Thioparib Inhibits Homologous Recombination Repair, Activates the Type-I IFN Response, and Overcomes Olaparib Resistance (2022)
Figure 5

Fig. 5. Thioparib treatment led to aberrant homologous recombination (HR) repair.

(A) HR repair assays in U2OS-DR-GFP cells treated with increasing concentrations of thioparib. Cells were incubated with the indicated drugs at the time of I-SceI introduction for 48 h. GFP-positive cells were analyzed after I-SceI transfection for 72 h by flow cytometry. The ATR inhibitor VE-821 was used as a positive control. Three independent experiments were performed and data are expressed as mean ± SD. Statistical analysis was performed by one-way ANOVA, and all groups were compared with control. ***P < 0.0001, *P = 0.0205, 0.0101 (from left to right).

(B) Correlation between the cytotoxicity of PARPi in 12 hematologic cancer cell lines (Appendix Table S5) and HR repair inhibition rates. Pearson test was performed to calculate the correlation.

(C) Representative images of RAD51 foci in U2OS-DR-GFP cells with or without radiation. Graphs (right panel) show quantification of cells with ≥ 5 RAD51 foci in vehicle- and drug-treated cells. Scale bar: 5 μm. Data are obtained from three biological replicates and presented as mean ± SD. Statistical analysis was performed by one-way ANOVA. Left to right: P < 0.0001, = 0.0033, < 0.0001, = 0.0006, = 0.0031, = 0.0015.

(D) HR repair assays in U2OS-DR-GFP cells treated with thioparib with or without chloroquine. Cells were pretreated with chloroquine for 4 h, followed by thioparib treatment for 48 h. GFP-positive cells were analyzed by flow cytometry 72 h later. Data are expressed as the mean ± SD from three independent experiments. Statistical analysis was performed by one-way ANOVA. NS: P > 0.9999, = 0.9763, = 0.9972.

(E) and (F) Western blotting of γH2AX, phospho-RPA32, and phospho-Chk1 (E) or PI-staining-based flow cytometry analysis of the cell cycle (F) in cells exposed to thioparib with or without chloroquine. Cells were pretreated with chloroquine for 4 h followed by thioparib treatment (100 nM in JeKo-1 cells for 12 h and 1 μM in Capan-1/TP cells for 24 h).

(G) The effect of the thioparib and chloroquine combination on the viability of JeKo-1 cells. Cells were pretreated with chloroquine for 4 h followed by thioparib treatment for 72 h and subjected to CCK-8 assay. The combination index (CI) was calculated from three independent experiments using CompuSyn software and the average CI values are presented (CI < 1, synergism; CI = 1, additive effect; CI > 1 antagonism). Data are expressed as mean ± SEM. ThP: Thioparib, OP: Olaparib, TP: Talazoparib, 391: Cpd 391, CQ: Chloroquine.

(H) HR repair assay. U2OS-DR-GFP (WT) and PARP1 KO cells (KO1 and KO2) were treated with the indicated drugs for 48 h and collected after 72 h of I-SceI transfection as in (A). VE-821 served as a positive control. PARP1 knockout efficiency was evaluated by western blotting. Data from three biological replicates are presented as mean ± SD. Statistical analysis was performed by one-way ANOVA, and each group was compared with the control. ***P < 0.0001, *P = 0.0143, 0.0483, ns: P = 0.3061, 0.8269, 0.9997, 0.3906 (from left to right).