expression.
    • Fig. S10. Integrative analysis of ALK-induced phosphorylation signaling and transcriptional response.
    • Fig. S11. Kaplan-Meier event-free survival curves of 498 patients with neuroblastoma from the SEQC cohort.
    • Fig. S12. Phosphatase treatment of ETV3 and DUSP4.
    • Legends for Data files S1 to S6

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data File S1 (Microsoft Excel format). Phosphoproteomic responses 1 hour after ALK TKI treatment.
    • Data File S2 (Microsoft Excel format). Reactome GSEA on genes encoding differentially phosphorylated proteins.
    • Data File S3 (Microsoft Excel format). Expression responses 24 hours after ALK TKI treatment.
    • Data File S4 (Microsoft Excel format). Transcription factor GSEA results on 764 differentially expressed genes in the CLB-BAR cell line.
    • Data File S5 (Microsoft Excel format). Transcription factor nearest-neighborhood analysis results in CLB-BAR.
    • Data File S6 (Microsoft Excel format). Cell Signaling Technology Phosphoscan raw data.

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