Cooperation of the ATM and Fanconi Anemia/BRCA Pathways in Double-Strand Break End Resection

Abstract

Cells deficient in ataxia telangiectasia mutated (ATM) are hypersensitive to ionizing radiation and other anti-cancer agents that induce double-strand DNA breaks. ATM inhibitors may therefore sensitize cancer cells to these agents. Some cancers may also have underlying genetic defects predisposing them to an ATM inhibitor monotherapy response. We have conducted a genome-wide CRISPR screen to identify genetic vulnerabilities that sensitize lung cancer cells to ATM inhibitors. Knockout of genes in the Fanconi anemia (FA)/BRCA pathway results in hypersensitivity to the ATM inhibitor M3541. Knockdown of either an FA gene or of ATM results in reduced double-strand break end resection, enhanced non-homologous end joining (NHEJ) repair, and decreased homologous recombination repair. Knockout of both the FA/BRCA pathway and ATM strongly inhibits end resection and generates toxic levels of NHEJ, thereby elucidating a mechanism of cellular death by synthetic lethality. ATM inhibitors may therefore be useful for the treatment of tumors with a defective FA/BRCA pathway.

Keywords: ATM inhibitor; CRISPR sgRNA screening; Fanconi anemia pathway; NHEJ; end resection.

Figure 1.. Whole Genome-wide CRISPR Screens Identify Determinants of ATMi Sensitivity

(A) Schematic of the genome-wide CRISPR-Cas9 screens in lung cancer lines. (B) Venn diagram of all high-confidence hits in the two cell lines (left panel: STAR score at ≥ 4 in one line and STAR score at ≥ 2 in both lines). Gene Ontology (GO) terms significantly enriched in hits common to the two-cell lines (right panel). (C) esyN network analysis of interactions between hits common to the two cell lines. (D) Gene-centric visualization of average log2 fold change (LFC) in ATMi (M3541)- versus DMSO-treated in both cell lines. See also Figure S1.

Figure 2.. Loss of FA Pathway Confers Sensitivity to ATM Inhibition in Cancer Cells

(A–C) Clonogenic assays of control and knockout (KO) cell pools after treatment with ATM inhibitor (M3541) following transduction with FAAP24- (A), FAAP100- (B), and FANCF-targeting (C) sgRNAs (3 sgRNAs per gene). Survival assays in A549 cells (left panels) and H460 cells (right panels). #1, #2 and #3 indicate three independent sgRNAs for targeting each gene. (D) Survival assays of HeLa and U2OS cells after treatment with M3541 in stable clones of FANCA and FANCD2 knockout via CRIPSR-Cas9. Clonogenic survival assays in HeLa cells (left panel) and U2OS cells (right panel). Data are shown as mean ± SEM from three independent experiments. See also Figure S2.

Figure 3.. Inhibition of ATM Impairs RAD51 and RPA Focus Formation and Results in DNA Damage in FA-Deficient Cells

Immunofluorescence and quantification of the percentage of cells with more than 10 γ-H2AX (A), RAD51 (B), RPA (C), and 53BP1 (D) foci 3 h after treatment with irradiation (5 Gy) following 24 h exposure to DMSO or M3541 (1 μM) in control and FANCA-KO cells: representative images (left panels) and graphical quantitation of foci in HeLa cells (middle panels) and U2OS cells (right panels). One hundred nuclei were randomly counted. Data are shown as mean ± SEM from three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 on unpaired two-tailed Student’s t test. See also Figure S3.

Figure 4.. Inhibition of ATM Decreases DNA End Resection Machinery in FA-Pathway-Deficient Cells

(A) Immunofluorescence and quantification of the percentage of cells with BrdU-positive cells 6 h after treatment with irradiation (5 Gy) following 24 h exposure to DMSO or M3541 (1 μM) in control and FANCD2-KO U2OS cells: representative images (left panel) and graphical quantitation of BrDU-positive cells (right panel). One hundred nuclei were randomly counted. *p < 0.05, unpaired two-tailed Student’s t test. Data are shown as mean ± SD from three independent experiments. (B) The schematic of in vitro end-resection assay in U2OS cells (left panel). ssDNA was quantified by qPCR at 335 bp or 1,618 bp downstream of the AsiSI-induced break site after indicated siRNA transfection and DSB induction (with 4-hydroxytamoxifen) or no DSB induction (without 4-hydroxytamoxifen) (right panel). Data are shown as mean ± SEM compiled from three independent experiments. *p < 0.05, **p < 0.01. (C) Change in ssDNA length of control and FANCD2^−/− U2OS cells 3 h after treatment with irradiation (5 Gy) following overnight exposure to 1 μM ATMi. Representative images of SMART assay (top panel) and the quantification (relative to untreated, bottom panel). In total, 100 DNA fibers were assessed per condition. *p < 0.05, **p < 0.01, ***p < 0.001, unpaired two-tailed Student’s t test. NS, not significant (bottom panel). (D) Immunoblots of the indicated proteins in subcellular fractions of control and FANCD2^−/− U2OS cells after treatment with 1 μM ATMi for 6 h. Quantification of the ratio of total and chromatin bound CtIP in control versus FANCD2^−/− U2OS cells is shown. See also Figure S4.

Figure 5.. Inhibition of ATM Results in Increased NHEJ and Decreased HR in FA-Pathway-Deficient Cells

U2OS cells stably carrying the EJ5-GFP (A) and DR-GFP (B) reporter were transfected with the indicated siRNAs and were treated with 1 μM ATMi overnight 36 h after siRNA transfection. Subsequently, the cells were exposed to I-Scel adenovirus for 6 h and then cultured with fresh medium. GFP-positive cells were quantified 72 h later by FACS. siRNA knockdown efficiency was assessed by western blotting assay (left panels). Quantitation for the HR activity and NHEJ activity as % of GFP positive cells is shown (right panels). Data are shown as mean ± SEM from three independent experiments. *p < 0.05 and **p < 0.01 on unpaired two-way ANOVA test. See also Figure S5.

Figure 6.. Inhibition of ATM Results in Chromosomal Aberrations and Radials in FA-Pathway-Deficient Cells

(A and B) Representative images (left panels) and quantification (right panels) of chromosomal aberrations (A) and radial chromosomes (B, indicated by arrows) per cell on metaphase spreads of PD326 (FANCG-deficient) cells 48 h after treatment with 5 ng/ml Mitomycin C (MMC) following 24 h exposure to 1 μM DMSO, 1 μM ATMi, or 1 μM ATMi plus 5 μM Lig4 inhibitor (SCR7). At least 50 cells were counted in each experiment, and the average of 50 cells is shown. (C) Quantification of chromosomal aberrations (left panel) and radial chromosomes (right panel) per cell as a measure on metaphase spreads 48 h after treatment with 5 ng/ml MMC following 24 h exposure to 1 μM DMSO, 1 μM ATMi, or 1 μM ATMi plus 5 μM Lig4 inhibitor (SCR7) in PD326 FANCG-corrected cells. See also Figure S6.

Figure 7.. ATM Inhibition Shows Greater Cytotoxicity with FA Pathway Deficiency

(A) Western blot showing FANCC expression (left panel) and quantitation of CellTiter-Glo cell viability assay (right panel) in FANCC mutant PL11 cells and theisogenic FANCC-corrected cells 72 h after M3541 treatment. (B) Western blot showing FANCG expression (left panel) and quantitation of CellTiter-Glo cell viability assay (right panel) in FANCG mutant HS766T cells and theisogenic FANCG-corrected cells 72 h after M3541 treatment. (C) Western blot showing FANCF expression (left panel) and quantitation of CellTiter-Glo cell viability assay (right panel) in FANCF mutant TOV-21G cells and theisogenic FANCF-corrected cells 72 h after M3541 treatment. (D and E) Quantitation of the CellTiter-Glo cell viability assay in BRCA1-KO RPE (D) and MB436 (E) (parental and olaparib-resistant) cells exposed to graded concentrations of the PARP inhibitor olaparib in the presence or absence of the ATMi M3541 (1 μM). See also Figure S7.