Fig 2: MYCN expression increases sensitivity to ATR inhibitors: (A) Survival fraction of SHEP-Tet21/N NB cell line with MYCN ON or OFF as measured by clonogenic survival assay 14 days post treatment with ATR inhibitors VE-821 and AZD6783. Statistical significance was calculated using the Student’s t-test, comparing MYCN OFF and MYCN ON cells at 1 µM. Mean and standard deviation of 3 independent repeats are shown. *** = p < 0.001. (B) Western blot of CHK1 activation (CHK1 serine 345 phosphorylation) 24 h post co-treatment with hydroxyurea (HU) and ATR inhibitor in the SHEP-Tet21/N NB cell line with MYCN ON or OFF. (C) Percentage control growth of IMR5/75 shMYCN NB cell line in response to increasing concentrations of VE-821 and AZD6738 with MYCN-amplified (MNA ct) or depleted MYCN (MNA kd) as measured by XTT cell proliferation assay. Mean and standard deviation of 3 independent repeats are shown. * = p < 0.05 and ns = non significant.

Fig 3: PARP inhibition activates ATR and sensitises NB cells to ATR inhibitors independent of MYCN expression. (A) example images and mean cell intensity of phosph-ATR(Thr1989) in SHEP-Tet21/N cells with MYCN ON or MYCN OFF 24 h post treatment with 1 μM PARP inhibitor Olaparib or DMSO control. n > 80 cells. Scale bar = 10 μm. (B) Survival fraction of SHEP-Tet21/N NB cell line with MYCN ON or OFF as measured by clonogenic survival assay 14 days post treatment with ATR inhibitors VE-821 and (C) AZD6783 with or without 0.5 µM Olaparib. (D) Growth inhibition (% control growth) of the IMR5/75 shMYCN cell line in MYCN ON and knock down (KD) states measured by XTT cell proliferation assay post treatment with ATR inhibitors VE-821 and (E) AZD6783 with or without 1 µM Olaparib for 72 h. Statistical significance and sensitisation factor was calculated using the Student’s t-test, comparing MYCN OFF and MYCN ON cells at 1 µM. Mean and standard deviation of 3 independent repeats are shown. *, ** and *** = p < 0.05, 0.01 and 0.001 respectively. Sensitisation factor was calculated in each case as the fold change in survival or growth between with and without PARPi at 1 µM ATRi.

Fig 4: MYCN expression perturbs replication forks in NB cells and this is exacerbated upon ATR inhibition. (A) Number of RPA foci/cell in SHEP-Tet21/N cells with MYCN ON or MYCN OFF 24 h post treatment with 0.5 μM VE-821 or DMSO control. Data are pooled from three independent repeats for each repeat n > 50 cells, lines indicate median value, independent repeats are shown in Supplementary Figure S1. (B) Example RPA images. Scale bar = 10 μm (C) DNA fibre analysis of replication fork speed and stalling in VE-821 treated SHEP-Tet21/N cells with MYCN ON and MYCN OFF. Cells were incubated in 0.5 µM VE-821 or DMSO control and then pulse labelled with CldU, for 20 min, and label switched to IdU for 20 min. Example images of replication forks are shown. (D) DNA fibre length (µm) (CIdU). (E) Percentage fork stalling, calculated as a % of CIdU only labelled tracts (red) from continuous forks (CIdU (red) and IdU (green) labelled tracts) and (F) Percentage new origin firing, calculated as % of IdU only labelled tracts (green) from continuous forks (CIdU (red) and IdU (green) labelled tracts. For (D–F), at least 100 forks were counted on each of three separate occasions, (D) shows pooled data (means of individual repeats is shown in Supplementary Figure S2), (E,F) show means of 3 independent repeats. Statistical significance was calculated using the Mann–Whitney U test (pooled data) and Student’s t-test (means). Throughout, *, ** and **** represent p < 0.05, <0.01 and <0.0001, respectively and ns = non significant.

Fig 5: Co-treatment with PARP and ATR inhibitors increases replication stress and DNA damage. DNA fibre analysis of replication fork speed and stalling in VE-821/Olaparib treated SHEP-1 cells. Cells were incubated in 0.5 µM VE-821 and or 0.5 µM Olaparib or DMSO control and then pulse labelled with CldU, for 20 min, and label switched to IdU for 20 min. (A) DNA fibre length (µm) (CIdU), (B) Percentage fork stalling, calculated as a % of CIdU only labelled tracts (red) from continuous forks (CIdU (red) and IdU (green) labelled tracts) and (C) Percentage new origin firing, calculated as % of IdU only labelled tracts (green) from continuous forks (CIdU (red) and IdU (green) labelled tracts. For (A–C), at least 100 forks were counted on each of three separate occasions, (A) shows pooled data (means of individual repeats is shown in Supplementary Figure S4), (B,C) show means of 3 independent repeats. (D) DNA damage; alkaline COMET assay of VE-821/Olaparib treated SHEP-1 cells incubated in 0.5 µM VE-821 and or 0.5 µM Olaparib or DMSO control for 24 h. Tail moment was calculated using CometScore software where at least 50 cells were analysed on each of three occasions. Pooled data are shown (means of individual repeats are shown in Supplementary Figure S4). (E) Representative COMET assay images. Images were obtained using the full spectrum function of the CometScore software. Cells were originally stained with SYBR Safe DNA gel stain. Statistical significance was calculated using the Mann–Whitney U test (pooled data) and Student’s t-test (means). Throughout, *, **, *** and **** represent p < 0.05, <0.01, <0.001 and <0.0001, respectively.