Topoisomerase IIIα controls alternative lengthening of telomeres

Alternative lengthening of telomeres (ALT) is a homologous recombination-dependent telomere elongation mechanism utilized by at least 10%-15% of all cancers. Here, we identify that the DNA topoisomerase, topoisomerase III⍺ (TOP3A), is enriched at the telomeres of ALT cells but not at the telomeres of telomerase (Tel)-positive cancer cells. We demonstrate that TOP3A stabilizes the shelterin complex in ALT cancer cell lines but not in Tel cells and that long non-coding telomeric-repeat containing RNA (TERRA) enrichment at telomeres depends on TOP3A. TOP3A also promotes the generation of single-stranded telomeric C-strand (ssTeloC) DNA, a recently discovered marker for ALT. Additionally, we find that inducing break-associated TOP3A-DNA-protein crosslinks in ALT cells suppresses TERRA enrichment and destabilizes the shelterin complex. Taken together, these observations uncover unexplored functions of TOP3A at ALT telomeres and suggest the potential of developing an ALT-specific cancer therapeutic strategy targeting TOP3A.