Temporal DNA-PK activation drives genomic instability and therapy resistance in glioma stem cells

Abstract

Cancer stem cells (CSCs) - known to be resistant to genotoxic radiation and chemotherapy - are fundamental to therapy failure and cancer relapse. Here, we reveal that glioma CSCs are hypersensitive to radiation, but a temporal DNA repair mechanism converts the intrinsic sensitivity to genomic instability and treatment resistance. Transcriptome analysis identifies DNA-dependent protein kinase (DNA-PK) as a predominant DNA repair enzyme in CSCs. Notably, DNA-PK activity is suppressed after irradiation when ROS induce the dissociation of DNA-PKcs with Ku70/80, resulting in delayed DNA repair and radiosensitivity; subsequently, after ROS clearance, the accumulated DNA damage and robust activation of DNA-PK induce genomic instability, facilitated by Rad50-mediated cell-cycle arrest, leading to enhanced malignancy, CSC overgrowth, and radioresistance. Finally, we show a requisite in vivo role for DNA-PK in CSC-mediated radioresistance and glioma progression. These findings identify a time-sensitive mechanism controlling CSC resistance to DNA-damaging treatments and suggest DNA-PK/Rad50 as promising targets for CSC eradication.

Keywords: Brain cancer; Cancer; DNA repair; Oncology; Stem cells.

Figure 1. Time-dependent radioresistance is associated with increased DNA damage and delayed γ-H2AX and DNA repair after irradiation in glioma CSCs.

(A) Human patient-derived IN528 CSCs and the matched non–cancer stem cells (non-CSCs) were cultured in serum-free stem cell medium and either irradiated by 5-Gy x-ray or were not irradiated. The number of viable cells was determined using cell viability assays. The data are presented as a percentage based on the number of viable nonirradiated cells of the same type (mean ± SEM, n = 3). (B) IN528 CSCs and matched non-CSCs were irradiated with 5-Gy x-ray. Cells were harvested at different times after irradiation. DNA damage was assessed using the single-cell gel electrophoresis comet assay with a neutral condition. Representative images and quantification of the percentage of DNA in the comet tails (mean ± SD). Scale bar: 50 μm. (C) IN528 CSCs and matched non-CSCs and glioma U87 cells were irradiated with 5-Gy x-ray. Cell lysates were immunoblotted with anti–P-H2AX-Ser¹³⁹ (γ-H2AX) and anti-GAPDH antibodies. Representative blots. γ-H2AX band intensity was quantified and expressed as a percentage of its expression level before radiation. (D) CD133⁺ CSCs and CD133^– non-CSCs were isolated from IN528 GBM cells and irradiated with 5-Gy x-ray. Cell lysates were immunoblotted with anti–P-H2AX-Ser¹³⁹ (γ-H2AX) and anti-GAPDH antibodies. (E) IN528 CSCs and matched non-CSCs were irradiated with 5-Gy x-ray. Nuclear extracts were incubated with linearized DNA in NHEJ reaction buffer, followed by electrophoresis and gel imaging. The reaction mixtures were immunoblotted using an anti-PCNA antibody. Band intensity of multimers was analyzed.

Figure 2. DNA-PK is selectively required for after radiation γ-H2AX and DNA repair in CSCs.

(A) RNA was isolated from IN528, T3961, and T4121 CSCs and from their corresponding matched non-CSCs and subjected to deep RNA-sequencing analysis. Approximately 200 genes encoding all known human proteins associated with DNA damage and repair were classified. Generated gene sets were ranked based on their mean percentage expression changes in CSCs versus non-CSCs and plotted as mean ± SEM. Genes with expression changes of more than 50% are annotated. (B) IN528 CSCs were irradiated with 5-Gy x-ray. Cell lysates were harvested at different time points after irradiation and subjected to immunoblot analysis. (C) IN528 CSCs and matched non-CSC cells were pretreated with 1 μM NU7441 (DNA-PK inhibitor), KU60019 (ATM inhibitor), or VE821 (ATR inhibitor) and irradiated by 5-Gy x-ray. At 48 hours after irradiation, cell lysates were collected and immunoblotted with anti–P-H2AX-Ser¹³⁹ and anti-GADPH antibodies. (D–F) IN528 CSCs were transduced with lentivirus to express shRNAs targeting control GFP, DNA-PK, ATM, or ATR, followed by puromycin selection for stable expression cell lines. Cells were irradiated with 5-Gy x-ray. (D) Cells were harvested at 48 hours after irradiation. Cell lysates were subjected to immunoblot analysis. (E) Cells were harvested 48 hours after irradiation. Nuclear extracts were incubated with linearized DNA in NHEJ reaction buffer, followed by electrophoresis and gel imaging. The reaction mixtures were immunoblotted with anti-PCNA antibody. (F) Cells were harvested at different time points after irradiation. DNA damage was assessed using a single-cell gel electrophoresis comet assay. Quantification of the percentage of DNA in the comet tails is shown (mean ± SD).

Figure 3. DNA-PK is preferentially expressed in CSCs and is critical for after radiation survival.

(A) RNA was isolated from IN528, T3961, and T4121 CSCs and their matched non-CSCs and subjected to deep RNA-sequencing analysis. Heat map of the expression values for different genes and quantified percentages of each expressed gene (CSCs versus non-CSCs, mean ± SEM, n = 3). (B) IN528 and T4121 CSCs and their matched non-CSCs were subjected to immunoblot analysis. (C) IN528 CSCs and (D) matched non-CSCs were transduced with lentivirus that express shRNAs targeting GFP, DNA-PK, or ATM, followed by puromycin selection for stable expression cells. Cell were either irradiated 5-Gy x-ray or were not irradiated. The number of viable cells was determined using cell viability assays. The data are presented as a percentage based on the number of viable nonirradiated cells of the same type (mean ± SEM, n = 3).

Figure 4. ROS induces time-dependent DNA-PK inhibition by disrupting Ku70/80 binding to DNA-PKcs, leading to delayed H2AX phosphorylation and DNA repair.

(A) IN528 CSCs and matched non-CSCs were irradiated with 5-Gy x-ray. Intracellular ROS levels (H₂O₂) were measured by luminol-based luminescence analysis at different time points after irradiation (mean ± SEM, n = 3). (B–E) IN528 CSCs were pretreated with TEMPOL (10 mM) and irradiated with 5-Gy x-ray. (B) Cells were harvested at different time after irradiation and were subjected to immunoblot analysis. (C) At 4 hours after irradiation, cell lysates were immunoprecipitated with anti–DNA-PKcs or anti-Ku80 antibodies, followed by immunoblot analysis with anti-Ku80 or anti–DNA-PKcs antibody. (D) Cells were harvested at different times after irradiation and analyzed by immunoblot with anti–P-H2AX-Ser¹³⁹ (γ-H2AX) and anti-GAPDH antibodies. (E) Cells were harvested different time after radiation. Nucleic extracts were incubated with linearized DNA in NHEJ reaction buffer, followed by electrophoresis and gel imaging. The reaction mixtures were immunoblotted with anti-PCNA antibody.

Figure 5. Delayed DNA-PK activation induces genomic instability and enhances cell malignancy in CSCs.

(A–C) IN528 CSCs and matched non-CSCs were either irradiated with 5-Gy x-ray or were not irradiated (sham control). (A) At 2 days after irradiation, the cells were subjected to micronucleus analyses. Representative images and quantification of data (mean ± SD, n = around 1,000 cells). Scale bar: 50 μm. (B and C) Cells were analyzed by flow cytometry for aneuploidy. (B) Representative sorting. (C) Quantified data (mean ± SEM, n = 3). (D) IN528 CSCs were pretreated with 1 μM KU60019 (inhibitor of ATM [iATM]), NU7441 (inhibitor of DNA-PK [iDNA-PK]), or control 0.1% DMSO, and irradiated with 5-Gy x-ray. At different times after irradiation, cells were subjected to aneuploidy analysis by flow cytometry (mean ± SEM, n = 3). (E–I) IN528 CSCs were transduced with lentivirus that expresses shRNAs targeting control GFP, DNA-PK, Rad50, or ATM, followed by puromycin selection for stable expression cell lines. Cells were irradiated by 5-Gy x-ray. (E) Cells were analyzed by immunoblot. (F) 24 hours after irradiation, cells were stained with propidium iodide and analyzed by flow cytometry for cell cycle. Representative sorting and quantitative results (mean ± SEM, n = 3). (G) 72 hours after irradiation, cells were stained with propidium iodide and analyzed by flow cytometry for cell aneuploidy (mean ± SEM, n = 3). (H) Cells were subjected to viability analysis at different time points after radiation (mean ± SEM, n = 3-6). (I) At 2 weeks after irradiation, cells were subjected to neurosphere formation/survival fraction analysis (mean ± SEM, n = 3–5).

Figure 6. DNA-PK is critical for genomic instability and radioresistance in CSCs in vivo.

Human IN528 CSCs that stably express luciferase and shRNAs targeting control GFP, ATM, or DNA-PK were injected into the brains of nude mice. Using guided micro-CT, mice were locally irradiated with stereotactic x-ray radiation (3 Gy, single dose). (A) Experimental procedure. (B) Animal survival was monitored for 90 days after injection (n = 7–9 mice). P values were determined by log-rank (Mantel-Cox) tests. MS, median survival. (C) Tumor growth was analyzed by bioluminescence. Representative images and quantitative analysis of integrated luminescence in tumors at day 41 (mean ± SEM). P values were determined by t tests. (D) Tumor sections were stained with H&E. Representative images are shown (n = 4–5 mice). Scale bar: 100 μm. (E) Tumor sections were stained by TUNEL. Representative images are shown, and TUNEL⁺ cells were counted (mean ± SEM, n = 3 mice). Scale bar: 100 μm. (F) Single-cell suspensions derived from tumors were stained with propidium iodide and subjected to aneuploid analysis by flow cytometry. Representative sorting and quantified data (mean ± SEM, n = 4–9 mice) are shown. P values were determined by t tests.

Figure 7. Combined radiotherapy with DNA-PK inhibition eradicates CSCs and improves mouse survival.

Primary GBM in Ntv-a;Ink4a^–/–;Pten^fl/fl;LSL-Luc donor mice is induced by RCAS-mediated somatic gene transfer. Recipient mice were wild-type B6 mice. Mice were treated with 10 mg/kg NU7441 (iDNA-PK) or KU60019 (iATM) for 8 days and simultaneously irradiated with x-ray (2.5-Gy doses, 4 times). (A) Experimental procedure. (B) Animal survival was monitored for 65 days after injection (n = 10–11 mice). P values were determined by log-rank (Mantel-Cox) tests. MS, median survival. (C) Tumor growth was analyzed by bioluminescence. Representative images and quantitative analysis of integrated luminescence in tumors at day 24 (mean ± SEM). P values were determined by t tests. (D and E) Tumor sections were probed with anti-CD133 antibody and counterstained with hematoxylin. (D) Representative images. Scale bar: 100 mm. (E) Quantitative results (mean ± SD, n = 5 mice). (F) Tumor sections were stained with H&E. Representative images are shown (n = 4–5 mice). Scale bar: 100 μm.

Figure 8. Schematic model.

A schematic model for DNA-PK–mediated temporal control of radioresistance in CSCs.