Background: Prior studies suggest 10-20% of high-risk neuroblastomas (NB) use the alternative lengthening of telomeres (ALT) mechanism (associated with a poor prognosis) to maintain telomeres. We identified 3 ALT NB cell lines in screening a panel of 112 human NB cell lines for TERT expression and c-circle content (extrachromosomal telomeric DNA) , ALT was confirmed by TRAP assay and telomere FISH.
Methods: Cytotoxicity was assessed using DIMSCAN, gene expression and c-circle content by qPCR, DNA damage by immunostaining (53BP1 foci), telomere damage by IF-FISH, and ATM knockdown by lentiviral shRNA transduction.
Results: The 3 ALT NB cell lines had a higher mean IC 90 for DNA damaging drugs relative to 79 comparator telomerase+ NB cell lines and higher expression of multiple DNA damage response (DDR) genes (29 of 59 genes analyzed, p<0.05) when compared to 9 comparator multidrug resistant telomerase+ NB cell lines. Numerous baseline DNA damage foci were observed in the nuclei of the 3 ALT NB cell lines and greater than 90% of these DNA damage foci co-localized to telomeres, indicating spontaneous telomere damage. DNA damage foci were not observed in telomerase+ NB cell lines. We assessed activation of both ATM/ATR kinases (which are involved in DNA damage signaling at telomeres) and observed a marked increase in phosphorylation of ATM kinase and its downstream target CHK2 in ALT lines but not in telomerase+ lines. Knockdown of ATM in 2 ALT NB cell lines reduced DNA damage foci (p<0.05), reduced C-circle (ALT marker) content (p<.05), downregulated 13 of 25 DDR genes that are commonly induced by genotoxic stress (p<0.05), and sensitized ALT NB cells to melphalan, 4-hydroperoxy-cyclophosphamide, doxorubicin and irradiation (p<0.05).
Conclusions: ATM kinase is constitutively activated in ALT NB and may be necessary for the ALT phenotype. Our data suggest that activation of ATM kinase contributes to DNA damaging agent resistance by upregulating DDR expression. These data identify ATM kinase as a potential therapeutic target in ALT NB.