Figure 6. ETS transcription factors induce DNA damage which is potentiated by PARP inhibition.

(A) γ-H2A.X immunofluorescence staining shows that ERG induces the formation of γ-H2A.X foci. Top row, benign prostate epithelial cells(PrEC) were infected with lentiviruses expressing LACZ or ERG. Bottom row, VCaP cells treated with control siRNA or ERG siRNA.

(B) Quantification of γ-H2A.X and 53BP1 immunofluorescence staining in PrEC or VCaP cells. For all experiments mean +/− SEM shown, * p < 0.05, ** p < 0.01.

(C) ETS overexpression or BRCA2 knockdown (with shRNA) induces DNA damage as assessed by neutral COMET assay in VCaP cells. Cells were treated with or without 10µM Olaparib for 48 hours. Cells with DNA damage have an extended “tail moment” of fragmented DNA shown in red. Relative tail length is shown in white. Representative images showing quantification of head and tail height, length and fluorescence intensity are shown (as indicated).

(D) Quantification of average COMET tail moments following treatment as noted in the box plot. Statistical tests were performed using the two-way ANOVA test (described in S.O.M.) to determine if the increase in DNA damage in Olaparib-treated ETS overexpressing cells (PC3-ERG, PC3-ETV1 and VCaP) was statistically greater than the increase observed in Olaparib-treated control cells with low ETS expression (PrEC-LACZ, PC3-LACZ or VCaP treated with ERG siRNA) as indicated in the text. Similar statistical tests were used to compare the increase in BRCA2 shRNA expressing cells to PC3 cells transduced with control shRNA ** p < 0.01

(E) Proposed model to therapeutically target ETS gene fusions via their interacting enzyme, PARP1.

All bar graphs are shown with +/− SEM unless otherwise indicated.

See also Figure S6.