*2.2. PDGFR-Beta, EGFR and ERK1/2 Kinases Are Highly Phosphorylated in Cells Harboring c.1681C>T (p.R561C) Mutation in PDGFRB*

Given that both siblings and NSTS-47 cells harbor the c.1681C>T (p.R561C) mutation in *PDGFRB* and that PDGFR-beta c.1681C>T (p.R561C) mutants are constitutively phosphorylated and can activate various signaling pathways [21], we assessed the phosphorylation level of 49 RTKs and 26 other signaling proteins in tumor samples as well as in NSTS-47 cells. NSTS-47 cells were harvested, and phosphorylation levels were analyzed after cultivation for 24 h in Dulbecco's modified Eagle's medium (DMEM) without fetal calf serum (FCS) to eliminate the effects of various serum growth factors on the phosphorylation of the studied proteins. The screening of all 75 proteins showed that PDGFR-beta, EGFR (Figure 1) and ERK1/2 (Figure 2) kinases exhibited very high levels of phosphorylation in all samples. High levels of phosphorylation were also observed for ROR2, AXL (Figure 1), HSP27 and p38-gamma (Figure 2). These results confirmed that some kinases (namely, PDGFR-beta, EGFR and ERK1/2) were constitutively activated, as the high phosphorylation levels of these proteins were easily detectable in both tumor samples and in NSTS-47 cells after cultivation under serum-free conditions for 24 h.

**Figure 1.** Phospho-receptor tyrosine kinases (RTK) array analysis. The relative phosphorylation of 49 RTKs was analyzed in tumor tissue obtained from the boy when he was 3.5 months old (Tumor sample 1), in the NSTS-47 cell line (derived from a tumor tissue of the boy obtained when he was 1 year and 7 months old) and in the tumor tissue of his 8-year-old sister (Tumor sample 2). platelet-derived growth factor receptor beta (PDGFR-beta) and epidermal growth factor receptor (EGFR) exhibited high levels of phosphorylation in all cases. Phosphorylation in NSTS-47 cells was measured after 24 h of serum-free cultivation. The array images captured using X-ray film are shown for each sample, and the five most phosphorylated receptor tyrosine kinases (RTKs) are marked. The upper part of the figure (Tumor sample 1) was already published in our previous case report [8] under the Creative Commons Attribution 4.0 International License.

**Figure 2.** Phospho-mitogen-activated protein kinase (MAPK) array analysis. The relative phosphorylation of 26 signaling proteins, including 9 MAPKs, was detected in tumor tissue obtained from the boy when he was 3.5 months old (Tumor sample 1), in the NSTS-47 cell line (derived from a tumor tissue of the boy obtained when he was 1 year and 7 months old) and in the tumor tissue of his 8-year-old sister (Tumor sample 2). ERK1/2 exhibited high levels of phosphorylation in all cases. Phosphorylation levels in NSTS-47 cells was measured after 24 h of serum-free cultivation. The array images captured using X-ray film are shown for each sample, and the five most phosphorylated proteins are marked.

### *2.3. NSTS-47 Cells Are Sensitive to Sunitinib and Erlotinib*

It was confirmed that cells with the mutation c.1681C>T (p.R561C) in *PDGFRB* are sensitive to the tyrosine kinase inhibitors imatinib, nilotinib and ponatinib [21]. Given the phosphorylation profile in the NSTS-47 cell line, whether specific tyrosine kinase inhibitors could affect the proliferation of this cell line was assessed. NSTS-47 cells were first treated with sunitinib. Sunitinib was chosen for several reasons: (1) The NSTS-47 cell line harbors a c.1681C>T (p.R561C) mutation in *PDGFRB*, and PDGFR-beta was substantially phosphorylated in these cells; (2) sunitinib treatment inhibits PDGFR-beta phosphorylation [25]; and (3) sunitinib was successfully used to treat the boy with IM whose tumor tissue was used to generate the NSTS-47 cell line [8].

Cells were treated for six days with various concentrations of sunitinib, and after incubation, the proliferative activity was determined using the MTT assay. At sunitinib concentrations of 50 and 100 nM, which can be achieved in the plasma of children treated with sunitinib [26], the proliferative activity of NSTS-47 cells was significantly decreased (Figure 3A). In addition, 50 nM and 100 nM sunitinib decreased the proliferative activity of NSTS-47 cells to 75% and 73%, respectively, after six days.

To verify whether the observed effect of sunitinib is robust, NSTS-47 cells were cultivated with sunitinib in medium supplemented with PDGF-BB. A significant decrease in proliferative activity was observed after sunitinib treatment even when the cells grew in medium supplemented with PDGF-BB at a high concentration of 10 ng/mL (Figure 3B). In some experiments, the cultivation medium was changed every 24 h, and new medium with fresh inhibitor and fresh PDGF-BB was added (at medium changes) to prevent the potential degradation of sunitinib and PDGF-BB (Figure 3C).

**Figure 3.** Proliferative activity of NSTS-47 cells after various experimental treatments. Proliferative activity was measured using an MTT assay after 6 days of incubation. The data represent the mean ± SD. Experiments were repeated three times in hexaplicate (**A**,**D**–**H**) or in triplicate (**B**,**C**). \* *p* < 0.05 indicates a significant difference compared to control cells. (**A**) Sunitinib significantly decreased the proliferative activity of NSTS-47 cells. (**B**) NSTS-47 cells were sensitive to sunitinib, and this effect was not influenced by the presence of PDGF-BB at a high concentration (10 ng/mL). (**C**) Medium containing inhibitor and PDGF-BB was changed every 24 h during cultivation, which had no significant effect on the efficacy of the inhibitor. (**D**) NSTS-47 cells were also sensitive to erlotinib, as this inhibitor significantly affected cell proliferation. (**E**) No significant effect was observed after U0126 treatment. (**F**) FR180204 also did not significantly affect proliferative activity. (**G**) The combination of erlotinib and sunitinib significantly decreased the proliferative activity of NSTS-47 cells. (**H**) The combination of U0126 and FR180204 did not have a significant effect on NSTS-47 cell proliferation.

Next, NSTS-47 cells were treated with erlotinib, U0126 and FR180204. These three inhibitors were chosen based on EGFR and ERK1/2 phosphorylation in NSTS-47 cells (Figures 1 and 2). The ability of the combination of sunitinib and erlotinib to block both highly phosphorylated RTKs was tested, and a combination of U0126 and FR180204 was used to block the MEK/ERK signaling pathway.

At an erlotinib concentration of 1 μM, which can be achieved in the plasma of children treated with erlotinib [27], the proliferative activity of the NSTS-47 cell line was significantly decreased to 75% after 6 days of cultivation (Figure 3D). In contrast, NSTS-47 cells were not sensitive to U0126 and FR180204 because treatment of the NSTS-47 cell line with these inhibitors did not induce a significant decrease in proliferative activity (Figure 3E,F).

The combination of erlotinib and sunitinib also significantly decreased the proliferative activity of NSTS-47 cells (Figure 3G), but the effect of this combined treatment was similar to the effects of sunitinib or erlotinib alone. For instance, 100 nM sunitinib and 100 nM erlotinib decreased the proliferative activity to 70% (Figure 3G), but 100 nM sunitinib alone decreased the proliferative activity to 73% (Figure 3A). Another example is the combination of 1 μM erlotinib and 1 μM sunitinib; this treatment decreased the proliferative activity to 61% (Figure 3G), but 1 μM erlotinib alone decreased the proliferative activity to 75% (Figure 3D), and 1 μM sunitinib decreased the proliferative activity to 76% after six days (Figure 3A). Therefore, the combination of sunitinib and erlotinib did not have a significant additional effect on the reduction of NSTS-47 cell proliferation. In addition, the combination of U0126 and FR180204 did not show any significant effect on proliferative activity (Figure 3H).

Taken together, our results demonstrate that sunitinib and erlotinib can significantly decrease the proliferative activity of NSTS-47 cells, which harbor a c.1681C>T (p.R561C) mutation in *PDGFRB*, at concentrations that are achievable for these inhibitors in children plasma. However, the combination of sunitinib and erlotinib did not show an additional significant effect on cell proliferation. The inhibitors FR180204 and U0126 also did not have a significant effect on NSTS-47 cell proliferation.

#### *2.4. PDGFR-Beta and EGFR Exhibited Ligand-Dependent Tyrosine Phosphorylation*

Considering that only some kinase inhibitors significantly decreased the proliferative activity of the NSTS-47 cell line, detailed analyses of target kinases that should be affected by previously used inhibitors were performed using Western blotting. First, it was observed that the constitutively phosphorylated receptors PDGFR-beta and EGFR in NSTS-47 cells can respond to their ligands: Our results show that phosphorylation of both receptors was considerably increased in response to PDGF-BB or EGF (Figure 4A,B). Cell populations were serum starved for 24 h and then stimulated for 15, 30 or 60 min using two different concentrations of PDGF-BB or EGF. The cells that were serum starved for only 24 h and cells that were cultivated with FCS were used as negative controls. Receptor phosphorylation was significantly increased after 15 min, and then decreased in a time-dependent manner. Surprisingly, serum-starved cells that were not stimulated with PDGF-BB or EGF also exhibited an increase in receptor phosphorylation, in comparison to serum-cultivated cells. These experiments demonstrated that both receptors were functional and were able to activate downstream signaling molecules.

**Figure 4.** Analysis of protein phosphorylation. (**A**) PDGFR-beta phosphorylation is increased in response to PDGF-BB. Cells were stimulated for 15, 30 or 60 min using two different concentrations (10 ng/mL and 30 ng/mL) of PDGF-BB. (**B**) EGFR phosphorylation is increased in response to epidermal growth factor (EGF). Cells were stimulated for 15, 30 or 60 min using two different concentrations (40 ng/mL and 100 ng/mL) of EGF. (**C**) Sunitinib was able to decrease PDGFR-beta and Akt phosphorylation but not MEK1/2 and ERK1/2 phosphorylation. (**D**) Erlotinib decreased EGFR and Akt phosphorylation but had no effect on MEK1/2 and ERK1/2 phosphorylation. (**E**) U0126 treatment did not decrease MEK1/2 phosphorylation. (**F**) FR180204 treatment did not cause any changes in ERK1/2 phosphorylation. (**G**) The combination of sunitinib and erlotinib decreased PDGFR-beta, EGFR and Akt phosphorylation, but MEK1/2 and ERK1/2 phosphorylation was not affected.
