Data on quantification of signaling pathways activated by KIT and PDGFRA mutants

The present data are related to the article entitled “Insights into ligand stimulation effects on gastro-intestinal stromal tumors signaling” (C. Bahlawane, M. Schmitz, E. Letellier, K. Arumugam, N. Nicot, P.V. Nazarov, S. Haan, 2016) [1]. Constitutive and ligand-derived signaling pathways mediated by KIT and PDGFRA mutated proteins found in gastrointestinal stromal tumors (GIST) were compared. Expression of mutant proteins was induced by doxycycline in an isogenic background (Hek293 cells). Kit was identified by FACS at the cell surface and found to be quickly degraded or internalized upon SCF stimulation for both Kit Wild type and Kit mutant counterparts. Investigation of the main activated pathways in GIST unraveled a new feature specific for oncogenic KIT mutants, namely their ability to be further activated by Kit ligand, the stem cell factor (scf). We were also able to identify the MAPK pathway as the most prominent target for a common inhibition of PDGFRA and KIT oncogenic signaling. Western blotting and micro-array analysis were applied to analyze the capacities of the mutant to induce an effective STATs response. Among all Kit mutants, only Kit Ex11 deletion mutant was able to elicit an effective STATs response whereas all PDGFRA were able to do so.


a b s t r a c t
The present data are related to the article entitled "Insights into ligand stimulation effects on gastro-intestinal stromal tumors signaling" (C. Bahlawane, M. Schmitz, E. Letellier, K. Arumugam, N. Nicot, P.V. Nazarov, S. Haan, 2016) [1]. Constitutive and ligandderived signaling pathways mediated by KIT and PDGFRA mutated proteins found in gastrointestinal stromal tumors (GIST) were compared. Expression of mutant proteins was induced by doxycycline in an isogenic background (Hek293 cells). Kit was identified by FACS at the cell surface and found to be quickly degraded or internalized upon SCF stimulation for both Kit Wild type and Kit mutant counterparts. Investigation of the main activated pathways in GIST unraveled a new feature specific for oncogenic KIT mutants, namely their ability to be further activated by Kit ligand, the stem cell factor (scf). We were also able to identify the MAPK pathway as the most prominent target for a common inhibition of PDGFRA and KIT oncogenic signaling. Western blotting and microarray analysis were applied to analyze the capacities of the mutant to induce an effective STATs response. Among all Kit mutants, only Value of the data Expression of different mutant proteins (Kit and PDGFRA) in an isogenic background to allow a direct comparison of their signalling capacities, without the complex patient specific-background. The constructs and/or the Hek293 cell lines, could be used for further molecular characterization, protein-protein interaction experiments or protein localization studies. Identified mutations in GIST could easily be investigated by insertion of the newly discovered mutation in the WT constructs by site-directed mutagenesis. Plasmids are available upon request.
Identification of a new feature specific to Kit mutants: their ability to be further stimulated by their natural ligand, in addition to their constitutive activation derived from the mutations observed in GIST.
A new MEK inhibitor (PD0325901) was identified to be efficient in inhibiting GIST cell proliferation in the nanomolar range.

Data
The data presented here derived mainly from western blot analysis for the quantification of signaling pathways activated by KIT and PDGFRA mutants, from micro-array analysis for quantification of changes in gene expression levels induced by the different mutations and from flow cytometry analysis for the detection of KIT at the cell surface.
Doxycycline induces Kit expression by a factor 100 in all cell lines and comparable mRNA expression levels were observed for all mutants (Fig. 1a). However, some differences in the protein expression levels were observed between the different constructs (Fig. 1b).
The ratio of surface ( Fig. 1a in [1]) to total KIT expression ( Fig. 1b in [1]) indicates that KIT WT is expressed almost exclusively at the surface, while this is the case for 70% of KIT Ex9. This value drops to 50% for both Ex11 mutants. The results of the FACS analysis after SCF stimulation (Fig. 2) indicate the decrease of KIT expression at the cell surface for all KIT mutants and wild type following stimulation with KIT ligand, SCF.    Nuclear extracts were prepared as previously done [4], diluted in 4 times Laemmli buffer and subjected to Western blot analysis. Phosphorylation status of STAT5, STAT3 and STAT1 is shown for nuclear extract prepared from Hek293 cells expressing KIT WT, KIT Ex11, KIT Ex9 and KIT V559D. (b) Induction of known STAT target genes by GIST mutants. Gene expression level of known STAT target genes, previously identified to be induced in PDGFRA GIST mutants [3], was retrieved from micro-array data and presented as heat map. Grey boxes indicate that the genes are not part of the DEG list for the corresponding mutant (FDR 40.05 or AbsFC o 0.5). The intensity of the red color corresponds to the value of the ratio to the background (Hek293 KIT WT non-stimulated for KIT WT and mutants and Hek293 PDGFRα non-stimulated for PDGFRα mutants). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article) .
The Mean Fluorescence Intensity (MFI) of Kit staining in the different Kit mutants are indicated in Table 1.
While PDGFRα, Akt, and Erk phosphorylation were induced by PDGFAA in PDGFRaWT, phosphorylation of PDGFRα, Akt, Erk and STAT5 remained identical to the non-stimulated control for the PDGFRA mutant V591D, as previously shown [3]. In contrast, Kit mutants exhibit all constitutive phosphorylation of kit at tyrosine 703 but the signal intensities for Erk, Akt and STAT5 phosphorylation was further increased upon SCF stimulation ( Fig. 3a and Fig. 3b).
STATs translocation to the nucleus was identified for Kit Ex11 deletion mutant and for Kit Ex9 duplication mutant to a lower extend. Induction of gene expression known to be part of the STAT pathway was found for Kit Ex11 deletion mutant after SCF stimulation as well as for Kit Ex9 duplication mutant upon SCF stimulation to a lower extend ( Fig. 4a and Fig. 4b).
We investigated KIT Ex11 specific gene signature, comparing KIT Exon 11 deletion mutant regulated genes to other GIST mutants (PDGFRA mutants and KIT Exon 9). 277 genes (Table 3) were differentially expressed in KIT Ex11 deletion mutant only. As noted in [1], these genes were associated with "cell cycle" and "insulin signalling pathway".
Both inhibitors were added to the medium 24 h after seeding for 30 h. Both drugs were added individually and in combination with a constant ratio of 1:1 (Fig. 5). Viability was assessed using PrestoBlue following the manufacturer's recommendation. Results were analysed using the Compusyn software [5] and the combination Index (CI) values are represented as a function of the percentage of inhibition. CI values below 1 indicates a synergy between the two compounds, while CI values above 1 indicates antagonism.
qPCR primers are listed in Table 2.

Flow cytometry analysis
Cell surface expression of KIT wild type and mutants was analyzed by flow cytometry using a FACS CantoII Instrument (Becton Dickinson, Heidelberg, Germany) either without ligand (blue lines) or 15 minutesmin after stimulation with 100ng/ml100 ng/ml SCF (pink lines). Cells were then either incubated with 10 μL KIT primary antibody (anti-CD117-APC conjugated; C7244; Dako, Belgium) for cell surface expression. Specificity was controlled using an isotype-matched/ APC conjugated antibody (grey).

Microarray analysis
293FR cells expressing KIT-WT, KIT Ex11 deletion mutant and KIT Ex9 mutant were incubated with 5 ng/ml doxycycline and 100 ng/ml SCF for 21 h in DMEM with 1%FCS. Cells were then starved for 3 h (without FCS) and further stimulated with SCF. RNAs of three biological replicates were isolated using the miRNeasy Mini KIT (Qiagen) according to manufacturer's instructions with additional on-column DNase I digestion. RNA quality and purity were assessed using a Nanodrop Spectrophotometer (Thermo Scientific) and Agilent 2100 Bioanalyzer (Nano KIT). Gene Expression analysis was performed using GeneChip Human Gene ST 2.0 arrays (Affymetrix). Quality control and data normalization were performed as previously reported [3,7]. We focused on differentially expressed genes (DEG) across the mutants comparing with non-stimulated KIT-WT. To exclude non-relevant lowly expressed transcript clusters, only those showing log2 expression above 4.5 were kept for further analysis. Transcript clusters were further summarized in order to obtain a single expression value for each gene in each experiment. The differentially expressed genes were statistically evaluated by two-factor linear model with empirical Bayes statistics approach using limma package of R/Bioconductor [8]. In order to correct for the false discovery rate (FDR), the Benjamini and Hochberg step-up method correction was applied. Probe-sets with FDR o0.05 and absolute fold change 40.5 were considered to be significantly differentially expressed (DEGs). Microarray data are available in the ArrayExpress database (www.ebi.ac. uk/arrayexpress) under accession number E-MTAB-4548. KIT Ex11 specific gene signature was determined by comparing KIT Exon 11 deletion mutant regulated genes to other GIST mutants (PDGFRA mutants and KIT Exon 9). 277 genes.

Nuclear extract preparation
Nuclear extracts were prepared as previously done [4]. In brief, cells were washed with ice-cold PBS, harvested gently with cell scraper and centrifuged at 4°C for 5 min, 4000 rpm. The pellet was then resuspend in Buffer A (10 mM Hepes/KOH pH7.9, 1.5 mM MgCl2 and 10 mM KCl). Following 10 min incubation on ice, samples were centrifuged at maximum speed, 4°C for 5 min. The operation was performed a second time and the pellets were finally resuspended in Buffer C for nuclear extraction (20 mM Hepes/KOH pH7.9, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA and 25% glycerol). Protein concentrations were determined using Bradford reagent before analysis by western blotting.

Synergy analysis
Synergy between Receptor Tyrosine kinase (RTK) and MEK inhibitors were performed at constant ratio, as recommended by Chou et al. [5,9]. GIST primary cells were seeded in 96 well plates 24 h prior Table 3 KIT Ex11 deletion mutant specific DEG, with FDRo 0.05 and absolute logFC4 0.5.