Research ArticleCELL BIOLOGY

The chromatin remodeler ALC1 underlies resistance to PARP inhibitor treatment

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Science Advances  18 Dec 2020:
Vol. 6, no. 51, eabb8626
DOI: 10.1126/sciadv.abb8626
  • Fig. 1 A genome-wide CRISPR knockout screen reveals ALC1 as a gene conveying PARP inhibitor resistance.

    (A) Schematic of the CRISPR screen. (B) Dot plot showing the enrichment of 20 Gene Ontology processes. The size of the dots represents the number of significant genes associated with the Gene Ontology term, and the color of the dots represents the P value. (C) Scatterplot of DrugZ analysis result of the genes showing synthetic interactions with olaparib. Genes annotated with functions in DNA repair are colored red. (D) Clonogenic cell survival assay of U2OS WT, ALC1KO, PARP1KO, and ALC1KO PARP1KO double knockout cells after a 24-hour treatment with olaparib. (E) Clonogenic cell survival assay of U2OS WT and ALC1KO expressing mCherry-ALC1 variants after a 24-hour treatment with olaparib. Graphs in (D) and (E) include all data points (n = 3 to 5) and fitted curves with 95% confidence intervals (gray shading). Asterisks indicate P values obtained by polynomial regression (n.s., not significant; ***P < 0.001). Model summary is provided in table S2.

  • Fig. 2 PARP1 inhibition induces chromosome aberrations and cell cycle arrest in ALC1KO cells.

    (A) Representative flow cytometry profiles of cells with the indicated genotypes with or without 1 μM olaparib treatment for 24 hours. The distribution of cells in G1, S, or G2-M is indicated in the inserted boxes. PI, propidium iodide; a.u., arbitrary units. (B) Cells were grown for 24 hours in the presence or absence of 1 μM olaparib. After 24 hours, cells were treated with colchicine to arrest the cells in M phase and collected after 6 hours. Left: Chromosome aberrations were counted in 40 chromosome spreads per sample. Right: Representative images of chromosome spreads of WT and ALC1KO cells with or without olaparib treatment. Scale bar, 2 μm. (C) Relative γH2AX intensity in ALC1KO and/or PARP1KO U2OS cells. The intensity of γH2AX signal was measured in untreated (NT) or olaparib-treated cells after 12 hours. (D) Representative images of the level of γH2AX in ALC1KO and/or PARP1KO U2OS cells after olaparib treatment. Scale bar, 30 μm. Graphs in (B) and (C) include all data points and mean ± SEM (n = 3). Asterisks indicate P values obtained by linear regression (***P < 0.001). Models in (C) were fitted independently for each concentration. Model summary is provided in table S2.

  • Fig. 3 The hypersensitivity of ALC1-deficient cells to DNA-damaging agents is enhanced by PARP1 inhibition.

    (A) Clonogenic cell survival assay of ALC1KO and/or PARP1KO U2OS cells. DNA damage was induced by MMS for 1 hour. (B) Clonogenic cell survival assay of ALC1KO and/or PARP1KO U2OS cells. PARP1 inhibition was induced by olaparib (OP) treatment for 24 hours, and then DNA damage was induced by MMS for 1 hour. NT, non-treated. (C and D) Quantification of γH2AX foci in ALC1KO and/or PARP1KO U2OS cells. Where indicated, cells were treated with 1 μM olaparib for 1 hour before DNA damage induction with x-ray irradiation (2 Gy). γH2AX foci were counted at different time points after irradiation. NT, non-treated. Graphs in (A) and (B) include all data points (n = 3) and fitted curves with 95% confidence intervals (gray shading). Asterisks indicate P values obtained by linear or polynomial regression, respectively (*P < 0.05, **P < 0.01, and ***P < 0.001). P values in (B) correspond to three-way interaction terms comparing genotypes. Graphs in (C) and (D) include all data points and mean ± SEM (n = 3). Asterisks indicate P values obtained by linear regression fitted independently for each time point. Statistical summary is provided in table S2.

  • Fig. 4 ALC1 deficiency increases olaparib-induced PARP1 trapping.

    (A) PARP1 at UV-induced DNA damage sites was quantified in WT and ALC1KO cells treated or not with 1 μM olaparib. (B) Representative images from (A). Scale bar, 10 μm. (C) Representative images showing GFP-PARP1 accumulation at sites of laser-induced DNA damage in WT and ALC1KO cells treated or not with 30 nM olaparib. Scale bar, 5 μm. (D and E) Quantified accumulation of GFP-PARP1 at DNA damage in WT or ALC1KO cells untreated (D) or treated with 30 nM olaparib (E). (F) Normalized FRAP curves of GFP-PARP1 at sites of DNA damage 30 min after irradiation in WT and ALC1KO cells. (G) Recovery time and (H) the immobile fraction of GFP-PARP1 at the break were calculated from the FRAP curves. (I) Quantified accumulation of GFP-PARP1 E988K at DNA damage in U2OS WT or ALC1KO cells expressing iRFP670-ALC1 variants. (J) Quantified accumulation of mCherry-PARP1 at DNA damage in ALC1KO cells ± hypotonic treatment. Graphs include all data points and mean ± SEM. Asterisks in (A) indicate P values obtained by linear regression (***P < 0.001). Model summary is provided in table S2. P values for (D) to (J) were obtained using an unpaired Student’s t test with Bonferroni correction (***P < 0.001).

  • Fig. 5 ALC1 acts upstream of DSB repair pathway choice.

    (A) Clonogenic cell survival assay of WT and ALC1KO U2OS cells transfected with siCtrl or siBRCA1 and/or si53BP1 treated or not with olaparib for 24 hours. (B) Clonogenic cell survival assay of DLD1-BRCA2+/Δ11 (BRCA2+/−) and DLD1-BRCA2Δ11/Δ11 (BRCA2−/−) cells transfected with siCtrl or siALC1 and treated or not with olaparib for 24 hours. (C to F) Quantification and representative images of Rad51 or Ku70 localization to UV-induced DNA damage sites in WT, PARP1KO, ALC1KO, and PARP1KO ALC1KO double knockout cells, treated or not with olaparib (1 μM). Scale bar, 10 μm. (G) Clonogenic cell survival assay of WT and YFP-ALC1 overexpressing U2OS cells transfected with siCtrl or siBRCA1 and treated or not with olaparib for 24 hours. (H) Clonogenic cell survival assay of DLD1-BRCA2+/Δ11 (BRCA2+/−) and DLD1-BRCA2Δ11/Δ11 (BRCA2−/−) cells transfected with GFP-ALC1 and treated or not with olaparib for 24 hours. Graphs in (A), (B), (G), and (H) include all data points (n = 3) and fitted curves with 95% confidence intervals (gray shading). Graphs in (C) and (E) include all data points and mean ± SEM (n = 3). Asterisks indicate P values obtained by polynomial (A, B, G, and H) or linear regression (C and E) (*P < 0.05, **P < 0.01, and ***P < 0.001). Model summary is provided in table S2.

  • Fig. 6 A model for the role of ALC1 in olaparib-mediated synthetic lethality.

    PARP1 is recruited to sites of DNA damage where it PARylates proteins in and around the break site, including itself. PARP1 removal from sites of damage involves a combination of autoPARylation and ALC1-dependent mobilization, which allows the recruitment of essential subsequent repair actors such as Ku70 or Rad51. While the impairment of either of the two modes of PARP1 mobilization does not have major deleterious consequences, inhibiting autoPARylation in ALC1-deficient cells fully blocks PARP1 release from DNA lesions, thus preventing the recruitment of downstream repair factors and ultimately leading to cell death.

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