1. Fig. 5. Mutant KRAS increases HR capacity and MEKi decreases HR capacity in RAS mutant cells, causing increased DNA damage.
Mutant KRAS increases HR capacity and MEKi decreases HR capacity in RAS mutant cells, causing increased DNA damage. (A) Microarray data from HPDE-iKRASG12D cell lines with or without Dox induction were analyzed by unsupervised clustering for HRD gene signatures. Heat maps of clusters indicate that cells with KRASG12D induction are more likely HR-intact (left). Quantification of HRD scores of HPDE-iKRASG12D with or without Dox induction was calculated on the basis of correlation to HRD gene signatures (right; higher scores are more likely to have HR defects). (B) Western blot showing dose-dependent protein changes in OVCAR8 after 24 hours of treatment with the indicated concentrations of AZD6244. (C) Immunofluorescence staining of FOXO3a/BRCA1 and RAD51/γ-H2AX after treatment with BMN673 (1 μM)/AZD6244 (5 μM)/combination therapy in OVCAR8 for 48 hours. Scale bars, 20 μm. (D) Comet assay in HPDE-iKRASG12D cell lines after treatment with BMN673 (200 nM)/AZD6244 (200 nM)/combination therapy for 72 hours with or without Dox induction. DNA damage was quantified via % DNA in tails. Each data point represents at least 50 cells. (E) Comet assay in OVCAR8 cell lines after treatment with BMN673 (1 μM)/AZD6244 (5 μM)/combination therapy for 72 hours. DNA damage was quantified via % DNA in tails. Each data point represents at least 50 cells. (F) Direct repeat (DR)–GFP assay to measure HR-mediated DNA DSB repair in DR-GFP U2OS. Frequency of GFP+ cells by flow cytometry after infection with the I-Sce1 endonuclease and incubation for 48 hours with or without 100 nM AZD6244 (top). NHEJ-mediated DNA DSB repair in EJ5-GFP U2OS. Frequency of GFP+ cells by flow cytometry after infection with the I-Sce1 endonuclease and incubation for 48 hours with or without 100 nM AZD6244 (bottom). (G and H) Immunofluorescence staining for RAD51, γ-H2AX, and MRE11 before and after IR in inducible HPDE-iKRASG12D (G) and OVCAR8 (H). Right: Images for MRE11 foci staining and quantification of MRE11 foci-positive cells at 2 hours after IR. Left: Because of relatively late recruitment of RAD51, quantification of RAD51 staining was performed at different times (0, 2, 4, 6, and 8 hours after IR), and representative images are shown. Scale bars, 20 μm. DAPI, 4′,6-diamidino-2-phenylindole. Error bars represent SEM of three independent experiments. Student’s t test: **P < 0.01 and ***P <
Chaoyang Sun et al., Sci Transl Med 2017;9:eaal5148
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