**8. Effect of NB MYCN status on susceptibility to AZD8055**

Amplification of the MYCN oncogene is the most powerful single predictor of adverse outcome of NB [46]. MYCN amplification is observed in about 20% of all NB patients and is usually associated with fatal outcome of the disease [47]. Schramm et al. demonstrated transcriptomal upregulation of mTOR-related genes by MYCN [48]. MYCN-driven NB in mice displayed activation of the mTOR pathway on the protein level, and activation of MYCN in NB cells resulted in high sensitivity toward mTOR inhibition [48]. Therefore, it is examined whether MYCN

#### **Figure 1.**

*Effect of MYCN status on susceptibility to AZD8055. (A) MYCN gene in tested 12 NB cell lines. MYCN status was defined as previously [53]. No alteration: cells with two MYCN signals and two CEP2 signals; amplification: the number of MYCN signals is at least 10 copies greater than the control probe signals; loss/ imbalance: presence of at least two MYCN signals and increased CEP2 signals; gain: the number of MYCN signals is 1–9 copies more than CEP2 signals. (B) Fifteen NB cell lines were treated with AZD8055 at different concentrations in RPMI1640 + 10% FBS. Cell growth was evaluated as cell numbers at 72 hours. Data are expressed as the mean ± SD. IC50 (half maximal inhibitory concentration) of the 15 NB cell lines was calculated depending on the MTT results.*

status of NB cell lines affects susceptibility to AZD8055. Using fluorescence in situ hybridization (FISH), we observed MYCN amplification in all investigated nuclei of NB19, IMR32, TGW, OZAWA, LAN-1, SMS-KAN, and SCMC-N4 cell lines (**Figure 1A**). As shown in **Figure 1A**, MYCN amplification was not observed in INDEN (loss/ imbalance), SK-N-SH (gain), KP-N-SI (loss/imbalance), KP-N-SIFA (gain), and SJ-N-KP (loss/imbalance). As shown in **Figure 1B**, the relationship between MYCN status and susceptibility to AZD8055 was not found.
