The alternative lengthening of telomeres phenotype in human neuroblastoma is associated with resistance to DNA-damaging



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Alternative Lengthening of Telomeres (ALT) is a telomerase-independent telomere length maintenance mechanism (TMM) that enables the unlimited proliferation of cancer cells. Recent data suggests that ALT is prevalent in high-risk neuroblastoma (NB) tumors that present at a later age of diagnosis, and in NBs with mutations in the ATRX gene. To further our understanding of ALT in NB, we report the identification of 4 ALT NB cell lines: CHLA-90, SK-N-FI, LA-N-6, and COG-N-291. These 4 cell lines lack detectable telomerase activity and TERT mRNA expression (which encodes the catalytic component of telomerase), but have overall increased telomere content (TC), indicative long telomeres and the presence of ALT. 2 of these cell lines also contain extrachromosomal DNA C-Circles (CCs), which are highly specific for ALT. All 4 ALT NB cell lines lack MYCN genomic amplification and 3 out of the 4 ALT cell lines are p53 non-functional. LA-N-6, the sole p53-functional ALT NB cell line harbors a p14(ARF) deletion. Only CHLA-90 (ATRX-mutated) showed loss of ATRX protein expression suggesting that inactivation of other genes may also facilitate the development of ALT in NB. In a pilot study, we found that 3 out of 24 ATRX wild type NB tumors were CC positive, further indicating that ALT-based telomere maintenance can occur in NB tumors lacking ATRX mutations. We compared mRNA expression for 58 proteins involved in DNA repair using TaqMan low-density array (TLDA) cards between 4 ALT+ cell lines and 8 telomerase-positive (TA+) NB cell lines. We found that mean mRNA levels for 28 out of 58 genes in our panel were significantly (p<.05) elevated in the ALT+ cell lines. Furthermore, the ALT lines showed a high degree of multi-drug resistance to DNA-damaging agents and radiotherapy. These data suggest that in NB, the ALT phenotype is not limited to tumors with ATRX mutations, is associated with a lack of MYCN genomic amplification, and with alterations in p53/MDM2/p14(ARF) pathways. Furthermore, ALT in NB manifests enhanced expression of DNA repair enzymes, and is associated with resistance to DNA-damaging therapy. We conclude that p53-independent therapies should be considered for ALT+ NB tumors.



Telomeres, Telomerase, Neuroblastoma