Targeting OGG1 arrests cancer cell proliferation by inducing replication stress

Abstract

Altered oncogene expression in cancer cells causes loss of redox homeostasis resulting in oxidative DNA damage, e.g. 8-oxoguanine (8-oxoG), repaired by base excision repair (BER). PARP1 coordinates BER and relies on the upstream 8-oxoguanine-DNA glycosylase (OGG1) to recognise and excise 8-oxoG. Here we hypothesize that OGG1 may represent an attractive target to exploit reactive oxygen species (ROS) elevation in cancer. Although OGG1 depletion is well tolerated in non-transformed cells, we report here that OGG1 depletion obstructs A3 T-cell lymphoblastic acute leukemia growth in vitro and in vivo, validating OGG1 as a potential anti-cancer target. In line with this hypothesis, we show that OGG1 inhibitors (OGG1i) target a wide range of cancer cells, with a favourable therapeutic index compared to non-transformed cells. Mechanistically, OGG1i and shRNA depletion cause S-phase DNA damage, replication stress and proliferation arrest or cell death, representing a novel mechanistic approach to target cancer. This study adds OGG1 to the list of BER factors, e.g. PARP1, as potential targets for cancer treatment.

Figure 1.

OGG1 knockdown is selectively toxic in oncogene-expressing cells and cancer cell lines. (A) Viability of BJ fibroblast cells immortalized with telomerase (BJ-Tert) or telomerase, SV40 large T protein and HRAS G12V oncogene (BJ-Ras) after OGG1 depletion for 5 days. Data are average ±SD of 10 technical replicates representative of two independent experiments. (B) Clonogenic survival of BJ-Tert and BJ-Ras cells following OGG1 depletion. Data are average ±SD of 8–10 replicates from three independent experiments. (C) Representative immunoblot analysis of OGG1 expression in BJ-Tert and BJ-Ras cells 48 h after transfection with siRNA. (D) Clonogenic survival of H460 cells stably transfected with doxycycline inducible shRNA constructs targeting OGG1 (sh1–sh3) or a non-specific sequence (shNT). Cells were seeded in the presence or absence of 500 ng/ml doxycycline and colonies were enumerated after 10–12 days. Data are average ±SD of six technical replicates from two independent experiments. All values are normalized to the number of colonies in medium free of doxycycline. (E) Proliferation of A3 cells stably transfected with doxycycline-inducible shRNA constructs targeting OGG1 (sh2–3) or a non-specific sequence (NT). Cells were seeded in medium with or without 250 ng/ml doxycycline and counted daily. Cultures reaching a density of more than one million cells per ml were added fresh medium to maintain cell growth and normalized to the starting density. (F) Representative immunoblot analysis of OGG1 expression in H460 and A3 cells stably transfected with shRNA constructs targeting OGG1 and treated with doxycycline as in D and E. Data are average ±SD of four technical replicates from two independent experiments. Statistical significance was determined using unpaired, two-sided t-tests (**P < 0.01, ***P < 0.001 and ****P < 0.0001), in all cases comparing against the distribution of the corresponding non-specific RNAi-sequence.

Figure 2.

OGG1 knockdown reduces tumor growth in vivo. (A) A3 cells harbouring luciferase and a doxycycline-inducible shRNA construct targeting OGG1 or a non-specific sequence were injected subcutaneously in mice. Doxycycline was added to the drinking water at day 7, and tumor growth were monitored twice a week thereafter. Data are average ± SD, n = 8 per group. (B) Bioluminescence of luciferase expressing A3 cells in five representative mice imaged 28 days after grafting. (C) Survival of animals grafted with A3 cells. Mice were euthanized when tumor size reached 1000 mm³.

Figure 3.

The OGG1 inhibitor TH5487 is selectively toxic to oncogene-expressing cells and cancer cell lines. (A) Close-up view of ligand TH5487 (green) binding to human OGG1. Important residues in the binding site are marked, hydrogen bond interactions are shown in black dashed line, water (red sphere)-mediated interactions in grey dashed line. (B) Comparison between the binding of ligand TH5487 (green) to human OGG1 (gray) with the structure of TH5675 (pink) bound to mouse OGG1 (yellow, PDB 6G3Y). (C) Viability of BJ-Tert and BJ-Ras cells exposed to the indicated concentrations TH5487 for five days. Data are average ±SD of four technical replicates from two independent experiments. (D) Clonogenic survival of cell lines exposed to TH5487. The cancer cell lines ACHN and H460, and the non-transformed cell lines MRC5 and Ogg1^−/− mouse embryonic fibroblasts (MEF) were incubated for 6–11 days in the presence of the indicated concentrations TH5487, followed by colony enumeration. Data are average ±SD values of four technical replicates, representative of three independent experiments. (E) TH5487 selectively decreases viability of cancer cell lines. EC50-values of 34 cancer (red) and 7 non-transformed cell lines (blue). Cells were exposed to a dilution series of TH5487 for five days followed by a viability assessment using resazurin. All cell lines were tested in two to ten independent experiments. Each point represents the EC50-value from one experiment (average of two or three technical replicates). (F) Comparative analysis of EC₅₀ values for TH5487 in cancer- and non-transformed cell lines, and CD34+ hematopoietic stem cells, CD34- fraction from cord blood or PBMCs from healthy donor blood. All primary blood cells were tested upon activation with Dynabeads/Phytohemagglutinin or not and exposed to a dilution series of TH5487 to calculate EC₅₀ values by resazurin assay. Data for blood cells are average ±SD from two to four independent donors, and are significantly different from the hematological cancer cell lines tested, using a two-sided unpaired t-test (*P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001, ns, non-significant).

Figure 4.

Anticancer properties overlap between OGG1 loss and OGG1 inhibitors. (A) Viability of T-cell acute leukoblastic leukemia cell lines treated for 5 days with the indicated doses TH5487. Data are average ±SD of independent experiments (A3, n = 5; Jurkat, n = 3; MOLT-4, n = 4; CCRF-CEM, n = 3). (B) Relative cell numbers and (C) viability (%) for A3 cells treated for 5 days with 10 μM TH5487. Data are average ±SD of six replicates from three independent experiments. (D) Induction of apoptosis in A3 cells treated for 72 h TH5487 and stained for Annexin V. Data are average ±SD of three independent experiments. (E) A3 cells transfected with shRNA targeting OGG1 were treated with doxycycline for 6 days and stained for Annexin V. Data are average ±SD of two independent experiments. (F) Scheme for recovery experiment in which A3 OGG1-shRNA cells or A3 cells were silenced or inhibited for 6 days with doxycycline (200 nM) or TH5487 (10 μM). Then, 250 000 cells/ml were seeded in fresh media and recovered for 24 h and 144 h by measuring cell number (G) and viability (H). Data are average ±SD of 2–6 technical replicates representative of two independent experiments (I). Scheme for off-target effect evaluation in which A3 OGG1-shRNA cells were exposed to doxycycline for six days and after that, cells were exposed to TH5487 (10 μM) for 48 h followed by measurements of cell numbers (J) and viability by trypan blue exclusion (K). Data are average ±SD of two technical replicates from four independent experiments. Statistical significance was determined using unpaired, two-sided t-tests (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns, non-significant).

Figure 5.

Effect of TH5487 on DNA lesions, DNA damage markers and DNA replication. (A) 8-oxodG accumulation in A3 cells. A3 cells were treated with 20 mM KBrO₃ for 1 h or with 10 μM TH5487 for the indicated times, and the amount of genomic 8-oxodG was quantified with LC-MS/MS. Data are average ±SD of five replicates from two independent experiments. (B) Comet assay. A3 cells were treated with 10 μM TH5487 for 72 h and strand-breaks and OGG1 substrate lesions were analyzed with the OGG1-modified Comet assay. Representative images of cells are shown. (C) Violin blot of Comet tail moment. Cells were treated as in B, and the tail moment of the cells were analyzed using blinded automatic analysis (n = 200 per condition from two independent experiments). The full line indicates median, and the dotted lines quartiles. (D) Violin blot of phosphorylated γH2AX intensity. A3 cells were treated with 0.1% DMSO or 10 μM TH5487 for the indicated times and stained for phosphorylated γH2AX. Treatment with 2 mM Hydroxyurea for 1 h was used as positive control. At least 33 000 nuclei per group were quantified from three independent experiments. The full line indicates median, and the dotted lines quartiles. All values are normalized to that of the mean value of the 1 h non-treated sample. (E) Relative induction of positive γH2AX cells in OGG1 shRNA depleted cells for 72 h or OGG1 inhibited with 10 μM of TH5487 for 24, 48 and 72 h. Data are average ±SD of two technical replicates representative of 1–2 independent experiments (F) Relative cell cycle distribution of positive γH2AX gated cells along G1, S or G2/M cell cycle phases after 72 h treatment with 10 μM TH5487. Data are average ±SD of two technical replicates from two independent experiments. (G) Experimental setup of DNA fiber assay. A3 cells were treated with 0.1% DMSO or 10 μM TH5487 for 48 h or 72 h, alternatively A3 shOGG#2 were treated with doxycycline for 48 or 96 h followed by addition of 5-chloro-2′-deoxyuridine (CldU) or 5-iodo-2′-deoxyuridine (IdU) to the medium. Representative images of DNA replication fibers are shown. (H) Distribution of fork speed in CldU-labelled A3 cells treated with DMSO or TH5487 for 48 or 72 h. (I) Total fork speed in DMSO and TH5487-treated cells. (J) Distribution of fork speed in CldU-labelled A3 shOGG1#2 cells treated or not with doxycycline for 48 or 96 h. (K) Total fork speed in A3 shOGG1#2 cells treated or not with doxycycline. Data shown as average ± SD from three independent experiments. At least 300 forks were scored per condition. Statistical significance was determined using unpaired, two-sided t-tests (**P < 0.01, ***P < 0.001 and ****P < 0.0001; ns, non-significant).

Figure 6.

TH5487 treatment induces a downregulation of DNA replication genes (A) Gene Set Enrichment analysis plot of the DNA Replication Gene Ontology Gene Set. A3 shNT cells were treated with 10 μM TH5487 for 24 h or DMSO as control, and RNA sequencing was performed. Genes with a mean normalized count > 5 were ranked by log2 fold change. Genes towards the top of the ranked gene list are upregulated after TH5487 treatment, genes with higher ranks are downregulated. NES: normalized enrichment score, P value has been adjusted for multiple testing using the Benjamini–Hochberg method. (B) Log₂ fold changes in expression level of DNA Repair genes of TH5487 treated A3 control cells compared to DMSO treated cells. Only genes with a mean normalized count >5 were analyzed. Visualization of the KEGG DNA Replication pathway. (C) Heatmap of the leading edge genes from the DNA Replication Gene Ontology Gene Set. Genes and samples were unsupervisedly clustered using hierarchical clustering (euclidean distance, complete linkage). Rows indicate technical replicates (n = 3). A negative z-score indicates relative downregulation, a positive relative upregulation of the gene. (D) Relative mRNA expression levels of replication-associated genes in OGG1-depleted cells (normalized to A3 shNT and expressed in percentage). A3 shNT or sh2-cells were treated with doxycycline for 72 h. Data shown as average ± SEM from four technical replicates from two independent experiments. (E) Oxidation at MCM4 promoter. The indicated cells were treated with 10 μM TH5487 or 250 ng/ml doxycycline for 72 and 96 h, respectively, DNA was purified and treated or not with OGG1 followed by PCR amplification of the MCM4 promoter. Data shown are the difference in Ct values caused by OGG1 enzymatic treatment or buffer. Data represent average ± SEM of three technical replicates from two independent experiments. (F) Relative expression of MCM4 mRNA levels in OGG1 perturbed cells expressed in percentage. Cells were treated as in D. and data represent average ± SEM 3–4 technical replicates from two independent experiments. Statistical significance was determined using a two-sided unpaired t-test (ns, non-significant, *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001).

Figure 7.

OGG1 inhibitors does not inhibit tumor growth in vivo. (A) Lack of TH5487 efficacy in vivo. A3 cells were injected subcutaneously in mice, allowed to graft for three days and then administered 20–40 mg/kg TH5487 orally, once a day on weekdays. Twelve days into the treatment, the dose was increased to 40 mg/kg. Data are average ± SD (n = 9 per treatment group). (B) TH5487 target engagement in A3 xenograft. Tumors were excised following termination, and TH5487 target engagement was analyzed by cellular thermal shift assay, using an antibody specific for the human OGG1 protein. Data are average ± SD (n = 4 different tumors per group). (C) Effect of bovine serum albumin on OGG1 inhibition by TH5487. A dilution series of TH5487 was incubated with OGG1 enzyme and a DNA reporter oligonucleotide containing an OGG1-substrate, in the presence or absence of 1% (w/v) bovine serum albumin. Data are average ± SD of four technical replicates from two independent experiments.