Fig. 6.

The 5-Azacytidine–mediated impairment of DSB repair sensitizes NSCLC to combination treatment with talazoparib and radiation. (A) Colony forming assay in H460 cells transfected with Ku80 siRNA or treated with 5-AZA (250 nM), followed by single 2 Gy (RT) or fractionated 3 × 2 Gy (FRT) irradiation (n = 9 from 3 experimental replicates performed in triplicate). Data are represented as mean number of colonies ± SEM. P value is calculated using 1-way ANOVA. (B) Colony forming assay in H460 cells pretreated with 5-AZA (250 nM; 4 d) and nucleofected with Ku80-wild-type or empty vector, followed by fractionated 3 × 2 Gy irradiation (n = 9 from 3 experimental replicates performed in triplicate). Data are represented as mean number of colonies ± SEM. P value is calculated using 1-way ANOVA. (C) Colony forming assay in H460 FANCD2-wild-type or FANCD2-KD cells, pretreated with 5-AZA (250 nM; 4 d) or transfected with Ku80 siRNA (or nontargeting control [NTC]), followed by fractionated 3 × 2 Gy irradiation (n = 9 from 3 experimental replicates performed in triplicate). Data are represented as mean number of colonies ± SEM. P value is calculated using 1-way ANOVA. (D) Colony forming assay in H460 NSCLC cell lines in presence of vehicle (DMSO), TAL (2 nM), 5-AZA (250 nM), or combination, followed by single 2 Gy (RT) or fractionated 3 × 2 Gy (FRT) irradiation (n = 9 from 3 experimental replicates performed in triplicate). Data are represented as mean number of colonies ± SEM. P value is calculated using 1-way ANOVA. (E) Detection of γH2AX foci in H460 after 2 Gy irradiation, following pretreatment with vehicle (DMSO), TAL (2 nM), 5-AZA (250 nM), or combination (day 4; n = 100 per condition; 25 cells counted per condition from 4 experimental replicates). Data are represented as mean number of cells with >20 foci ± SEM. P value is calculated using 1-way ANOVA. (F) Detection of RAD51 foci in H460 at 4 h after 2 Gy irradiation, following treatment with vehicle (DMSO), TAL (2 nM), 5-AZA (250 nM), or combination (day 4, n = 100 per condition; 25 cells counted per condition from 4 experimental replicates). Data are represented as mean number of cells with >20 foci ± SEM. P value is calculated using 1-way ANOVA. (G) Tumor volume in in vivo H460 NSCLC model. H460 xenograft (10⁷ cells per mouse) was delivered via flank injection (n = 8 per group). Treatment with 5-AZA (0.5 mg/kg SC) and TAL (0.3 mg/kg PO) was initiated once tumor volume reached 100 mm³ and continued until endpoint. Fractionated radiation was delivered at days 7, 8, and 9 after initiation of TAL+5-AZA. *P < 0.05 from day 18 onward as indicated by arrow. (H) Kaplan–Meier survival plot of in vivo H460 NSCLC model treated with 5-AZA+TAL and/or fractionated radiation (n = 8 per group).