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Cited by 184DNA and Nucleic Acid ChemistryGlioma Diagnosis and TreatmentDNA Repair Mechanisms

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G-quadruplex DNA drives genomic instability and represents a targetable molecular abnormality in ATRX-deficient malignant glioma

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Wang, Yuxiang, Yang, Jie, Wild, Aaron T., Wu, Wei H., Shah, Rachna, Danussi, Carla, Riggins, Gregory J., Kannan, Kasthuri, Sulman, Erik P., Chan, Timothy A., Huse, Jason T.·2019·Nature Communications
DOI 10.1038/s41467-019-08905-8PMC PMC6391399

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Abstract

Mutational inactivation of ATRX (α-thalassemia mental retardation X-linked) represents a defining molecular alteration in large subsets of malignant glioma. Yet the pathogenic consequences of ATRX deficiency remain unclear, as do tractable mechanisms for its therapeutic targeting. Here we report that ATRX loss in isogenic glioma model systems induces replication stress and DNA damage by way of G-quadruplex (G4) DNA secondary structure. Moreover, these effects are associated with the acquisition of disease-relevant copy number alterations over time. We then demonstrate, both in vitro and in vivo, that ATRX deficiency selectively enhances DNA damage and cell death following chemical G4 stabilization. Finally, we show that G4 stabilization synergizes with other DNA-damaging therapies, including ionizing radiation, in the ATRX-deficient context. Our findings reveal novel pathogenic mechanisms driven by ATRX deficiency in glioma, while also pointing to tangible strategies for drug development.

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Figures

Fig. 1 ATRX deficiency promotes G4 formation. a Western blots for ATRX in parental (Par), shControl (shCon), and shATRX NHA (Vinculin loading control). Left panel shows constitutive NHA lines (shATRX1) and right panel shows the inducible lines post Doxycycline induction (shATRX2) and withdrawal (Dox-off). b Immunofluorescent staining of G4 (1H6) in constitutive (upper panel) and inducible (lower panel) NHA lines (DAPI counterstain). c Quantified relative G4 signal intensity (50 nuclei counted in all cases). d, e Immunofluorescent staining of G4 (BG4) in ATRX-intact (TS 603) and ATRX-mutant (08-0537) GSCs (d) and in TS 543 GSCs (e) subjected to ATRX knockdown (sh590) versus control (shCon; DAPI counterstain). f G4-containing DNA fragments from shCon and shATRX1 NHAs (three replicates each) were pulled down from sheared genomic DNA with purified hf2 antibodies. Recovered DNA was subjected to real-time PCR for telomeric sequences or G4-rich promoter regions of MYC and ZNF618 (ESR1 used as negative control). Graphs show enrichment over GAPDH scaled to shCon levels. g ATRX and G4 immunofluorescence in parental NHAs showing no significant colocalization. Where applicable, error bars reflect SEM; P values determined by unpaired, two-tailed t-test (*P < 0.05, **P < 0.01, ***P < 0.001); scale bars represent 10 µm
Fig. 1 ATRX deficiency promotes G4 formation. a Western blots for ATRX in parental (Par), shControl (shCon), and shATRX NHA (Vinculin loading control). Left panel shows constitutive NHA lines (shATRX1) and right panel shows the inducible lines post Doxycycline induction (shATRX2) and withdrawal (Dox-off). b Immunofluorescent staining of G4 (1H6) in constitutive (upper panel) and inducible (lower panel) NHA lines (DAPI counterstain). c Quantified relative G4 signal intensity (50 nuclei counted in all cases). d, e Immunofluorescent staining of G4 (BG4) in ATRX-intact (TS 603) and ATRX-mutant (08-0537) GSCs (d) and in TS 543 GSCs (e) subjected to ATRX knockdown (sh590) versus control (shCon; DAPI counterstain). f G4-containing DNA fragments from shCon and shATRX1 NHAs (three replicates each) were pulled down from sheared genomic DNA with purified hf2 antibodies. Recovered DNA was subjected to real-time PCR for telomeric sequences or G4-rich promoter regions of MYC and ZNF618 (ESR1 used as negative control). Graphs show enrichment over GAPDH scaled to shCon levels. g ATRX and G4 immunofluorescence in parental NHAs showing no significant colocalization. Where applicable, error bars reflect SEM; P values determined by unpaired, two-tailed t-test (*P < 0.05, **P < 0.01, ***P < 0.001); scale bars represent 10 µmCC BY · PubMed Central
Fig. 2 ATRX deficiency promotes replication stress and DNA damage. a γ-H2AX immunofluorescence in constitutive (upper panel) and inducible (lower panel) NHAs (DAPI counterstain). b Quantified percentage of cells with >10 immunopositive γ-H2AX foci (50 nuclei counted in all cases). c NHAs with ATRX knockdown were double stained for G4 and 53BP1, showing extensive colocalization. d Western blots of p-KAP1 and p-CHK1 show activation of replication stress arising with ATRX knockdown. e γ-H2AX and BLM ChIP in shCon and shATRX1 NHAs (three replicates each) showing that ATRX deficiency promotes enrichment for both proteins at putative G4-rich promoter regions (MYC and ZNF618) far exceeding that seen at a negative control site (ESR1). Where applicable, error bars reflect SEM; P values determined by unpaired, two-tailed t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s.: P > 0.05); scale bars represent 10 µm
Fig. 2 ATRX deficiency promotes replication stress and DNA damage. a γ-H2AX immunofluorescence in constitutive (upper panel) and inducible (lower panel) NHAs (DAPI counterstain). b Quantified percentage of cells with >10 immunopositive γ-H2AX foci (50 nuclei counted in all cases). c NHAs with ATRX knockdown were double stained for G4 and 53BP1, showing extensive colocalization. d Western blots of p-KAP1 and p-CHK1 show activation of replication stress arising with ATRX knockdown. e γ-H2AX and BLM ChIP in shCon and shATRX1 NHAs (three replicates each) showing that ATRX deficiency promotes enrichment for both proteins at putative G4-rich promoter regions (MYC and ZNF618) far exceeding that seen at a negative control site (ESR1). Where applicable, error bars reflect SEM; P values determined by unpaired, two-tailed t-test (*P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, n.s.: P > 0.05); scale bars represent 10 µmCC BY · PubMed Central
Fig. 3 ATRX deficiency induces chromosome breaks. a ATRX-deficient NHAs (shATRX1, passage 15) showed significantly increased chromosome breaks by cytogenetic analysis relative to ATRX-intact controls (shCon). b ATRX-mutant GSCs (08-0537) showed significantly increased chromosome breaks by cytogenetic analysis relative to ATRX wild-type GSCs (TS 603). c Quantified chromosome breaks in Atrx-intact and Atrx-KO mNPCs (also Tp53-/-, passage 10). In all cases, 10 sets of chromosomes were quantified. Error bars reflect SEM; P values determined by unpaired, two-tailed t-test
Fig. 3 ATRX deficiency induces chromosome breaks. a ATRX-deficient NHAs (shATRX1, passage 15) showed significantly increased chromosome breaks by cytogenetic analysis relative to ATRX-intact controls (shCon). b ATRX-mutant GSCs (08-0537) showed significantly increased chromosome breaks by cytogenetic analysis relative to ATRX wild-type GSCs (TS 603). c Quantified chromosome breaks in Atrx-intact and Atrx-KO mNPCs (also Tp53-/-, passage 10). In all cases, 10 sets of chromosomes were quantified. Error bars reflect SEM; P values determined by unpaired, two-tailed t-testCC BY · PubMed Central
Fig. 4 ATRX deficiency induces clinically relevant copy number alterations. a Size distribution of CNAs in parental (P0), shCon (P5 and P15), and shATRX1 (P5 and P15) NHAs (three replicates each). In ATRX knockdown cells, large CNAs (>1 Mb) arise over passage number. b Size distribution of CNAs in IDHmut-noncodel gliomas (TCGA)2. c, d IGV plots for chromosome 12 (c) and 14 (d) comparing CNA regions in IDHmut-noncodel gliomas (above dotted line) to NHAs (below dotted line). Color scales indicate log2 copy number. e, f Kaplan–Meier curves for IDHmut-noncodel glioma patients with or without either 12p gain or 14q loss, showing significant differences in overall survival. P values determined by Log-rank (Mantel–Cox) test
Fig. 4 ATRX deficiency induces clinically relevant copy number alterations. a Size distribution of CNAs in parental (P0), shCon (P5 and P15), and shATRX1 (P5 and P15) NHAs (three replicates each). In ATRX knockdown cells, large CNAs (>1 Mb) arise over passage number. b Size distribution of CNAs in IDHmut-noncodel gliomas (TCGA)2. c, d IGV plots for chromosome 12 (c) and 14 (d) comparing CNA regions in IDHmut-noncodel gliomas (above dotted line) to NHAs (below dotted line). Color scales indicate log2 copy number. e, f Kaplan–Meier curves for IDHmut-noncodel glioma patients with or without either 12p gain or 14q loss, showing significant differences in overall survival. P values determined by Log-rank (Mantel–Cox) testCC BY · PubMed Central
Fig. 5 ATRX-deficient glioma models are selectively sensitive to G4 stabilization. a, b Cell viability (CellTiter-Glo) of constitutive (a) and inducible (b) shCon and shATRX NHAs (four replicates each) treated with CX-3543 from 0-300nM. c Clonogenic assay of inducible shATRX2 NHAs demonstrates enhanced and reversible sensitivity to CX-3543 (50 nM) with ATRX deficiency. d Clonogenic assay of TS 543 with (sh590) and without (shCon) ATRX knockdown demonstrates enhanced sensitivity to CX-3543 (50 nM) with ATRX deficiency. e, f γ-H2AX immunofluorescence in constitutive (e) and inducible (f) shATRX NHAs showing increased DNA damage with CX-3543 treatment (100 nM), particularly in the setting of ATRX knockdown. g, h Western blots showing increased phosphorylation of replication stress pathway constituents (CHK1 and KAP1) in constitutive (g) and inducible (h) shATRX NHAs following CX-3543 treatment (100 nM). Where applicable, error bars reflect SEM; P values determined by two-way ANOVA; scale bars represent 10 µm
Fig. 5 ATRX-deficient glioma models are selectively sensitive to G4 stabilization. a, b Cell viability (CellTiter-Glo) of constitutive (a) and inducible (b) shCon and shATRX NHAs (four replicates each) treated with CX-3543 from 0-300nM. c Clonogenic assay of inducible shATRX2 NHAs demonstrates enhanced and reversible sensitivity to CX-3543 (50 nM) with ATRX deficiency. d Clonogenic assay of TS 543 with (sh590) and without (shCon) ATRX knockdown demonstrates enhanced sensitivity to CX-3543 (50 nM) with ATRX deficiency. e, f γ-H2AX immunofluorescence in constitutive (e) and inducible (f) shATRX NHAs showing increased DNA damage with CX-3543 treatment (100 nM), particularly in the setting of ATRX knockdown. g, h Western blots showing increased phosphorylation of replication stress pathway constituents (CHK1 and KAP1) in constitutive (g) and inducible (h) shATRX NHAs following CX-3543 treatment (100 nM). Where applicable, error bars reflect SEM; P values determined by two-way ANOVA; scale bars represent 10 µmCC BY · PubMed Central
Fig. 6 G4 stabilization selectively promotes apoptosis and synergizes with DNA-damaging therapies. a, b Time course study in shCon and shATRX1 NHAs (three replicates each) treated with 100 nM CX-3543 showing parallel kinetics of apoptosis (Annexin V positivity) (a) and p-CHK1/p-KAP1 levels by western blot (b). c, d Analogous time course study in inducible shATRX2 cells (three replicates each) documenting p-CHK1 an p-KAP1 levels (d) by western blot and Annexin V positivity (c) over time. e, f Soft agar colony counts for NHAs (3 replicates each) treated with either vehicle control (Veh) or 50 nM CX-3543 and either increasing doses of IR (e) or HU (f). Colony counts measured at 21 days scaled to that of shCon, Veh NHAs. Where applicable, error bars reflect SEM; P values determined by two-way ANOVA (*P < 0.05, **P < 0.01)
Fig. 6 G4 stabilization selectively promotes apoptosis and synergizes with DNA-damaging therapies. a, b Time course study in shCon and shATRX1 NHAs (three replicates each) treated with 100 nM CX-3543 showing parallel kinetics of apoptosis (Annexin V positivity) (a) and p-CHK1/p-KAP1 levels by western blot (b). c, d Analogous time course study in inducible shATRX2 cells (three replicates each) documenting p-CHK1 an p-KAP1 levels (d) by western blot and Annexin V positivity (c) over time. e, f Soft agar colony counts for NHAs (3 replicates each) treated with either vehicle control (Veh) or 50 nM CX-3543 and either increasing doses of IR (e) or HU (f). Colony counts measured at 21 days scaled to that of shCon, Veh NHAs. Where applicable, error bars reflect SEM; P values determined by two-way ANOVA (*P < 0.05, **P < 0.01)CC BY · PubMed Central
Fig. 7 G4 stabilization markedly slows the growth of ATRX-mutant glioma xenografts. a representative image of mice bearing JHH-273 xenografts following treatment with either vehicle (veh) or 12.5 mg/kg CX-3543 for 42 days. b Average xenograft volume over time in 10 mice randomized (5 and 5) into two groups, receiving either 12.5 mg/kg CX-3543 or vehicle control (veh). c Kaplan–Meier analysis of survival in a separate cohort of 10 mice randomized (5 and 5) into two groups, receiving either 12.5 mg/kg CX-3543 or vehicle control (Veh). d Representative H&E-stained, immunostained (Ki67 and γ-H2AX), and TEL-FISH stained sections of xenografts from vehicle (Veh) and CX-3543-treated mice. CX-3543-treated tumors showed decreased cellularity, decreased proliferative activity, and increased DNA damage. P values were determined by two-way ANOVA; scale bars represent 200 μm in staining panels and 25 μm in Tel-FISH panels
Fig. 7 G4 stabilization markedly slows the growth of ATRX-mutant glioma xenografts. a representative image of mice bearing JHH-273 xenografts following treatment with either vehicle (veh) or 12.5 mg/kg CX-3543 for 42 days. b Average xenograft volume over time in 10 mice randomized (5 and 5) into two groups, receiving either 12.5 mg/kg CX-3543 or vehicle control (veh). c Kaplan–Meier analysis of survival in a separate cohort of 10 mice randomized (5 and 5) into two groups, receiving either 12.5 mg/kg CX-3543 or vehicle control (Veh). d Representative H&E-stained, immunostained (Ki67 and γ-H2AX), and TEL-FISH stained sections of xenografts from vehicle (Veh) and CX-3543-treated mice. CX-3543-treated tumors showed decreased cellularity, decreased proliferative activity, and increased DNA damage. P values were determined by two-way ANOVA; scale bars represent 200 μm in staining panels and 25 μm in Tel-FISH panelsCC BY · PubMed Central
Fig. 8 Efficacy of G4 stabilization in vivo is dependent on ATRX deficiency. a TS 543 (ATRX intact) xenografts exhibited similar rates of growth when treated with either vehicle control (Veh, n = 3) or 12.5 mg/kg CX-3543 (n = 3) as reflected by tumor volume over time. b Kaplan–Meier analysis of survival In a separate cohort of 10 mice randomized (5 and 5) into two groups, receiving either 12.5 mg/kg CX-3543 or vehicle control (Veh). c ATRX knockdown in TS 543 cells (sh590) restored the sensitivity of xenografts to CX-3543 treatment as shown by tumor volume over time in 10 mice randomized (5 and 5) into two groups, receiving either 12.5 mg/kg CX-3543 or vehicle control (Veh). d Kaplan–Meier analysis of survival In a separate cohort of 10 mice randomized (5 and 5) into two groups, receiving either 12.5 mg/kg CX-3543 or vehicle control (Veh). e, f Representative H&E-stained, immunostained (Ki67 and γ-H2AX), and TEL-FISH stained sections of xenografts from vehicle (Veh) and CX-3543-treated mice harboring either TS 543-shCon (e) or TS 543-sh590 (f) xenografts. CX-3543-associated histopathological effects were limited to TS 543-sh590 xenografts. Evidence of ALT was not seen in either GSC line. P values were determined by two-way ANOVA for tumor growth and Log-rank (Mantel–Cox) test for survival curve comparison; scale bars represent 200 μm in staining panels and 25 μm in Tel-FISH panels
Fig. 8 Efficacy of G4 stabilization in vivo is dependent on ATRX deficiency. a TS 543 (ATRX intact) xenografts exhibited similar rates of growth when treated with either vehicle control (Veh, n = 3) or 12.5 mg/kg CX-3543 (n = 3) as reflected by tumor volume over time. b Kaplan–Meier analysis of survival In a separate cohort of 10 mice randomized (5 and 5) into two groups, receiving either 12.5 mg/kg CX-3543 or vehicle control (Veh). c ATRX knockdown in TS 543 cells (sh590) restored the sensitivity of xenografts to CX-3543 treatment as shown by tumor volume over time in 10 mice randomized (5 and 5) into two groups, receiving either 12.5 mg/kg CX-3543 or vehicle control (Veh). d Kaplan–Meier analysis of survival In a separate cohort of 10 mice randomized (5 and 5) into two groups, receiving either 12.5 mg/kg CX-3543 or vehicle control (Veh). e, f Representative H&E-stained, immunostained (Ki67 and γ-H2AX), and TEL-FISH stained sections of xenografts from vehicle (Veh) and CX-3543-treated mice harboring either TS 543-shCon (e) or TS 543-sh590 (f) xenografts. CX-3543-associated histopathological effects were limited to TS 543-sh590 xenografts. Evidence of ALT was not seen in either GSC line. P values were determined by two-way ANOVA for tumor growth and Log-rank (Mantel–Cox) test for survival curve comparison; scale bars represent 200 μm in staining panels and 25 μm in Tel-FISH panelsCC BY · PubMed Central
Fig. 9 Selectively targeting G4s in the ATRX-deficient context. G4s are normally resolved by both ATRX-dependent and ATRX-independent mechanisms to mitigate DNA damage in cells (a). In the setting of ATRX deficiency, DNA damage increases but its cytotoxic effects are dampened by ATRX-independent G4 resolution, maintaining cellular viability (b). However, concurrent G4 stabilization impairs these salvage pathways (c, d) further enhancing DNA damage and inducing cell death in the ATRX-deficient context
Fig. 9 Selectively targeting G4s in the ATRX-deficient context. G4s are normally resolved by both ATRX-dependent and ATRX-independent mechanisms to mitigate DNA damage in cells (a). In the setting of ATRX deficiency, DNA damage increases but its cytotoxic effects are dampened by ATRX-independent G4 resolution, maintaining cellular viability (b). However, concurrent G4 stabilization impairs these salvage pathways (c, d) further enhancing DNA damage and inducing cell death in the ATRX-deficient contextCC BY · PubMed Central

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Metadata

Authors
Wang, Y., Yang, J., Wild, A. T., Wu, W. H., Shah, R., Danussi, C., Riggins, G. J., Kannan, K., Sulman, E. P., Chan, T. A., & Huse, J. T.
Affiliations
  • Memorial Sloan Kettering Cancer Center (US)
  • The University of Texas MD Anderson Cancer Center (US)
  • Johns Hopkins University (US)
  • New York University (US)
Corresponding author
Huse, Jason T.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2019-02-26
Volume
10
Issue
1
Pages
943-943
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-019-08905-8
PMC
PMC6391399
OpenAlex
W2950531883
Cited by
184
References
90
Citations by year
2026: 11 · 2025: 16 · 2024: 21 · 2023: 24 · 2022: 34 · 2021: 35 · 2020: 31 · 2019: 11
FWCI
9.18
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, other-oa)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • American Cancer Society (RSG-16-179-01- DMC)
  • Sontag Foundation
  • Memorial Sloan-Kettering Cancer Center (P30-CA08748)
  • Sidney Kimmel Foundation
  • Cycle for Survival
  • National Institutes of Health (CA- 016672, CA08748)
  • National Cancer Institute (CA 016672, P30-CA-08748, CA08748, P30CA008748, P30CA016672)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA and Nucleic Acid Chemistry
  • Health Sciences › Medicine › Oncology › Glioma Diagnosis and Treatment
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
Keywords
high-grade glioma, G-quadruplex, genomic instability, replication stress, DNA damage, copy number alterations, G-quadruplex stabilization, ionizing radiation, combination therapy, therapeutic targeting
MeSH terms
X-linked Nuclear Protein, Animals, Brain Neoplasms, DNA Damage, DNA Replication, DNA, Neoplasm, Glioma, Humans, Mice, Nude, Mutation, Genomic Instability, Cell Line, Tumor, Mice, G-Quadruplexes, Gene Knockdown Techniques, DNA Copy Number Variations, Heterografts

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