EWS/FLI overexpression and TRIM8 degradation increase DNA damage and sensitize cells to PARP inhibitors
(A) TC71 and TC32 cells expressing dox-inducible EWS/FLI-HA were treated with doxycycline (dox) (500 ng/mL) for 48 h and immunoblotted with the indicated antibodies.
(B) TC71 and TC32 C-dTAG TRIM8 cells were treated with either DMSO control or dTAGV-1 (1 μM) for 48 h and immunoblotted with the indicated antibodies.
(C) TC32 and TC71 cells were infected with lentivirus encoding either vector control, TRIM8-V5, or TRIM8ΔRING-V5 and immunoblotted with the indicated antibodies.
(D–I) TC32 and TC71 C-dTAG TRIM8 cells were pre-treated with either DMSO control or dTAGV-1 (1 μM) for 48 h and assessed for sensitivity to olaparib (D and E), cisplatin (F and G), and etoposide (H and I). Mean of eight technical replicates ±SD of relative viability are shown. Statistical significance calculated using unpaired two-tailed Student's t test. Data in (A–I) representative of three independent experiments.
(J) Heatmap showing area under the curve values of PARP inhibitor sensitivity of Ewing sarcoma cells in the PRISM Repurposing Secondary Screen at the Broad Institute (https://depmap.org/portal/).
(K–O) Scatterplots showing the correlation between TRIM8 dependency and sensitivity to PARP inhibitors: talazoparib (K), niraparib (L), olaparib (M), rucaparib (N), and veliparib (O). Spearman correlation (R) and p value from one-sided exact t test are shown.