Figure 1 | RNAi screen reveals genetic determinants of ATRi sensitivity. (a) Structure of VE-821 and schematic representation describing workflow for parallel VE-821 chemosensitization screens in MCF12A and HCC1143 cells. (b) Scatter plots of VE-821 Drug Effect (DE) Z-scores from all 1280 siRNAs SMARTpools used in the chemosensitization screens. The DE Z-score threshold of 2 (dotted red line) was used for defining candidate/unvalidated synthetic lethal interactions. (c) Venn diagram of DEo2 VE-821 sensitization hits in MCF12A and HCC1143 cells. Numbers shown indicate number of sensitization genes. Amongst the 30 genes with DE Z-scores o2 in both cell lines, seven well-established tumour suppressor genes were identified.
(d) Bar charts illustrating DE Z-scores for control, non-targeting, siRNAs (siALLSTAR, siCon1, siCon2) and ARID1A SMARTPool siRNAs in the chemosensitization screens. Values shown are medians from triplicate screens. Error bars represent s.d. (e) Three-hundred eighty-four-well plate cell survival data from HCC1143 cells transfected with siRNA targeting ARID1A (red) or siCon (blue). Twenty four hours after transfection, cells were exposed to VE-821 for 5 continuous days. Error bars represent s.d. (n¼16) and results are representative of three biological replicates. Survival curve siARID1A versus siCon P value o0.0001, ANOVA. (f) Western blot illustrating ARID1A protein silencing from experiment (e). (g) Bar chart illustrating the Log2 surviving fractions (Log2(SF)) of HCC1143 cells transfected with the indicated individual siRNAs and exposed to VE-821 (1 mM) for 5 days. Error bars represent s.d.
and P values of o0.001, Student’s t-test, for each siRNA compared with siCon. (h)Western blot illustrating ARID1A protein silencing from experiment (g).
目的：To uncover clinically actionable genetic determinants of singleagent ATRi response
方法：:performed a series of high-throughput RNAi chemosensitization screens where cells were transfected with a library of SMARTPool short interfering (si)RNAs and then exposed to the highly potent and selective ATR catalytic inhibitor VE-821
why:selected the p53 mutant, triple negative (ERa negative, PR negative and ERBB2 negative) breast tumour cell line HCC1143, based on previous work suggesting that ATRi might have utility in TP53 mutant cancers
scree in differt cells
目的：To identify clinically actionable effects, the RNAi library we used encompassed 1,280 siRNA SMARTPools (four siRNAs per gene in each pool) targeting either recurrently mutated genes in cancer28, kinases, due to their inherent tractability as drug targets, and DDR genes
Figure 2 | In vitro ARID1A/ATR synthetic lethality. (a) Chemical structure of VX-970. (b) Western blot of ARID1A in human ARID1A isogenic HCT116 cells. (c) Image of colonies in six-well-plate clonogenic assay. HCT116 ARID1A isogenic (þ/þ and /) cell lines were exposed to increasing concentrations of VE-821 (0, 0.1, 1 mM) or VX-970 (0, 0.01, 0.05 mM) for 14 days. (d–f) Dose–response clonogenic survival curves of HCT116 ARID1A isogenic (þ/þ and /) cell lines exposed to increasing concentrations of VE-821 (d), VX-970 (e) and AZ-20 (f) for 14 days. Error bars represent s.d.(n¼3), ANOVA P value ofo0.0001, results are representative of triplicate biological experiments. (g–i) Dose–response survival curves of HCT116 ARID1A isogenic (þ/þ and /) cell lines exposed to increasing concentrations of VE-821 (g), VX-970 (H) or AZ-20 (I) for 5 days. Cell viability was estimated by CellTitre-Glo reagent. Error bars represent s.d. (n¼16), ANOVA P value of o0.001, results are representative of triplicate biological experiments.(j) Area under curve (AUC) box whisker comparison plot for human tumour cell lines exposed to VX-970 for 5 days. ARID1A wild-type tumour cell lines (n¼15) were compared with ARID1A mutant cell lines (n¼9). P¼0.00594 median permutation test. (k). Dose–response clonogenic survival curve of mouse Arid1a isogenic ES cell lines. Experiment was performed as per (c). Error bars represent s.d. (n¼3), ANOVA P value of o0.0001, results are representative of triplicate biological experiments. (l). Western blot of Arid1a protein expression in mouse ES Arid1a isogenic cells.
目的：To assess whether constitutive loss of ARID1A function, as would occur in tumours, also resulted in ATRi sensitivity, we used human colorectal HCT116 isogenic cells containing either wildtype ARID1A or homozygous loss-of-function ARID1A mutations
To address whether the ATR/ARID1A synthetic lethal interaction was limited to human cell line models, we also studied isogenic Arid1aþ/þ and Arid1a/ mouse embryonic stem (ES) cells