Loss of p53 suppresses replication stress-induced DNA damage in ATRX-deficient neuroblastoma

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.

Replication Stress: A Review of Novel Targets to Enhance Radiosensitivity-From Bench to Clinic

“…The RS response shares many biological pathways with DDR. They are widely intertwined and thus hard to completely distinguish (124). Here, we grouped the proteins that are typically related to the DDR pathway but are not necessarily involved in the RS response.…”

Regulation of Replication Stress in Alternative Lengthening of Telomeres by Fanconi Anaemia Protein

“…Studies found higher replication levels of stress and damage to telomeres compared to telomerase-dependent tumour cells, which is thought to be an activation signal of the ALT pathway and can promote elongation of ALT telomeres [ 37 ]. Numerous studies show that there are multiple sources of replication stress in ALT cells and that these cells are more likely to accumulate replication stress [ 11 , 12 , 13 , 38 , 39 ].…”

“…ALT can be engaged in a wide range of tumor types, and is a common telomere-lengthening mechanism among certain sarcomas (e.g., osteosarcomas and liposarcomas), as well as in subsets of central nervous system tumors, including astrocytomas; however, ALT is relatively rare in the epithelial malignancies [10]. Adopting the ALT pathway confers many unique molecular characteristics to tumors, some of which are commonly used as markers of ALT activity, including (1) high-frequency ATRX mutations, (2) abundant telomeric secondary structures such as R-loops, G4-quadruplexes and telomeric fragility such as extrachromosomal Telomere Repeat (ECTR), (3) active telomeric sister chromatid exchange (T-SCE), (4) ALT-associated PML bodies (APBs) and (5) heterogeneous telomere length [8,9,[11][12][13]. The accumulation of R-loops and G-quadruplexes at telomeres may impede the progression of DNA replication, leading to the collapse of replication forks and the formation of one-ended DSBs, which activate BIR-independent telomeric DNA synthesis, consist of DNA end reaction for the formation of 3 hangout, RAD51/RAD52-dependent invastion, telomere extension mediated by polδ or polη, and Holliday junction resolution named D-loop resolution [14][15][16].…”

E2F3 gene expression is a potential negative prognostic marker for localised and MYCN not‐amplified neuroblastoma: Results of in silico analysis of 786 samples

“…[6][7][8][9][10][11] Many other genes or pathways have been identified that can explain the extremely divergent and heterogeneous clinical phenotypes of NB, including telomerase maintenance mechanisms, the adrenergic or mesenchymal developmental origin and the presence of ALK-RAS or TP53 pathway mutations. [12][13][14] In the last decades, the association between many potential oncogenes and patient's survival has been largely investigated. [15][16][17] A recent study reported a high expression of E2 transcription factor 3 (E2F3) in association with poor survival in NB patients with a normal MYCN copy number diagnosed in stage 4S.…”

“…The main recognised prognostic factors include metastatic disease, age >18 months, recurrent segmental chromosomal aberrations (SCA) and/or genomic amplification of the proto‐oncogene MYCN 6–11 . Many other genes or pathways have been identified that can explain the extremely divergent and heterogeneous clinical phenotypes of NB, including telomerase maintenance mechanisms, the adrenergic or mesenchymal developmental origin and the presence of ALK‐RAS or TP53 pathway mutations 12–14 …”