2021 Fiscal Year Annual Research Report
Study of ATRX mediated telomere maintenance in neuroblastoma by using genome editing technology (CRISPR/Cas9)
| Project/Area Number |
18K15256
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| Research Institution | Research Institute for Clinical Oncology Saitama Cancer Center |
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Principal Investigator |
AKTER JESMIN 地方独立行政法人埼玉県立病院機構埼玉県立がんセンター(臨床腫瘍研究所), 臨床腫瘍研究所, 技師 (70795830)
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| Project Period (FY) |
2018-04-01 – 2022-03-31
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| Keywords | Neuroblastoma / ATRX / Replication stress / G-quadruplex / p53 / FANCD2 |
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| Outline of Annual Research Achievements |
Genetic aberrations are present in the ATRX gene in older high-risk neuroblastoma (NB) patients with very poor clinical outcomes. Its loss-of-function (LoF) facilitates the alternative lengthening of telomeres (ALT) pathway in tumor cells and is strongly linked to replication stress (RS) and DNA damage through G-quadruplex (G4) DNA secondary structures. However, limited information is available on ATRX alteration-related NB tumorigenesis.
Previously, we knocked out (KO) ATRX in MYCN amplified (NGP) and single copy (SK-N-AS) NB cells with wild type (wt) and mutant TP53, respectively, using CRISPR-Cas9 system. ATRX loss increased DNA damage and G4 formation related to RS in TP53 wt isogenic ATRX KO NGP cells, but not in SK-N-AS clones. The accumulation of DNA damage activated the ATM/CHK2/p53 pathway, leading to cell cycle arrest in NGP clones. Interestingly, ATRX loss did not induce RS related to DNA damage response in TP53-truncated SK-N-AS cells. p53 inactivation abrogated cell cycle arrest and reduced G4 accumulation in NGP clones. The loss of p53 also induced G4 DNA helicases or Fanconi anemia group D2 protein (FANCD2) with ATRX deficiency, suggesting that ATRX maintained genome integrity and p53 deficiency attenuated replication stress-induced DNA damage in NB cells featuring inactivated ATRX by regulating DNA repair mechanisms and replication fork stabilization. We also studied the hallmarks of ALT, including ALT-associated PML bodies and presence of extrachromosomal telomeric DNA (e.g. c-circle) in these subclones.
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