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

Supplementary Materials for

Phosphoproteome and gene expression profiling of ALK inhibition in

neuroblastoma cell lines reveals conserved oncogenic pathways

Jimmy Van den Eynden, Ganesh Umapathy, Arghavan Ashouri, Diana Cervantes-Madrid, Joanna Szydzik, Kristina Ruuth, Jan Koster, Erik Larsson, Jikui Guan, Ruth H. Palmer*, Bengt Hallberg*

*Corresponding author. Email: bengt.hallberg@gu.se (B.H.); ruth.palmer@gu.se (R.H.P.)

Published 20 November 2018, Sci. Signal. 11, eaar5680 (2018)

DOI: 10.1126/scisignal.aar5680

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

Other Supplementary Material for this manuscript includes the following: (available at www.sciencesignaling.org/cgi/content/full/11/557/eaar5680/DC1)

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.

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

Two ALK-positive (CLB-BAR and CLB-GE) and one Ras-positive (SK-N-AS)

neuroblastoma cell line was treated with either crizotinib or lorlatinib for 1h. Cell lysates were

immunoblotted for pALK, ALK, AKT, pAKT, ERK1/2 and pERK1/2. Actin was used as a

loading control. Bar plots show normalized quantification results for pERK1/2 as indicated

(mean ± SD). P values were calculated using a paired two-sided Student's t-test; ***P<0.005.

n=3 biological replicates.

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

(A) CLB-BAR, CLB-GE and SK-N-AS cells were treated with increasing concentrations of

crizotinib alone, linsitinib alone or a combination of both, respectively. (B) Cells were treated

with increasing concentrations of lorlatinib alone, linsitinib alone or a combination of both

respectively. (C, D) CLB-BAR, CLB-GE and SK-N-AS cells were treated with (C) a DDR1/2

inhibitor (DDR1-IN-1) or (D) an EGFR inhibitor (afatinib). Cells were treated for 72hrs. The

concentrations of the different inhibitors are indicated in the figures. Results are shown as

mean ± SD, n=3 independent experiments.

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

(A) Cell cycle analysis of CLB-BAR, CLB-GE and SK-N-AS cells treated with lorlatinib (30

nM) or crizotinib (250 nM) for 1h or 24h. Data were normalized to the respective (1h or 24h)

control. N=3 independent experiments. (B) CLB-BAR, CLB-GE, and SK-N-AS cells were

grown on six-well plates with complete growth medium, starved, and treated with crizotinib

(250 nM) and lorlatinib (30 nM) for 24h. Cell lysates were immunoblotted with antibodies

against PARP to analyze apoptosis. ERK was used as a loading control. Bar plots show

normalized quantification results for PARP as indicated (mean ± SD). P values were

calculated using a paired two-sided Student's t-test; *P<0.05, ***P<0.005. n=3 biological

replicates.

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

lines.

Correlation between the effects of both ALK inhibitors on protein phosphorylation states in

CLB-BAR (A), CLB-GE (B) and SK-N-AS (C). Each dot represents one phosphorylation

site. Tyrosine phosphorylation sites on ALK are indicated in blue. Thresholds used to

determine differential phosphorylation (-1.5/1.5) are indicated by dotted lines. Bar plots in the

right panels from (A) and (B) show the proportions of proteins with hypophosphorylated sites

belonging to the indicated reactome pathways. P values were determined using Fisher’s exact

test. (D) Venn diagram shows the overlap of proteins with decreased phosphorylation

(proteins with sites below the -1.5 log FC thresholds in response to treatment with both drugs)

between the three cell lines. Analysis based on our phosphoproteomic datasets; n=1 biological

replicate for each cell line and treatment condition and n=2 technical replicates.

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

neuroblastoma cell line.

(A) Fifty-six proteins contained minimal one site that was considered hypophosphorylated

(log FC < -1.5) after treatment with both crizotinib (left bars) and lorlatinib (right bars). If a

protein contained multiple hypophosphorylated sites, the value of the site with the lowest log

FC value is shown. (B) Sites that were only measured for one drug (meaning that data was

missing for the other drug). (C) Eighteen additional proteins were defined as being

dephosphorylated after drug treatment if data were missing for the other drug and based on a

higher log FC threshold (< -4). (D) Reactome gene set enrichment analysis on all 74 (56 + 18)

proteins that were identified to show decreased phosphorylation upon ALK inhibition. The 10

most significantly enriched pathways are shown and ranked on FDR values. Analysis based

on our phosphoproteomic datasets; n=1 biological replicate for each cell line and treatment

condition and n=2 technical replicates.

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

(A) Bar plots show the ALK mRNA normalized counts for all three cell lines in control or

ALK inhibitor conditions as indicated. (B) Correlation between the effects of both ALK

inhibitors on gene expression. ALK (blue), genes encoding factors known to be involved in

ALK signaling (green) and genes with the largest expression changes (black) are indicated.

Thresholds used to determine differential expression (-1.5/1.5) are indicated by dotted lines.

Pearson correlation coefficient is indicated on the top left. Analysis is based on our RNA-Seq

datasets, n=1.

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

neuroblastoma cell lines.

Correlation between the effects of both ALK inhibitors on gene expression states in CLB-

BAR (top), CLB-GE (middle) and SK-N-AS (bottom) cells. ALK and genes encoding factors

known to be involved in ALK signaling are labeled in blue and green respectively. Thresholds

used to determine differential phosphorylation (-1.5/1.5) are indicated by dotted lines.

Analysis is based on our RNA-Seq datasets, n=1 biological replicate.

Fig. S8. Changes in expression of genes encoding TRKs upon treatment with ALK

inhibitors in different neuroblastoma cell lines.

Bar plots indicate RNA-Seq gene expression responses (log2FC values) for all tyrosine

receptor kinases 24h after treatment with crizotinib (left) and lorlatinib (right) for CLB-BAR

(top), CLB-GE (middle) and SK-N-AS (bottom) cells as indicated. Analysis is based on our

RNA-Seq datasets, n=1 biological replicate.

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

Time course of RET phosphorylation and protein expression in CLB-BAR cells treated with

crizotinib or lorlatinib for the indicated time (h, hours). pAKT and pERK1/2 were employed

as readout of ALK signaling activity in response to treatment with either crizotinib or

lorlatinib. Bar plots show normalized quantification results for RET as indicated (mean ± SD).

P values were calculated using a paired two-sided Student's t-test; *P<0.05, **P<0.01,

***P<0.005. n=3 biological replicates..

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

transcriptional response.

(A) HPRD shortest path analysis between the 53 identified transcription factors (rows,

indicated in blue) and the 74 dephosphorylated proteins (columns, indicated in red). FDR

values are indicated by colors for each pair. Proteins with no significant connections are not

shown. Dots indicate direct connections. Color scale is shown on top left. (B) Each of 53

identified transcription factors was analyzed for enrichment of proteins with decreased

phosphorylation in their first- and second-degree connections in the HPRD network using

Fisher’s exact test. Seven transcription factors were significant at FDR 10%. For each factor

the dephosphorylated proteins in their neighborhoods are shown. For visualization purposes,

linker proteins that do not show decreased phosphorylation are not shown. Analysis based on

our phosphoproteomic datasets (n=1 biological replicate for each cell line and treatment

condition and n=2 technical replicates) together with our RNA-Seq datasets (n=1 biological

replicate).

Fig. S11. Kaplan-Meier event-free survival curves of 498 patients with neuroblastoma

from the SEQC cohort.

An ALK gene expression signature was generated from our RNA-Seq data (N=1) as a proxy

of ALK activity to segregate the SEQC cohort into two groups; a group of patients with less

predicted ALK (low, red) activity and a group with more predicted ALK (high, blue) activity.

As expected, this analysis showed that high expression of ALK correlates with poor prognosis,

but in addition suggests that this is worsened with high expression levels of (A) ETV3, (B)

ETV4 and (C) DUSP4 or low expression levels of (D) FOXO3.

Fig. S12. Phosphatase treatment of ETV3 and DUSP4.

Immunoblot analysis of CLB-BAR cell lysates treated with either lambda phosphatase

(1500U) or lorlatinib (30 nM) for 30 min. Lysates were immunoblotted for ALK, ETV3,

ERK1/2 and phospho-ERK1/2 as indicated. Tubulin was used as loading control for DUSP4.

n=3 biological replicates.

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

Log2 FC (treated/untreated) values were calculated for every analyzed amino acid site.

Columns represent protein gene names, amino acid, site location, information on whether this

site has been published or not and log2 FC values for crizotinib and lorlatinib respectively.

Sites are ranked from low to high based on log FC values after crizotinib treatment. Tab

names indicate the cell lines.

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

A reactome gene set enrichment analysis was performed on all genes encoding proteins that

had hypophosphorylated sites after treatment with both ALK TKIs in each neuroblastoma cell

line (first 3 tabs), or on all genes encoding proteins that had either hypo- or

hyperphosphorylated sites after treatment with either drug (last 12 tabs). Columns represent

the number of genes in the reactome pathway (total, not retrieved and retrieved respectively),

the percentages of not retrieved and retrieved genes that are known genes for the indicated

pathways, P values, Q values and the retrieved genes know to be active in the specified

pathway. Reactome pathways are ranked based on increasing enrichment of P values. Cell

lines and conditions are indicated by tab names.

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

Log2 FC (treated/untreated) expression values were calculated for all genes that were expressed in

each cell line. Column names represent log2 FC values after crizotinib and lorlatinib treatment

respectively. Expression responses are ranked from low to high based on log2 FC values after

crizotinib treatment. Tab names indicate the cell lines.

Data File S4. Transcription factor GSEA results on 764 differentially expressed genes in

the CLB-BAR cell line.

The different transcription factor target databases that were used are indicated by tab names.

Columns represent the number of targets (total, not differentially expressed and differentially

expressed respectively), the proportion of not differentially expressed and differentially

expressed genes that are known targets for the indicated transcription factor, P values, Q

values and the differentially expressed genes that are known targets. Transcription factors are

ranked based on increasing enrichment P values.

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

Each of 48 predicted transcription factors that were present in the HPRD network were

analyzed for enrichment of proteins with decreased phosphorylation in their first- and second-

degree connections in the HPRD network using Fisher’s exact test. Columns represent the

proportion of proteins with or without decreased phosphorylation that are first- or second-

degree connections of the transcription factor, P values, Q values and the proteins with

decreased phosphorylation that are first- or second-degree connections. Transcription factors

are ranked based on increasing enrichment P values.

Data File S6. Cell Signaling Technology Phosphoscan raw data.

Raw signal intensities and log2 fold change values for each drug (crizotinib or lorlatinib) and

antibody (pTyr or pSer/Thr) as indicated by tab names.