Fig. 7: CX-5461 and BMN-673 induce markers of replication stress and DNA damage in OVCAR8 and OVCAR8 RAD51C KO cells.

a OVCAR8 cells were incubated with 10 μM EdU, before being treated with vehicle, 100 nM CX-5461, 100 nM BMN-673 or the combination of both for 24 h. Co-IF for γH2AX and RAD51 was performed. Cells were incubated for 30 minutes at room temperature with Click-IT reaction, washed with PBS and then counterstained with DAPI. Representative images of three biologically independent experiments. b Quantitation of γH2AX foci counts. n = 554 OVCAR8 cells and n = 708 OVCAR8 RAD51C KO cells per treatment condition were analysed over three biologically independent experiments. Error bars represent mean ± SD. Quantitation of RAD51 foci counts in EdU positive cells. n = 223 EdU +ve OVCAR8 cells and n = 221 OVCAR8 RAD51C KO cells per treatment condition analysed over three independent experiments. Error bars represent mean ± SD. Statistical analysis was performed using a two-sided one-way ANOVA, Tukey’s multiple comparisons test (adjusted p-values are shown). c Western blot analysis of cells treated as in (a). Representative of n = 2 biologically independent experiments. The blots shown are of samples derived from the same experiment and were processed in parallel. Full scan sizes of western blots are provided in Supplementary Fig. 10. d A schematic of molecular response to CX-5461. CX-5461 inhibits the Pol I transcription complex by binding to the selectivity complex 1 (SL-1) and preventing Pol I from binding to rRNA gene promoters. Displacement of Pol I and inhibition of Pol I transcription initiation are associated with R-loops stabilization, recruitment of RPA to single strand rDNA, rDNA replication stress and activation of DDR at the nucleoli. CX-5461 also induces global replication stress associated with stalling and destabilization of replication forks via MRE11 activity leading to DNA damage, S-phase and G2/M cell cycle arrest. The HR pathway and PARP activity are necessary to counteract DNA replication stress. CX-5461 co-operates with HRD and inhibition of PARP activity in exacerbating replication stress and DNA damage, promoting cell death.