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Integrated proximal proteomics reveals IRS2 as a determinant of cell survival in ALK-driven neuroblastoma

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Science Signaling  20 Nov 2018:
Vol. 11, Issue 557, eaap9752
DOI: 10.1126/scisignal.aap9752
  • Fig. 1 Multilayered proteomics approach to study potentially druggable ALK signaling in NB cells.

    (A) Cell viability of NB1 NB cells in response to treatment (48 hours) with different concentrations of crizotinib, LDK378, and TAE684. Data are presented as means ± SEM of n = 3 to 6 independent experiments. (B and C) Lysates from NB1 cells treated with either dimethyl sulfoxide (DMSO) or LDK378 (250 nM) for different times and immunoprecipitated (IP) for ALK (B) or immunoblotted (B and C) as indicated (n = 3 independent experiments). p, phospho. Arrows indicate protein variants as previously described (8486). (D) Schematic representation of the proteomics strategy using crizotinib, LDK378, and TAE684 to inhibit constitutive ALK signaling in NB1 cells. Drug-induced changes in the ALK interactome and phosphoproteome were measured after 30 min of inhibitor treatment including mapping of the ALK phosphotyrosine interactome and proteome analysis of untreated NB1 cells. (E) Overview of results from ALK interactome (yellow; n = 2 independent experiments for each inhibitor; significance B test, P < 0.05), phosphotyrosine interactome [green; n = 4 pull-downs for each pY-peptide (bait) and non–p-peptide (control); t test for significance, P < 0.05, S score > 1], phosphoproteome analysis (blue; n = 2 independent experiments for each inhibitor), and proteome (pink; n = 2 independent experiments). aa, amino acids. (F) Number of total regulated phosphorylation sites by amino acid distribution [determined as previously described (28)]. (G) Overlap between the TKI-regulated phosphoproteome and the identified and quantified proteome (top) and adaptors with decreased ALK association (interactome) upon TKI treatment (bottom). See also figs. S1 and S2 and data files S1 to S4.

  • Fig. 2 Proximal signaling proteomics reveals drug-sensitive ALK adaptors.

    (A) Four integrated heatmaps representing results from the ALK interactome, phosphotyrosine interactome, phosphoproteome, and proteome analysis. The list includes ALK and the 30 most TKI-sensitive (as assessed by decreased association) ALK adaptor proteins (shown by gene name) and displays the median log2 SILAC ratio (yellow) from the ALK interactome analysis, the number of phosphorylation sites (blue; phosphoproteome) with significantly decreased ratios in response to at least two inhibitors, the relative protein abundance by iBAQ value (pink; proteome), and the significantly associated with phosphotyrosine-specific binding (green; phosphotyrosine interactome; P < 0.05 and in red S score > 1). (B) Overview of site-specific phosphotyrosine interactions. Data are a graphical summary of fig. S3. TKI-sensitive and TKI-insensitive adaptors are highlighted. Only proteins containing SH2, PTB, and PI domains are included. Each square indicates a median SILAC ratio for the indicated ALK tyrosine residue. (C) Functional association network based on STRING and visualized by Cytoscape. ALK is gray, and IRS2 has a pink halo. (D) Significantly overrepresented GO terms for biological process among proteins listed in (A). (E and F) Lysates from NB1 cells treated with either DMSO, crizotinib, TAE684, or LDK378 for 30 min and immunoprecipitated for ALK (E) or IRS2 (F) and immunoblotted as indicated (n = 3 independent experiments). (G) Volcano plot showing the phosphotyrosine-specific interactors of the phosphorylated IRS2 Tyr675-containing peptide. SH2, PI, PTB, and Cbl-like PTB domain–containing proteins are indicated by gene name and a star. Log2 ratios of fold change of the median intensities of pull-downs (n = 4 independent experiments) of phosphorylated peptide (bait) versus nonphosphorylated peptide (control) (x axis) are plotted versus −log10 of the P values derived from a t test. Significant associations are represented above the S curve. See also fig. S3 and data files S1 and S2.

  • Fig. 3 Phosphoproteomics identifies ALK-driven phosphorylation of FoxO3.

    (A) Overlap in number of identified and quantified phosphorylation sites in NB1 cells treated with TAE684, LDK378, and crizotinib. (B) Kinase substrate motif enrichment analysis (Fisher’s exact test) including phosphorylation sites common to at least two of three inhibitors and displaying either decreased (697 sites) or increased (634 sites) ratio in response to inhibitors. (C) Sequence motif analysis by iceLogo of the ±6 amino acid residues flanking the regulated phosphorylation site (left: tyrosine specific; right: serine/threonine) compared to sites (tyrosine, serine, and threonine) that are not regulated. (D) KEGG pathway enrichment analysis (Fisher’s exact test) for proteins with sites displaying a decreased and increased ratio in response to TKI treatment. (E) Overview of phosphorylation regulation of transcription factors ranked according to their five most prominently decreased phosphorylation sites in response to ALK TKIs. Each square corresponds to ALK inhibitor–induced changes in phosphorylation sites as indicated. (F) Lysates from NB1 cells treated with either DMSO, crizotinib, TAE684, or LDK378 for 30 min and immunoblotted as indicated (n = 3 independent experiments). See also fig. S4. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

  • Fig. 4 ALK inhibition by lorlatinib and ALK depletion by siRNA in NB1 cells reduces IRS2, Akt, FoxO3, and ERK phosphorylation.

    (A) Cluster dendrogram of the TMT 11-plex phosphoproteomics data showing the relation between analyzed samples: 10 nM lorlatinib–treated (lorlatinib low conc.) cells, 10 μM lorlatinib–treated (lorlatinib high conc.) cells, and ALK-depleted cells to siRNA control and DMSO-treated cells (see also fig. S7C). Hierarchical clustering was performed in Perseus using quantile-based normalized intensities for identified and quantified phosphorylated peptides. (B and C) Volcano plots of −log10 transformed false discovery rate (FDR)–corrected P values versus log2(fold change) of median phosphorylation site intensities measured for NB1 cells upon low-dose lorlatinib (10 nM) (B) and siRNA depletion of ALK (C) as measured by TMT multiplexing analysis and MS. Fold change in (B) represents low-dose lorlatinib–treated NB1 cells (n = 2) compared to DMSO-treated cells (n = 3). Fold change in (C) represents ALK depletion by two different siRNA ALK-targeting sequences as well as their mix (n = 3) compared to control siRNA (siCTRL) cells (n = 3). Statistical analysis was performed for n = 2 to 3 independent experiments by two-sided t test, and significance was determined on the basis of an FDR of <0.05 and hyperbolic curve threshold of s0 = 0.1 using Perseus. (D) Overlap between significantly down-regulated phosphorylated sites for the conditions comparing 10 nM lorlatinib–treated (lorlatinib low conc.) cells to DMSO-treated cells, 10 μM lorlatinib–treated cells to DMSO-treated cells, and ALK-depleted cells to siRNA control cells. See also fig. S7 and data file S5.

  • Fig. 5 ALK regulates NB cell survival through the IRS2-FoxO3 axis.

    (A) Lysates from NB1, SH-SY5Y, and CLBGA cells depleted for IRS2 using siRNA and immunoblotted as indicated in (A) and quantified in (B). Blots are representative of n = 3 to 4 independent experiments. (C and D) Cell viability (C) and caspase activity normalized to cell viability (D) upon siRNA-mediated depletion of IRS2. (E) Immunoblots of cleaved caspase-3 and quantification for NB1 and SH-SY5Y cells upon IRS2 depletion. Blots are representative of n = 3 to 4 independent experiments. Data are represented relative to siRNA control (C or siCTRL) for each cell line, and values are presented as means ± SD of n = 3 to 4 (A, B, and E) or n = 4 to 5 (C and D) independent experiments. *P < 0.05, **P < 0.01, and ***P < 0.001 compared with siRNA control (one-sample t test on log-transformed fold changes relative to siRNA control). See also fig. S9.

  • Fig. 6 The constitutive ALK signaling network in NB1 cells.

    Model summarizing main findings from our integrated proteomics approach to unravel oncogenic ALK signaling in NB1 cells linking IRS2 to survival signaling (highlighted in dark orange). ALK interactors belonging to the GO terms “transmembrane RTK signaling pathway” and “insulin receptor signaling pathway” (Fig. 2D) are represented (light orange) together with proteins belonging to the KEGG pathways “ErbB signaling,” “neurotrophin signaling,” and “insulin signaling” from the down-regulated phosphoproteome (Fig. 3D). Transcription factors are represented on the basis of their relation to the KEGG pathways or their phosphorylation regulation (Fig. 3E; more than two regulated phosphorylation sites). ALK TKI–sensitive nodes are highlighted by red boxes. Arrows indicate activation, T-bars indicate inhibition, and dotted arrows indicate translocation.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/11/557/eaap9752/DC1

    Fig. S1. Effect of crizotinib and TAE684 on NB1 cells and experimental workflow.

    Fig. S2. Quality control for ALK interactome, phosphoproteome, and proteome data.

    Fig. S3. Volcano plot representation of ALK phosphotyrosine interactome data.

    Fig. S4. Phosphoproteomics analysis of pathway regulation and sequence motif enrichment analysis.

    Fig. S5. Differential responses to LDK378, lorlatinib, U0126, and LY294002 in ALK mutant cell lines.

    Fig. S6. Effects of dasatinib and KD025 treatment alone and in combination with LDK378.

    Fig. S7. Analysis of signaling changes upon siRNA depletion of ALK or lorlatinib treatment in NB1 cells.

    Fig. S8. Gene expression data for ALK, IRS2, and FOXO3 in various cancer cell lines.

    Fig. S9. Effects of IRS2 depletion in NB cells.

    Data File S1. Summary of ALK interactome data from NB1 cells treated with crizotinib, TAE684, and LDK378 for 30 min.

    Data File S2. Summary of ALK and IRS2 phosphotyrosine interactome data.

    Data File S3. Summary of phosphoproteomics data from NB1 cells treated with crizotinib, TAE684, and LDK378 for 30 min.

    Data File S4. Summary of NB1 cell line proteome data.

    Data File S5. Summary of phosphoproteomics data by TMT 11-plex analysis upon siRNA depletion of ALK or ALK inhibition with low- and high-dose lorlatinib in NB1 cells.

  • The PDF file includes:

    • Fig. S1. Effect of crizotinib and TAE684 on NB1 cells and experimental workflow.
    • Fig. S2. Quality control for ALK interactome, phosphoproteome, and proteome data.
    • Fig. S3. Volcano plot representation of ALK phosphotyrosine interactome data.
    • Fig. S4. Phosphoproteomics analysis of pathway regulation and sequence motif enrichment analysis.
    • Fig. S5. Differential responses to LDK378, lorlatinib, U0126, and LY294002 in ALK mutant cell lines.
    • Fig. S6. Effects of dasatinib and KD025 treatment alone and in combination with LDK378.
    • Fig. S7. Analysis of signaling changes upon siRNA depletion of ALK or lorlatinib treatment in NB1 cells.
    • Fig. S8. Gene expression data for ALK, IRS2, and FOXO3 in various cancer cell lines.
    • Fig. S9. Effects of IRS2 depletion in NB cells.
    • Legends for data files S1 to S5

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Data File S1 (Microsoft Excel format). Summary of ALK interactome data from NB1 cells treated with crizotinib, TAE684, and LDK378 for 30 min.
    • Data File S2 (Microsoft Excel format). Summary of ALK and IRS2 phosphotyrosine interactome data.
    • Data File S3 (Microsoft Excel format). Summary of phosphoproteomics data from NB1 cells treated with crizotinib, TAE684, and LDK378 for 30 min.
    • Data File S4 (Microsoft Excel format). Summary of NB1 cell line proteome data.
    • Data File S5 (Microsoft Excel format). Summary of phosphoproteomics data by TMT 11-plex analysis upon siRNA depletion of ALK or ALK inhibition with low- and high-dose lorlatinib in NB1 cells.

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