Research ArticleCancer

ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma

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Science Translational Medicine  02 Mar 2016:
Vol. 8, Issue 328, pp. 328ra28
DOI: 10.1126/scitranslmed.aac8228
  • Fig. 1. SB mouse model represents ATRX-deficient GBM.

    (A) Constructs of SB plasmids, including the plasmid with short hairpin against ATRX. Purple brackets represent IR/DR (inverted repeat/direct repeat) sequences; the area between these IR/DR sequences is recognized by the SB transposase for insertion into the host genomic DNA. miR-30 sequences represent the 5′ and 3′ flanking sequences of the 300-nucleotide primary microRNA molecule. PGK, phosphoglycerate kinase; CMV, cytomegalovirus. (B) Plasmid insertion and tumor growth are monitored by in vivo luminescence. ROI, region of interest. (C and D) Hematoxylin and eosin staining of an 8-day-old mouse brain (7 days after injection) (C), including inset of subventricular zone lining the lateral ventricle (D). (E) Immunofluorescence of the region depicted in (D), with transfected cells (GFP-positive, to the right of the dotted line) showing ATRX reduction at 7 days after injection. Asterisks indicate a tumor cell that is positive for expression of GFP and negative for ATRX. DAPI, 4′,6-diamidino-2-phenylindole. (F) IHC staining of transfected cells/tumors for markers associated with neural stem cells, including GFAP, OLIG2, and Nestin in mice injected with shp53/NRAS/shATRX.

  • Fig. 2. ATRX loss decreases median survival in mice bearing SB-generated GBMs.

    (A) Kaplan-Meier survival curves of C57BL/6 mice bearing SB-induced tumors (P = 0.0032, Mantel log-rank test). (B) Representative brain tumors with histologic hallmarks of GBM: pseudopalisading necrosis and nuclear atypia (black arrows and blue arrow, respectively). (C) Tumors with addition of shATRX plasmid are larger at earlier time points than tumors with shp53/NRAS alone and show ATRX loss throughout the tumor. Asterisk in the left panel indicates a region in a p53/NRAS tumor with positive expression of ATRX. Asterisk in the right panel shows a region in a p53/NRAS/shATRX tumor which is negative for ATRX.

  • Fig. 3. ATRX loss increases MSI in mouse GBM and SNV frequency in human glioma.

    (A) Representative microsatellite lengths from a tumor with MSI-high positivity (log2 ratio of >4 for predominant tumor sample allele versus control mouse tail DNA) using marker D1Mit62. Plot shows a shift in populations of allele lengths in p53/NRAS/shATRX tumor (orange) compared to control mouse tail DNA (gray). Predominant allele size in this example is two nucleotides longer (orange arrow) than control mouse tail DNA allele (gray arrow). (B) Comparison of the percentage of tumors with MSI positivity using four independent MSI primer sets (mBat26, D7mit91, D1mit62, and D6mit59) (n = 48 tumor versus control DNA comparisons). Data sre means ± SEM; *P < 0.05 using two-sided χ2 analysis. (C) Analysis of matched human tumor/germline integrated sequencing data sets showing SNV frequency in tumors by ATRX mutational status (GBM, WHO grade IV; pediatric GBM excludes diffuse intrinsic pontine glioma; HGG, high-grade glioma, WHO grades III and IV). **P < 0.005 using unpaired Mann-Whitney test. (D) Analysis of significance of contribution to SNV rate by ATRX and TP53 mutational status, using a two-way analysis of variance (ANOVA) model in adult GBM (n = 290) and pediatric GBM (n = 128). Each data point represents an individual human tumor; line represents mean ± SEM.

  • Fig. 4. ATRX loss does not cause structural/chromosomal alterations or change chromosome count.

    (A) Analysis of chromosomal alterations, including copy number alterations, in the integrated pediatric GBM data sets, and percentage of genomic alterations in the adult TCGA data set show no difference by ATRX mutational status. Line represents mean ± SEM; P ≥ 0.05 using unpaired Mann-Whitney test; NS, not significant. (B) Chromosome count by metaphase preparation of independent GBM neurosphere cultures. Line represents mean ± SEM; P ≥ 0.05 using unpaired Mann-Whitney test. Both groups had similar coefficients of variation [31.4% in p53/NRAS tumor cells (n = 15) and 30.7% in p53/NRAS/shATRX tumor cells (n = 20)].

  • Fig. 5. ATRX-deficient mouse GBMs display ALT.

    (A) Detection of ALT using telomeric FISH assay showing characteristic ultrabright spots in human PanNETs (white arrows, positive control). A distinct population of cells with increased telomere signal is seen in ATRX-deficient tumors (white dotted circle). (B) CTCF in arbitrary units for telomeric FISH signal (data points represent individual cells from three tumors under each condition); black dotted circles denote cells qualitatively showing ultrabright spots, consistent with ALT. Line represents mean ± SD; *P < 0.05 using unpaired Mann-Whitney test.

  • Fig. 6. ATRX loss reduces NHEJ repair.

    (A) Reporter assay with GFP expression that is restored by NHEJ or HR in the appropriate plasmids. Addition of shATRX impairs NHEJ (assay performed in triplicate). (B) Flow cytometric quantification of HR and NHEJ activity as assessed by the percentage of GFP-positive cells normalized to control (differences in HR activity are nonsignificant). Line represents mean ± SD; **P < 0.005 using unpaired t test. (C) Loss of ATRX in mouse GBM decreases pDNA-PKcs immunostaining (showing representative results of three mouse tumors per condition). Dotted line represents distinction between tumor and nontumor brain.

  • Fig. 7. ATRX-deficient GBM cells are sensitive to double-stranded DNA-damaging treatments.

    (A) In vitro data showing proliferation of mouse GBM cell cultures (with or without shATRX) after exposure to escalating doses of cytotoxic agents. ATRX-deficient tumor cells have reduced proliferation only after exposure to agents that induce double-stranded breaks. Line represents mean ± SEM; *P < 0.05 , **P < 0.01, ***P < 0.001 using F test of logIC50 (median inhibitory concentration). (B) Schematic of whole-brain radiation for mice with GBM (with or without shATRX). Tumor growth assessed by in vivo luminescence is reduced in ATRX-deficient tumors (representative mice and their luminescence values are shown). Plot on right shows average tumor luminescence for all mice at early time points after radiation (n = 6 mice in each group). Line represents mean ± SEM; *P < 0.05 using unpaired t test.

  • Fig. 8. Pediatric patients with high-grade glioma and ATRX mutation survive longer.

    (A) Kaplan-Meier curve based on genome-wide data from multiple pediatric data sets (n = 293) showing the survival benefit of ATRX mutation in treated patients. (B) Schematic of impact of ATRX loss on GBM.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/328/328ra28/DC1

    Materials and Methods

    Fig. S1. SB-responsive shATRX plasmids are cloned to explore the impact of ATRX on GBM development.

    Fig. S2. Cells cotransfected with shp53, shATRX, and NRAS plasmids are characterized at 15 days after injection.

    Fig. S3. SB-mediated transfected cells are distinct from ependymal cells in mice.

    Fig. S4. Characterization of p53/NRAS/shATRX mice 7 days after injection shows tumor cells expressing GFAP and Nestin.

    Fig. S5. p53/NRAS/shATRX mice at 21 days after injection show tumor cells expressing OLIG2 and Nestin.

    Fig. S6. Moribund p53/NRAS/shATRX mice show tumor cells expressing OLIG2, Nestin, and pERK.

    Fig. S7. p53/NRAS/shATRX mice show loss of GFAP expression by 15 days after injection.

    Fig. S8. ATRX mutations do not cluster in SNF2/helicase domain in human glioma.

    Fig. S9. Primary GBM cell cultures are generated from SB-induced mouse tumors.

    Fig. S10. Telomere length measured by qPCR is not different in tumors with or without ATRX.

    Fig. S11. ALT was assessed by c-circle assay with DNA from mouse tumors and neurospheres.

    Fig. S12. Cells transfected with shATRX plasmid show reduction in NHEJ by flow cytometry.

    Fig. S13. NHEJ pathway is characterized by IHC.

    Fig. S14. HR pathway is characterized by IHC.

    Fig. S15. Mismatch repair pathway is characterized by IHC.

    Fig. S16. SB mouse tumor growth (without radiation treatment) is characterized by luminescence.

    Fig. S17. Tumors with ATRX loss show reduction in pDNA-PKcs before and after radiation treatment.

    Fig. S18. Mouse tumors with ATRX loss show increased expression of γH2A.X in vitro and in vivo.

    Fig. S19. Detailed schematic shows proposed impact of ATRX loss on GBM progression.

    Table S1. Animal models of glioma all have RTK-RAS-PI3K alterations.

    Table S2. Mice injected with p53/NRAS/shATRX have faster-growing tumors.

    Table S3. MSI rate is increased in p53/NRAS/shATRX tumors.

    Table S4. IDH1 and TP53 mutations do not alter SNV rate calculated by two-way ANOVA model.

    Table S5. Telomere qPCR average telomere length ratio worksheet and standard curve show similar results in all experimental groups.

    Table S6. Immunofluorescence analysis shows reduced pDNA-PKcs expression in ATRX-deficient mouse GBM.

    Table S7. Antibodies used for tissue analysis are summarized in table form.

  • Supplementary Material for:

    ATRX loss promotes tumor growth and impairs nonhomologous end joining DNA repair in glioma

    Carl Koschmann, Anda-Alexandra Calinescu, Felipe J. Nunez, Alan Mackay, Janet Fazal-Salom, Daniel Thomas, Flor Mendez, Neha Kamran, Marta Dzaman, Lakshman Mulpuri, Johnathon Krasinkiewicz, Robert Doherty, Rosemary Lemons, Jacqueline A. Brosnan-Cashman, Youping Li, Soyeon Roh, Lili Zhao, Henry Appelman, David Ferguson, Vera Gorbunova, Alan Meeker, Chris Jones, Pedro R. Lowenstein, Maria G. Castro*

    *Corresponding author. E-mail: mariacas{at}med.umich.edu

    Published 2 March 2016, Sci. Transl. Med. 8, 328ra28 (2016)
    DOI: 10.1126/scitranslmed.aac8228

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. SB-responsive shATRX plasmids are cloned to explore the impact of ATRX on GBM development.
    • Fig. S2. Cells cotransfected with shp53, shATRX, and NRAS plasmids are characterized at 15 days after injection.
    • Fig. S3. SB-mediated transfected cells are distinct from ependymal cells in mice.
    • Fig. S4. Characterization of p53/NRAS/shATRX mice 7 days after injection shows tumor cells expressing GFAP and Nestin.
    • Fig. S5. p53/NRAS/shATRX mice at 21 days after injection show tumor cells expressing OLIG2 and Nestin.
    • Fig. S6. Moribund p53/NRAS/shATRX mice show tumor cells expressing OLIG2, Nestin, and pERK.
    • Fig. S7. p53/NRAS/shATRX mice show loss of GFAP expression by 15 days after injection.
    • Fig. S8. ATRX mutations do not cluster in SNF2/helicase domain in human glioma.
    • Fig. S9. Primary GBM cell cultures are generated from SB-induced mouse tumors.
    • Fig. S10. Telomere length measured by qPCR is not different in tumors with or without ATRX.
    • Fig. S11. ALT was assessed by c-circle assay with DNA from mouse tumors and neurospheres.
    • Fig. S12. Cells transfected with shATRX plasmid show reduction in NHEJ by flow cytometry.
    • Fig. S13. NHEJ pathway is characterized by IHC.
    • Fig. S14. HR pathway is characterized by IHC.
    • Fig. S15. Mismatch repair pathway is characterized by IHC.
    • Fig. S16. SB mouse tumor growth (without radiation treatment) is characterized by luminescence.
    • Fig. S17. Tumors with ATRX loss show reduction in pDNA-PKcs before and after radiation treatment.
    • Fig. S18. Mouse tumors with ATRX loss show increased expression of γH2A.X in vitro and in vivo.
    • Fig. S19. Detailed schematic shows proposed impact of ATRX loss on GBM progression.
    • Table S1. Animal models of glioma all have RTK-RAS-PI3K alterations.
    • Table S2. Mice injected with p53/NRAS/shATRX have faster-growing tumors.
    • Table S3. MSI rate is increased in p53/NRAS/shATRX tumors.
    • Table S4. IDH1 and TP53 mutations do not alter SNV rate calculated by two-way ANOVA model.
    • Table S5. Telomere qPCR average telomere length ratio worksheet and standard curve show similar results in all experimental groups.
    • Table S6. Immunofluorescence analysis shows reduced pDNA-PKcs expression in ATRX-deficient mouse GBM.
    • Table S7. Antibodies used for tissue analysis are summarized in table form.

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