Centromeric footprints preserve telomere integrity in ALT cancers

Alternative lengthening of telomeres (ALT) is a specialized telomere extension mechanism associated with 5–10% of all cancers1. Although ALT has been linked to epigenetic dysregulation and genome instability, specific genomic and epigenetic rearrangements generated after ALT activation have not been identified. Here we report the insertion of centromeric α-satellite repeats and CENP-B boxes at telomeric locations specifically in ALT cancer cell lines and primary ALT paediatric neuroblastomas, indicating a pathological link for this alteration. Analysis using directed methylation with long-read sequencing (DiMeLo-seq) revealed discrete footprints of CENP-A chromatin assembled at telomeric locations on subsets of chromosomes. By modelling ALT activation, we show that epigenetic dysregulation due to ATRX loss and DNA hypomethylation facilitates the acquisition of these centromeric chromatin signatures. Functionally, interfering with HJURP-mediated CENP-A deposition compromises telomere integrity and ALT, leading to aberrant telomeric mitotic DNA synthesis (MiDAS). We propose that, while originally generated by illegitimate recombination, these centromeric signatures became integral by maintaining telomeric chromatin integrity in the unique context of ALT cancer cells. Centromeric DNA repeat insertions and CENP-A chromatin assembly are identified as genomic signatures that preserve telomere integrity in ALT cancer cells.