ATRX cooperates with TOP2B for replication fork stability and DNA damage response through G-quadruplex regulation

Abstract

G-quadruplexes (G4s) are noncanonical DNA structures that promote genomic instability, particularly in α-thalassemia/mental retardation X-linked (ATRX)-deficient gliomas. Although TOP2B has been implicated in chromatin remodeling, its role in G4 resolution remains poorly understood. Here, we identify TOP2B as a previously unrecognized regulator of G4 homeostasis and show that it functionally cooperates with ATRX to facilitate G4 resolution during DNA replication. Disruption of this pathway by CX-5461, a small molecule originally developed as an RNA polymerase I inhibitor, leads to G4 accumulation, replication stress, and DNA damage. Mechanistically, CX-5461 acts as a TOP2B poison that selectively impairs TOP2B binding at G4 sites, alters replication fork dynamics, and induces MRE11-dependent degradation of stalled forks. These effects are strongly enhanced in ATRX-deficient glioma cells, where TOP2B plays a dominant role in G4 regulation. While etoposide similarly induces G4-related DNA damage, it does not affect the ATRX-TOP2B interaction, highlighting CX-5461's unique mechanism. Our findings establish TOP2B as a critical player in G4 resolution, reveal CX-5461's dual function as a TOP2B poison and G4 stabilizer, and propose G4-associated replication stress as a potential therapeutic target in ATRX-deficient gliomas.

Graphical Abstract

Figure 1.

TOP2B is associated with G4 structures in glioma PDCs. (A) Workflow for generating patient-derived glioma cells (PDCs) from fresh surgical samples. Tumor tissues were minced, enzymatically dissociated with collagenase IV, filtered, and cultured. (B) Schematic of the CUT&Tag assay used to map G4 regions and TOP2B-bound chromatin regions. Antibody-bound pA-Tn5 transposase inserts sequencing adaptors at target loci in situ, followed by DNA extraction and sequencing. (C) Pie chart showing the percentage of TOP2B-enriched regions that overlap with G4 peaks (19 897/54 494, 36.51%). (D) Dot plots displaying TOP2B CUT&Tag average profiles in PDCs at G4 and non-G4 sites. Error bars, mean ± SEM; ***P < 0.001, Mann-Whitney test. (E) TOP2B coverage at TSS in G4-containing versus non-G4 regions. (F) Genome browser tracks illustrating TOP2B peaks overlapping with G4 peaks in chromatin. TOP2B exhibits higher enrichment at G4 regions compared to non-G4 regions. (G) Metagene analysis of TOP2B and G4 distribution across gene bodies. Heatmap shows read counts spanning ±3 kb around gene bodies. (H) Pie chart showing the genomic annotation of TOP2B and G4-enriched regions in glioma PDCs. (I) Motif discovery analysis of TOP2B binding regions using HOMER (±250 bp from summit), revealing sequence motif characteristics. (J) PLA of TOP2B and G4 in U87MG, U251 cell lines, and glioma PDCs; scale bars: 10 μm. Green, PLA signal (TOP2B + G4); red, TOP2B staining; blue, DAPI nuclear staining. (K) Co-immunofluorescence (Co-IF) analysis of TOP2B and EdU in control and TOP2B knockdown U87MG cells; scale bars: 10 μm. TOP2B intensity per nucleus was quantified in 100 cells per condition from three independent experiments. The percentage of EdU-positive cells was calculated from five independent experiments. Error bars, mean ± SEM. ***P < 0.001, Mann-Whitney test and Student’s t-test. (L) Co-IF analysis of G4 and EdU in control and TOP2B knockdown U87MG cells; scale bars: 10 μm. Quantification of G4 intensity per nucleus was performed in 100 EdU-positive and EdU-negative cells per condition across three independent experiments. Error bars, mean ± SEM. ns, not significant; ***P < 0.001, Kruskal-Wallis test.

Figure 2.

CX-5461 disrupts TOP2B chromatin binding and enhances G4 accumulation. (A) Schematic diagram of the rapid approach to DNA adduct recovery (RADAR) assay. Left: Nuclei contain DNA and proteins, some of which are covalently bound (e.g., TOP2B in DPCCs). Middle: DPCCs are isolated along with free DNA. Right: Specific DPCCs (TOP2Bcc) are detected using an antibody. Colored circles represent nuclear proteins. (B) Detection of TOP2B-DPCC (TOP2Bcc) by RADAR assay. Slot blot comparing TOP2B signal in whole-cell extract (WCE) or DPCCs isolated from U87MG cells treated with 5 μM CX-5461 for different durations (0, 0.5, 1, 2, and 3 h; left) or with increasing concentrations (0, 0.5, 1, 5, and 10 μM) of CX-5461 for an hour (right). (C) Band-depletion assay for CX-5461-treated U87MG and U251 cells. Lanes 1-4 contain proteins equivalent to 40, 20, 10, and 5 thousand cells, respectively. For all other lanes, protein equivalent to 40 thousand cells was loaded. (D) Band-depletion assay for etoposide-treated U87MG cells as a positive control. Lanes 1-4 contain proteins equivalent to 40, 20, 10, and 5 thousand cells, respectively. For all other lanes, protein equivalent to 40 thousand cells was loaded. (E) Venn diagram showing the overlap of TOP2B CUT&Tag peaks between control and CX-5461-treated glioma PDCs. Metagene analysis of TOP2B peak distribution across summits post-CX-5461 treatment. Heatmap displays read counts spanning ±3 kb around summits. (F) GO enrichment analysis of genes associated with TOP2B CUT&Tag differential peaks between control and CX-5461-treated PDCs. (Gand H) Co-IF analysis of TOP2B and G4 in U87MG cells treated with vehicle or 5 μM CX-5461 for 24 h; scale bars: 10 μm. Quantification of TOP2B and G4 intensity per nucleus in 50 cells per treatment condition across five independent experiments. Error bars, mean ± SEM. ns, not significant; ***P < 0.001, Student’s t-test. (I) Dot plots of TOP2B and G4 coverages after CX-5461 treatment, obtained from TOP2B CUT&Tag and G4 CUT&Tag in PDCs. ***P < 0.001, *P < 0.05, Mann-Whitney test. (J) Bar graph depicting the number of overlapping TOP2B and G4 CUT&Tag peaks after CX-5461 treatment in PDCs. (K) Dot plots showing TOP2B CUT&Tag peaks at G4 and non-G4 regions in control and CX-5461-treated PDCs. Error bars, mean ± SEM; ***P < 0.001, Kruskal-Wallis test. (L) Genomic tracks illustrating changes in TOP2B occupancy at G4 sites near the TSS of the Clta gene following CX-5461 treatment in PDCs.

Figure 3.

ATRX and TOP2B co-localize with G4 structures and regulate genome stability. (A) Venn diagram showing the overlap of ATRX and TOP2B CUT&Tag peaks in glioma PDCs. Metagene analysis of ATRX and TOP2B peak distribution across gene bodies. (B) Genome browser tracks illustrating ATRX peaks overlapping with TOP2B peaks in chromatin. (C) PLA of ATRX and TOP2B in U87MG and U251 glioma cells; scale bars: 10 μm. Red, PLA signal (TOP2B + ATRX); blue, DAPI nuclear staining. (D) Co-IP from U87MG and U251 extracts showing ATRX interaction with TOP2B but not TOP2A. Anti-ATRX was used to pull down TOP2B and TOP2A. (E) PLA of ATRX and TOP2B in glioma PDCs (top); scale bars: 10 μm. Green, PLA signal (TOP2B + ATRX); red, ATRX staining; blue, DAPI nuclear staining. The merged image demonstrates co-localization of PLA signals with ATRX in the nucleus. Insets highlight magnified regions of interest. PLA of ATRX and TOP1 in glioma PDCs (below, as negative control); scale bars: 10 μm. Green, PLA signal (TOP1 + ATRX); red, ATRX staining; blue, DAPI nuclear staining. (F) Venn diagram showing the overlap of ATRX, TOP2B, and G4 CUT&Tag peaks in glioma PDCs. (G) Metagene analysis of ATRX, TOP2, and G4 distribution across TSS in glioma PDCs. Heatmap displays read counts spanning ±3 kb around TSS. (H) Genome browser tracks illustrating ATRX peaks and TOP2B peaks overlapping with G4 peaks across the TSS of the gene STAT2. (I) PLA of ATRX and G4 in control and TOP2B knockdown U87MG cells; scale bars: 10 μm. Green, PLA signal (ATRX + G4); blue, DAPI nuclear staining. Quantification of PLA (ATRX + G4) foci per nucleus was performed in 100 cells per condition across three independent experiments. Error bars, mean ± SEM; ***P < 0.001, Mann-Whitney test. (J) PLA of TOP2B and G4 in control and ATRX knockdown U87MG cells; scale bars: 10 μm. Green, PLA signal (TOP2B + G4); blue, DAPI nuclear staining. Quantification of PLA (TOP2B + G4) foci per nucleus was performed in 100 cells per condition across three independent experiments. Error bars, mean ± SEM; ***P < 0.001, Mann-Whitney test.

Figure 4.

CX-5461 stabilizes G4 structures by disrupting ATRX-TOP2B chromatin interactions. (A) Co-IP from U87MG cells showing that ATRX interacts with TOP2B, and this interaction is reduced by CX-5461. Reciprocal IPs were performed using anti-ATRX and anti-TOP2B antibodies. (B) PLA of ATRX and TOP2B in control and CX-5461-treated U87MG and U251 glioma cells. Green, PLA signal (TOP2B + ATRX); blue, DAPI nuclear staining; scale bars: 10 μm. PLA (ATRX + TOP2B) foci per nucleus were performed in 100 cells per condition across three independent experiments. Error bars, mean ± SEM; ***P < 0.001, Mann-Whitney test. (C) Co-IP from glioma PDCs showing that ATRX interacts with TOP2B, and this interaction is reduced by CX-5461 treatment. Immunoprecipitation was performed using the anti-ATRX antibody. (D) PLA of ATRX and TOP2B in control and CX-5461-treated glioma PDCs. Green, PLA signal (TOP2B + ATRX); red, ATRX staining; blue, DAPI nuclear staining; scale bars: 10 μm. PLA (ATRX + TOP2B) foci per nucleus were performed in 100 cells per condition across three independent experiments. Error bars, mean ± SEM; ***P < 0.001, Mann-Whitney test. (E) Pie chart showing the percentage of ATRX and TOP2B CUT&Tag overlapped peaks in control and CX-5461-treated glioma PDCs. (F) Dot plots displaying ATRX CUT&Tag average profiles in control and CX-5461-treated PDCs. Error bars, mean ± SEM; ***P < 0.001, Mann-Whitney test. (G) PLA of ATRX and G4 in control, TOP2B KD, and CX-5461-treated U87MG cells; scale bars: 10 μm. Green, PLA signal (ATRX + G4); blue, DAPI nuclear staining. Quantification of PLA (ATRX + G4) foci per nucleus was performed in 100 cells per condition across three independent experiments. Error bars, mean ± SEM; ***P < 0.001, Kruskal-Wallis test. (H) Dot plots displaying ATRX CUT&Tag average profiles in control and CX-5461-treated PDCs at G4 and non-G4 sites. Error bars, mean ± SEM; ***P < 0.001, Kruskal-Wallis test. (I) PLA of TOP2B and G4 in control, ATRX KD, CX-5461-treated, and the combination in U87MG cells; scale bars: 10 μm. Green, PLA signal (TOP2B + G4); blue, DAPI nuclear staining. Quantification of PLA (TOP2B + G4) foci per nucleus was performed in 100 cells per condition across three independent experiments. Error bars, mean ± SEM; ***P < 0.001, *P < 0.05, Kruskal-Wallis test. (J) Pie chart showing the percentage of ATRX and TOP2B co-enriched regions overlapping with G4 peaks in control (11 913/17 515, 68.02%) and CX-5461-treated PDCs (6556/23 126, 28.35%). Bar graph depicting the number of overlapping ATRX, TOP2B, and G4 CUT&Tag peaks after CX-5461 treatment in PDCs. (K) Metagene analysis of ATRX, TOP2B, and G4 distribution across gene bodies in control and CX-5461-treated PDCs. (L) Genome browser tracks illustrating ATRX peaks and TOP2B peaks overlapping with G4 peaks, whose locations were altered by CX-5461 treatment. (M) GO enrichment analysis of genes related to G4 CUT&Tag differential peaks between control and CX-5461-treated PDCs.

Figure 5.

CX-5461 causes G4-associated DNA damage in ATRX-deficient glioma cells. (A) Comet assay analysis of DNA damage in control and ATRX knockdown (shATRX) U87MG cells treated with vehicle or 5 μM CX-5461 for 24 h; scale bars: 20 μm. Tail moment quantification was performed on 100 nuclei per condition using the Comet Assay Software Package (CASP). Error bars, mean ± SEM; ***P < 0.001, Kruskal-Wallis test. (Band C) IF analysis of γH2AX in control and shATRX U87MG cells at 0, 2, 8, and 24 h after 5 μM CX-5461 treatment; scale bars: 10 μm. Quantification of γH2AX foci per nucleus was performed in 100 cells per condition from three independent experiments. Error bars, mean ± SEM. ns, not significant; ***P < 0.001, two-way ANOVA. (D and E) Co-IF analysis of γH2AX and G4 in control and shATRX cells treated with vehicle or 5 μM CX-5461 for 24 h; scale bars: 10 μm. The percentage of G4 foci co-localizing with γH2AX foci was quantified in 100 nuclei per condition from three independent experiments. Error bars, mean ± SEM; *** P < 0.001, ** P < 0.01, one-way ANOVA. (F) KEGG enrichment analysis of downregulated pathways in U87MG cells following CX-5461 treatment. (G) Western blot analysis of p-ATR, ATR, p-ATM, ATM, p-CHK1, CHK1, p-CHK2, CHK2, p-RPA32, and RPA32 in control and shATRX U87MG and U251 cells treated with vehicle or 5 μM CX-5461 for 24 h.

Figure 6.

CX-5461 disrupts replication fork progression through TOP2Bcc accumulation. (A) Flow cytometry analysis of cell cycle in control and shATRX U87MG cells treated with vehicle or 5 μM CX-5461 for 24 h. (B) Co-IF analysis of γH2AX in EdU-labeled control and shATRX cells treated with vehicle or 5 μM CX-5461 for 24 h; scale bars: 10 μm. Quantification of γH2AX foci per nucleus was performed in EdU-positive and EdU-negative U87MG and U251 cells (n = 100 per condition) from three independent experiments. Error bars, mean ± SEM. ns, not significant; ***P < 0.001, Mann-Whitney test. (C) DNA fiber assay: schematic of CIdU and IdU pulse-labeling (top). Control and shATRX U87MG cells were sequentially labeled and treated with vehicle or 5 μM CX-5461 for 5 h. DNA fibers were analyzed to measure replication fork length. IdU track lengths (in μm) were converted to kilobases (1 kb = 2.59 μm). n = 100 replication tracks analyzed from three independent experiments. (D) DNA fiber analysis of U87MG cells labeled with CldU and IdU, then treated with 5 μM CX-5461, 50 mM mirin, or both for 3 h as indicated in the schematic (top). Fibers were processed and analyzed as described above. IdU and CldU track lengths were used to calculate the IdU/CldU ratio. n = 100 replication tracks analyzed from three independent experiments. (E) DNA fiber analysis of control and shATRX U87MG cells pre-treated with 5 μM CX-5461 for 24 h, washed, and sequentially labeled with CldU and IdU as indicated in the schematic (top). n = 100 replication tracks analyzed from three independent experiments. Statistical analyses (C-E) were performed using one-way ANOVA and the Kruskal-Wallis multiple comparisons test. ns, not significant; ***P < 0.001. (F) Schematic of CX-5461-induced TOP2Bcc obstructing the progression of replication forks.

Figure 7.

CX-5461 inhibits glioma proliferation by impairing DDR. (A) Comparison of TOP2B expression between ATRX WT and mutant glioma samples in the TCGA datasets; ***P < 0.001, Student’s t-test. (B) Representative images of GSC spheroids at 0, 24, 48, and 72 h after treatment with 5 or 10 μM CX-5461; scale bar: 100 μm. Quantification of 3D cell viability was performed 72 h post CX-5461 treatment in three independent experiments. Error bars, mean ± SEM; **P < 0.01, ***P < 0.001, one-way ANOVA. (C) Colony formation assay in control and siATRX U87MG cells treated with vehicle or 5 μM CX-5461. Colony numbers were quantified from five independent experiments. Error bars, mean ± SEM. **P < 0.001, one-way ANOVA. (D) Transwell migration assay in control and siATRX U87MG cells treated with vehicle or 5 μM CX-5461. Migration was quantified from five independent experiments. Error bars, mean ± SEM; ***P < 0.001, *P < 0.05, one-way ANOVA. (E) IF analysis of EdU in control and shATRX U87MG cells treated with 5 μM CX-5461 for 0, 2, 8, and 24 h; scale bars: 100 μm. The percentage of EdU-positive cells was quantified from three independent experiments. Error bars, mean ± SEM; ***P < 0.001, **P < 0.01, two-way ANOVA. (F) Western blot analysis of γH2AX, BRCA1, RAD51, 53BP1, and RIF1 in control and shATRX U87MG and U251 cells treated with vehicle or 5 μM CX-5461 for 24 h. (G) IF analysis of BRCA1, RAD51, 53BP1, and RIF1 in control and shATRX U87MG cells treated with vehicle or 5 μM CX-5461 for 24 h; scale bars: 10 μm. Quantification of foci per nucleus was performed in 100 cells per condition from three independent experiments. The percentage of cells with ≥5 BRCA1/RAD51 or RIF1/53BP1 co-localized foci was determined. Error bars, mean ± SEM; ***P < 0.001, **P < 0.01, *P < 0.05, Kruskal-Wallis test and one-way ANOVA.

Figure 8.

CX-5461 traps TOP2B and induces synthetic lethality in ATRX-deficient glioma. Schematic model showing how CX-5461 functions as a TOP2B poison to impair G4 resolution. In WT glioma cells, the ATRX and TOP2B cooperate to resolve G4 structures to ensure replication fork progression and genome stability. In ATRX-deficient cells, TOP2B becomes the primary resolver of G4s during replication. CX-5461 traps TOP2B cleavage complexes (TOP2Bcc), disrupts the ATRX-TOP2B interaction, and blocks G4 resolution, leading to G4 accumulation. This accumulation induces replication fork collapse, single-stranded DNA gaps, and DSBs, activating ATM/ATR signaling and downstream DDR pathways, including CHK1/CHK2-mediated cell cycle arrest. However, ATRX deficiency impairs key DDR components, particularly BRCA1-RAD51-mediated HR and RIF1-53BP1-mediated NHEJ, resulting in defective fork protection and repair. These vulnerabilities culminate in synthetic lethality upon CX-5461 treatment in ATRX-deficient glioma cells.