Fig. 5: Mitochondrial priming and response to BH3 mimetics in the transient olaparib-induced TIS phenotype in BRCA1-mutant breast epithelial cells.

A Left. Representative flow cytometry plots showing the gating of the cell cycle distribution of proliferative (untreated) and olaparib-treated IMEC BRCA1^+/+ and IMEC BRCA1^mut/+ cells. The histograms show the percentage (mean ± S.D., n = 3) of cells in the four cell cycle phases as a function of the treatment condition. Right. Expression levels of phospho-RB^{Ser807/Ser811} and p21^WAF1/Cip1 were detected by immunoblotting in whole cell lysates of proliferative (untreated) and olaparib-treated IMEC BRCA1^+/+ and IMEC BRCA1^mut/+ cells (7-day) using specific antibodies and β-actin/GAPDH as loading controls. The figure shows a representative immunoblot from multiple (n = 3) independent experiments. B. Left. Representative images of SA-β-gal staining in proliferative (untreated) and olaparib-treated IMEC BRCA1^+/+ and IMEC BRCA1^mut/+ cells (7-day) from three independent experiments (scale bar: 200 μm). Right top. Recovery of proliferative potential was evaluated by reseeding untreated and olaparib-treated IMEC BRCA1^+/+ and IMEC BRCA1^mut/+ cells (7-day) cultures (2000 cells/well) in drug-free conditions. After 10 days, cell growth was monitored by crystal violet staining. Shown are representative images from three technical replicates. Right bottom. Representative flow cytometry plots showing the gating of the cell cycle distribution olaparib-treated IMEC BRCA1^mut/+ cells reseeded in drug-free conditions for 3 days. C Top. The synthetic lethal interaction between the PARPi olaparib and DNA repair due to BRCA1 deficiency triggers breast epithelial cancer cell senescence. Bottom. BH3 profiling was performed to measure mitochondrial depolarization in proliferative (untreated) and olaparib-treated IMEC BRCA1^+/+ and IMEC BRCA1^mut/+ breast epithelial cells exposed to activator and sensitizer BH3 peptides. Figure shows a global heat map of % mitochondrial depolarization caused by increasing concentrations of the activator (BIM, BID, PUMA) and sensitizer (BMF, BAD, NOXA, HRK) peptides in proliferative (untreated) and olaparib-treated cells. Data shown are the mean of ≥3 independent experiments using three technical replicates for each peptide. D Proliferative and olaparib-treated IMEC BRCA1^+/+ and IMEC BRCA1^mut/+ cells (7-day) were incubated with serial dilutions of ABT-263/navitoclax, A1331852, and ABT-199/venetoclax. After 5 days, cell viability was measured using the alarmarBlue™ assay. Top. Graphs show the senolytic indexes obtained by dividing the IC₅₀ values of BH3 mimetics in proliferative IMEC (BRCA1^+/+ and BRCA1^mut/+) cells by those obtained in corresponding olaparib-treated IMEC (BRCA1^+/+ and BRCA1^mut/+) cells. Bottom. Graphs show the senolytic indexes obtained by dividing the IC₅₀ values of BH3 mimetics in proliferative IMEC (BRCA1^+/+ and BRCA1^mut/+) cells by those obtained in olaparib-treated IMEC BRCA1^+/+ cells. Data represent the mean (columns) ± S.D. (bars) of 3 or more independent experiments. Statistically significant differences (ANOVA analysis) between the means for untreated and olaparib-treated cells are shown. n.s. not statistically significant. E Global heat map of % mitochondrial depolarization in proliferative (untreated) IMEC BRCA1^+/+ (normal-like breast epithelium, 0), IMEC BRCA1^mut/+ (breast cancer-prone breast epithelium, 1), A549 (lung adenocarcinoma, 2), and LoVo (colon cancer cells, 3).