Figure 5. Targeting the ATR-CHK1 axis in BRCA1/2^MUT cell models.

(A) Viability of established ovarian cancer cells after treatment with 1 μM PARPi (AZD2281), 1 μM CHK1i (MK8776), 1 μM ATRi (AZD6738), and 30 μM carboplatin was assessed with an MTT assay. Cell models included PEO1 (BRCA2^MUT), JHOS4 (BRCA1^MUT), and the homologous recombination–proficient cells, PEO4 (BRCA2^REV). Cells were incubated in their respective drug concentrations for 5 days. PARPi was significantly more cytotoxic in BRCA^MUT relative to BRCA2^REV cells (32.9% ± 0.7% cell viability for BRCA2^MUT cells and 42.3% ± 2.0% for BRCA1^MUT cells vs. 76.7% ± 2.0% for BRCA2^REV cells; P < 0.00001 and P = 0.0002 for BRCA2 vs. BRCA^REV and BRCA1 vs. BRCA^REV, respectively). Carboplatin treatment appeared to have a similar effect to that of PARPi in all lines: BRCA2^MUT (41.2% ± 3.9% cell viability; P = 0.07 carboplatin compared with PARPi); BRCA1^MUT (55.0% ± 5.2% viability; P = 0.0003 carboplatin vs. PARPi); and BRCA2^REV (96.3% ± 4.6% viability; P = 0.01 carboplatin vs. PARPi). Only the BRCA2^MUT cell model was sensitive to 1 μM CHK1i (56.6% ± 1.1% cell viability). Both BRCA^MUT and BRCA2^REV cells were sensitive to ATRi monotherapy (8.6% ± 0.2%, 38.9% ± 1.4%, and 26.3% ± 0.5% cell viability for BRCA2^MUT, BRCA1^MUT, and BRCA2^REV cells, respectively; P < 0.001 for all lines ATRi vs. carboplatin treatment). The box-and-whisker plots show the median, with boxes extending from the 25th to 75th percentile and the whiskers extending from minimum and maximum values of the dataset (n = 5 per group). Two-way ANOVA was performed before the Tukey’s honestly significant difference test to determine whether there was an overall difference between the groups. The data shown are a single representative experiment with 5 determinations. Three independent experiments were performed. (B) Colony forming assay was performed with 1 μM PARPi, 1 μM CHK1i, 1 μM ATRi, and 30 μM carboplatin treatment in BRCA2^MUT (PEO1) and BRCA2^REV (PEO4) cell models. Cells were incubated in their respective drug concentrations for 13 days. Colony formation was decreased in BRCA2^MUT compared with BRCA2^REV cells with PARPi (3.3% ± 1.3% vs. 116.8% ± 5.2%; P < 0.00001), CHK1i (28.6% ± 3.1% vs. 94.1% ± 6.0%; P = 0.0001), and carboplatin (3.9% ± 1.2% vs. 43.3% ± 2.8%; P < 0.00001). All values are percentage colony formation relative to control. Both cell models showed decreased colony formation with ATRi (6.4% ± 1.6% for BRCA2^MUT vs. 10.9% ± 1.4% for BRCA2^REV; P = 0.08). (C) BRCA2^MUT (PEO1) and BRCA2^REV (PEO4) cells were treated with 1 μM PARPi, 1 μM CHK1i, and 1 μM ATRi and lysates were collected after 24 hours of treatment. Western blot for the indicated total and phosphoproteins is shown.