Unprocessed genomic uracil as a source of DNA replication stress in cancer cells

Abstract

Alterations of bases in DNA constitute a major source of genomic instability. It is believed that base alterations trigger base excision repair (BER), generating DNA repair intermediates interfering with DNA replication. Here, we show that genomic uracil, a common type of base alteration, induces DNA replication stress (RS) without being processed by BER. In the absence of uracil DNA glycosylase (UNG), genomic uracil accumulates to high levels, DNA replication forks slow down, and PrimPol-mediated repriming is enhanced, generating single-stranded gaps in nascent DNA. ATR inhibition in UNG-deficient cells blocks the repair of uracil-induced gaps, increasing replication fork collapse and cell death. Notably, a subset of cancer cells upregulates UNG2 to suppress genomic uracil and limit RS, and these cancer cells are hypersensitive to co-treatment with ATR inhibitors and drugs increasing genomic uracil. These results reveal unprocessed genomic uracil as an unexpected source of RS and a targetable vulnerability of cancer cells.

Keywords: ATR; BER; PrimPol; UNG; cancer therapy; gaps; lung cancer; permetrexed; replication fork; replication stress; ssDNA gaps; synthetic lethality; uracil.

Figure 1.. UNG2-mediated processing of genomic uracil reduces ATRi sensitivity.

(A) A schematic of RS induction by BER intermediates—AP sites and SSBs. (B) Quantification of 8-oxoG immunofluorescence in cells treated with DMSO or 10 μM TH5487 for 6 h (n>900 cells per condition). One of two independent experiments is shown. (C) U2OS cells were treated with DMSO or 10 μM TH5487 and indicated concentrations of ATRi (VE-821) for 5–7 days. Cell viability was determined using CellTiter-Glo. Data are shown as mean ± s.d. (n=2 independent experiments). (D) Levels of UNG in U2OS cells transfected with the indicated siRNAs for 48 h. MCM2 serves as a loading control. (E and F) Representative images for U-comet assay in U2OS cells transfected with indicated siRNAs. Dot plots represent comet tail moment in individual cells, and bars display the mean in cell populations (n>70 cells per condition). ns: non-significant. ***: P ≤ 0.001. One of three experiments is shown. (G) Viability of U2OS cells transfected with indicated siRNAs and treated with indicated concentrations of ATRi (VE-821). Data are shown as mean ± s.d. (n=2 independent experiments). (H) Levels of UNG2 in U2OS cells and two independent UNG2^−/− knockout (KO) clones. MCM2 serves as a loading control. (I) Viability of U2OS or UNG2 KO cells treated with indicated concentrations of ATRi (VE-821). Data are shown as mean ± s.d. (n=2 independent experiments). (J) Levels of the indicated proteins in U2OS cells transfected with UNG#1 and SMUG1 siRNAs for 48 h. MCM2 serves as a loading control. (K) U2OS cells transfected with UNG#1 and SMUG1 siRNAs were analyzed by U-comet assay (n>80 cells per condition). One of two experiments is shown. (L) Viability of U2OS cells transfected with UNG#1 and SMUG1 siRNAs and treated with indicated concentrations of ATRi (VE-821). Data are shown as mean ± s.d. (n=3 independent experiments).

Figure 2.. Unprocessed genomic uracil interferes with replication fork progression.

(A) U2OS cells were transfected with indicated concentrations of UNG siRNA#1 for 48 h. Levels of UNG2 were analyzed by western blot, and replication rate were analyzed by DNA fiber assay (n=100 fibers per condition). MCM2 serves as a loading control. One of three independent experiments is shown. ***: P ≤ 0.001. (B) The distribution of replication rate in U2OS cells treated with indicated siRNAs (n=125 fibers per condition). One of two experiments is shown. (C) Replication rate in U2OS cells treated with indicated concentrations of Pemetrexed (PMX) for 24 h (n=125 fibers per condition). One of two experiments is shown. (D and E) Replication rate in U2OS cells transfected with control or UNG#1 siRNA for 48 h and treated with 200 nM PMX (D) or 10 μM TAS-114 (E) for 24 h (n=125 fibers per condition). One of two experiments is shown. (F and G) U2OS cells were transfected with dUTPase (DUT) and UNG#1 siRNAs for 48 h. Levels of dUTPase and UNG2 were analyzed by western blot (F). MCM2 serves as a loading control. Replication rate was analyzed by DNA fiber assay (n=125 fibers per condition) (G). One of two experiments is shown.

Figure 3.. Genomic uracil induces PrimPol-dependent ssDNA gaps in nascent DNA.

(A and B) U2OS cells transfected with control or UNG#1 siRNA and treated with 100 nM PMX for 24 h were analyzed by PLA using anti-Pol ε catalytic subunit and anti-RPA32 antibodies. Representative images of PLA foci are shown (A) and PLA foci are quantified in individual cells (B) (n>400 cells per condition). Scale bar, 10 μM. **: P ≤ 0.01. ***: P ≤ 0.001. One of three independent experiments is shown. (C) U2OS cells were transfected with control or UNG#1 siRNA and treated with indicated concentrations of PMX for 24 h. Chromatin fractions were prepared and blotted for PrimPol. MCM2 serves as a loading control. (D) U2OS cells were transfected with control or UNG#1 siRNA for 48h and analyzed by DNA fiber assay. DNA fibers were treated with S1 nuclease (20 U/mL) for 30 min at 37°C, and the length of IdU tracts (n=125 fibers per condition) was measured. One of two experiments is shown. (E) U2OS cells were transfected with UNG#1 and PrimPol siRNA for 48 h and analyzed as in (D) (n=125 fibers per condition). One of two experiments is shown. Right, ratio of IdU tracts from S1-treated to -untreated fibers. Data are displayed as mean ± s.d. from two experiments. *: P ≤ 0.05. (F) U2OS cells transfected with control or UNG#1 siRNA were treated with 200 nM PMX for 24 h and analyzed as in (D) (n=125 fibers per condition). One of three experiments is shown. (G) U2OS cells transfected with control or UNG#1 siRNA were treated with 10 μM TAS-114 for 24 h and analyzed as in (D) (n=125 fibers per condition). One of two experiments is shown.

Figure 4.. Exogenous dUTP supplementation induces replication stress.

(A) A schematic of import of Cy3-dUTP into live cells by Bio-Tracker (BT). U2OS cells were treated with 2 μM BT and 2 μM Cy3-dUTP for 24 h in Leibovitz’s L-15 Medium and analyzed by fluorescence. After 24 h, cells were analyzed by DNA fiber to visualize the incorporation of Cy3-dUTPs to genomic DNA. (B) U2OS cells were treated with 2 μM BT and 2 μM Cy3-dUTP for 24 h and analyzed by U-comet assay (n>50 cells per condition). One of two experiments is shown. (C) Replication rate in U2OS cells treated with 2 μM BT and indicated concentrations of Cy3-dUTP for 24 h (n=125 fibers per condition). One of two experiments is shown. (D) Replication rate in U2OS cells treated with 2 μM BT, 2 μM Cy3-dUTP and indicated concentrations of dTTP for 24 h (n=125 fibers per condition). One of two experiments is shown. (E) Replication rate in U2OS cells treated with 2 μM BT, 2 μM unlabeled dUTP and indicated concentrations of dTTP for 24 h (n=125 fibers per condition). One of two experiments is shown. (F) U2OS cells transfected with control or PrimPol siRNA were treated with 2 μM BT and 2 μM Cy3-dUTP for 24 h. DNA fibers were treated with S1 nuclease and the length of IdU tracts (n=125 fibers per condition) was measured. One of two experiments is shown. (G) U2OS cells were transfected with control or UNG#1 siRNA, treated with 2 μM BT and 2 μM Cy3-dUTP for 24 h, and analyzed as in (F) (n=125 fibers per condition). One of two experiments is shown.

Figure 5.. ATRi prevents repair of uracil-induced ssDNA gaps and increases DNA damage

(A) U2OS cells were transfected with control or UNG#1 siRNA, sequentially labeled with CldU and IdU, and cultured in the presence of absence of 10 μM ATRi (VE-821) for 0, 8 or 24 h. DNA fibers were treated with S1 nuclease and the length of IdU tracts (n=125 fibers per condition) was measured. One of two experiments is shown. (B) U2OS cells transfected with control or UNG#1 siRNA and treated with 2 μM ATRi (VE-821) for 48 h. 53BP1 nuclear bodies (NBs) in Cyclin A-negative G1 cells were quantified (n>1200 cells per condition). One of two experiments is shown. (C) Model for the induction of DNA damage by ATRi in cells lacking UNG2. (D) U2OS cells were transfected with indicated siRNAs, labeled with 5 μM EdU for 15 min, and then treated with 10 μM ATRi (VE-821) for 24 h. γH2AX intensity in EdU-positive cells was quantified (n>250 cells per condition). One of two experiments is shown. (E) U2OS cells were transfected with UNG#1, labeled with 5 μM EdU for 15 min, then treated with 10 μM ATRi (VE-821) +/− 10 μM CDK1i (RO-3306) for 24 h, and analyzed as in (E) (n>250 cells per condition). One of three experiments is shown. (F) U2OS cells transfected with UNG#1 and PrimPol siRNA were treated and analyzed as in (D) (n>250 cells per condition). One of three experiments is shown. (G) Viability of U2OS cells transfected with UNG#1 and PrimPol siRNA and treated with indicated concentrations of ATRi (VE-821). Data are shown as mean ± s.d. (n=2 independent experiments).

Figure 6.. A subset of cancer cells rely on UNG2 to suppress replication stress.

(A) Levels of UNG2 in the indicated cell lines. α-tubulin serves as a loading control. (B) The indicated cell lines were transfected with control or UNG#1 siRNA and treated with indicated concentrations of ATRi (AZD6738). Cell viability was analyzed with CellTiter-Glo in 5–7 days. Data are shown as mean ± s.d. (n=4 independent experiments). (C) H1299 and H1838 cells transfected with control or UNG#1 siRNA were analyzed by U-comet assay (n>50 cells per condition). One of two experiments is shown. (D) H1299 and H1838 cells were transfected with control or UNG#1 and PrimPol siRNAs for 48 h. DNA fibers were treated with S1 nuclease and the length of IdU tracts (n=125 fibers per condition) was measured. One of two experiments is shown. (E) Viability of H1299 and H1838 cells transfected with UNG#1 and PrimPol siRNA and treated with indicated concentrations of ATRi (AZD6738). Data are shown as mean ± s.d. (n=2 independent experiments).

Figure 7.. Induction of genomic uracil sensitizes UNG-dependent cancer cells to ATRi.

(A) Viability of H1299 and H1838 cells treated with indicated concentrations of PMX and ATRi (VE-821). Data are shown as mean ± s.d. (n=2 independent experiments). (B) Viability of H1299 cells treated with 100 nM PMX, 50 μM THY, and indicated concentrations of ATRi (VE-821) (n=2 independent experiments). (C) Growth curves of H1299 tumors in control mice and mice treated with 50 mg/kg ATRi (AZD6738) and/or 75 mg/kg PMX. n=4 mice in each group. Two-way ANOVA with Tukey’s multiple comparisons test was used to measure the significance throughout. (ns) non-significant; (*) P ≤ 0.05; (**) P ≤ 0.01; (***) P ≤ 0.001. (D) Viability of MGH 707.1, MGH 134.1 and MGH 143–3B cells treated with 200 nM PMX and indicated concentrations of ATRi (AZD6738) (n=2 independent experiments). Levels of UNG2 in the indicated cells were analyzed by western blot. α-tubulin serves as a loading control. (E) Model for RS induction by different types of base lesions. (F) Model for RS induction by unprocessed uracil and the effects in the presence and absence of ATRi.