Temporal DNA-PK activation drives genomic instability and therapy resistance in glioma stem cells
Yanling Wang, Haineng Xu, Tianrun Liu, Menggui Huang, Param-Puneet Butter, Chunsheng Li, Lin Zhang, Gary D. Kao, Yanqing Gong, Amit Maity, Constantinos Koumenis, Yi Fan
Yanling Wang, Haineng Xu, Tianrun Liu, Menggui Huang, Param-Puneet Butter, Chunsheng Li, Lin Zhang, Gary D. Kao, Yanqing Gong, Amit Maity, Constantinos Koumenis, Yi Fan
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Research Article Oncology Stem cells

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.

Authors

Yanling Wang, Haineng Xu, Tianrun Liu, Menggui Huang, Param-Puneet Butter, Chunsheng Li, Lin Zhang, Gary D. Kao, Yanqing Gong, Amit Maity, Constantinos Koumenis, Yi Fan

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Figure 2

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

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DNA-PK is selectively required for after radiation γ-H2AX and DNA repair...
(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).