Fig. 3

(a) Representative dose response curve for AZD6738 in a panel of neuroblastoma cell lines (b) Results of 3 independent SF50 experiments in the panel of neuroblastoma cell lines. Cell lines are grouped and colour coded according to MYCN and ALT [36, 75] status. For SF₅₀experiments, cells were seeded into 96-well plates and the following day compound was added to wells in triplicate, across a concentration gradient including DMSO-only controls. After 5 days cell viability was assessed by Cell Titer Glo®assay. The SF₅₀was calculated as the drug concentration that inhibits viability/cell growth by 50% compared with controls, according to non-linear regression analysis, using Graphpad Prism. Statistical comparison of results is by unpaired t-test (c) MYCN expression by western blot in the panel of cell lines (N-MyC antibody: santa cruz sc-53,993, GAPDH antibody: cell signaling #2118) (d) Representative images of a Th-MYCN GEMM tumour prior to, and after 7 days treatment with 75 mg/kg AZD6738. (e) Waterfall plot documenting the relative changes in tumour volume following 7-day treatment with AZD6738 at three different dose levels. Preliminary studies of AZD6738 were performed in the Th-MYCN model of MYCN amplified neuroblastoma [76]. AZD6738 was supplied by AstraZeneca under a Material Transfer Agreement. It was diluted in 10% DMSO, 40% propylene glycol and 50% water as per manufacturers instructions. In a dose finding study three different doses (25 mg/kg, 50 mg/kg and 75 mg/kg) were trialled in 2 mice each for 7 days by oral gavage. Comparison of tumour response was made between animals receiving vehicle only and AZD6738. For response assessment, magnetic resonance imaging (MRI) of tumours was performed at day zero and after 7 days of administration of the compound