Extended Data Fig. 3. ALK inhibition suppresses the HR efficiency.
(a, b) Representative images of BRCA1 (a) and CtIP (b) with EdU/DAPI staining in PARP inhibitor (PARPi)-resistant ovarian cancer cells treated with 0.25 μM PARPi (talazoparib) or 0.5 μM ALK inhibitor (ALKi; lorlatinib), either alone or in combination, for 48 hours. Insets, 3.3× magnification. Bar, 20 µm. Data are representative of N = 3 independent experiments with similar results. (c) Quantification of EdU-positive cells with BRCA1 or CtIP foci in PARPi-resistant ovarian cancer cells treated with 0.25 μM PARPi (talazoparib) or 0.5 μM ALKi (lorlatinib), either alone or in combination, for 48 hours. Error bars represent mean ± SD of N = 3 independent experiments, one-way ANOVA analysis, n.s., not significant, Control (Con,○) vs ALK inhibitor (ALKi,●): *P = 0.0235 in BRCA1, ***P < 0.0001 in CtIP; PARP inhibitor + ALK inhibitor (PARPi+ALKi, ■) vs PARP inhibitor (PARPi, □): **P = 0.0091 in BRCA1, ***P < 0.0001 in CtIP. (d) Representative images (left) and quantification (right) of EdU positive cells with RAD51 foci in PARPi-sensitive (TNBC) treated with 0.25 μM PARPi (talazoparib) or 0.5 μM ALKi (lorlatinib), either alone or in combination, for 48 h. Insets, 3.3× magnification. Bar, 20 µm. Error bars represent mean ± SD of N = 3 independent experiments, one-way ANOVA analysis, n.s., not significant. (e) Top: Schematic of the DR-GFP reporter assay. Functional HR can repair DNA double-strand breaks and results in the expression of GFP. Bottom: HR efficiency in U2OS DR-GFP cells treated with 0.25 μM PARPi (talazoparib) or 0.5 μM ALKi (lorlatinib), either alone or in combination, for 48 hours. Flow cytometry analysis was used to measure the percentage of GFP + cells. Error bars represent mean ± SEM of N = 3 independent experiments, two-tailed unpaired t test, Control (Con,○) vs ALK inhibitor (ALKi, ■): *P = 0.0337; PARP inhibitor + ALK inhibitor (PARPi+ALKi, ■) vs PARP inhibitor (PARPi, □): *P = 0.0215. (f, g) Gating strategy to determine the percentage of GFP-positive cells in U2OS DR-GFP cells under (f) basal conditions or (g) after transfection with I-SceI expression plasmid.