Abstract
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. We herein knocked out (KO) ATRX in MYCN-amplified (NGP) and MYCN single copy (SK-N-AS) NB cells with wild-type (wt) and truncated TP53 at the C terminus, respectively, using CRISPR/Cas9 technologies. The loss of ATRX increased DNA damage and G4 formation related to RS in TP53 wt isogenic ATRX KO NGP cells, but not in SK-N-AS clones. A gene set enrichment analysis (GSEA) showed that the gene sets related to DNA double-strand break repair, negative cell cycle regulation, the G2M checkpoint, and p53 pathway activation were enriched in NGP 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 (DDR) 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 RS-induced DNA damage in NB cells featuring inactivated ATRX by regulating DNA repair mechanisms and replication fork stability.
Highlights
Neuroblastoma (NB) is a pediatric tumor of the sympathetic nervous system that accounts for 8–10% of all childhood cancers and 15% of pediatric oncology deaths
We revealed that alpha thalassemia mental retardation X-linked (ATRX) depletion in TP53 wild-type NB cells was associated with an increased frequency telomeres, we investigated γH2AX levels as a marker of stalled replication forks and double-strand breaks (DSBs). γH2AX levels were elevated in ATRX knocked out (KO)
Generation of ATRX KO cells by CRISPR/Cas9 genome editing We performed genome editing with the CRISPR/Cas9 system to recapitulate the cellular and molecular perspectives of ATRX deficiency in human NB using the NGP, NB-69, and SK-N-AS cell lines, which are wt for the gene
Summary
Neuroblastoma (NB) is a pediatric tumor of the sympathetic nervous system that accounts for 8–10% of all childhood cancers and 15% of pediatric oncology deaths. Received: 31 May 2021 Revised: 3 October 2021 Accepted: 11 October 2021 anemia (FA) pathway protein, FANCD2, plays a key role in limiting ATRX loss induces G4 formation and RS in TP53 wt NB cells RS by controlling the stability of stalled replication forks in cells or To clarify the biological consequences of ATRX deficiency, we tumors lacking BRAC1/2 [27, 28], and has been shown to initially examined the viability and clonogenic survival of ATRX KO cooperate with ATRX to limit RS and promote the homologous NGP and NB-69 cells. The present results indicate that p53 formation of γH2AX-associated telomere dysfunction-induced foci, deficiency limits ATRX loss-induced RS/genome integrity in NB TIF (Fig. 1D, E) This result suggests that ATRX functions to cells by regulating DNA repair mechanisms and replication fork protect against telomere DNA damage for telomere maintenance. IF showed that ATRX loss induced phospho-RPA32 foci (Supplementary Fig. 4A) and colocalization with G4 signal
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