Centromeric footprints preserve telomere integrity in ALT cancers

Article Centromeric footprints preserve telomere integrity in ALT cancers https://doi.org/10.1038/s41586-026-10598-1 Received: 8 January 2025 Accepted: 27 April 2026 Published online: xx xx xxxx Open access Check for updates Ragini Bhargava1, Megan A. Mahlke1,11, Tobias T. Schmidt2,11, Christoph Bartenhagen3,4, Baylee A. Smith1, Katherine L. Ramsey1, Takoda T. Zuehlke1, Ray W. Bowman II1, Michelle L. Lynskey1, Anne R. Wondisford1, Jean-Baptiste Ouriou1, Sandra Schamus-Hayes1, Michael J. Calderon5,6, Simon C. Watkins5,6, April E. Williams-Wehner2,7, Jennifer M. Bone8,9, Alok V. Joglekar8,9,10, Matthias Fischer3,4, Jan Karlseder2, Yael Nechemia-Arbely1,12 ✉ & Roderick J. O’Sullivan1,12 ✉ 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. Telomeres and centromeres are the most prominent chromosomal landmarks, designating the physical ends of chromosomes and sites for kinetochore formation, respectively2,3. Distinct repetitive DNA sequences define these domains: telomeric TTAGGG repeats4 and centromeric α-satellite (α-sat) higher-order repeat arrays5, typically containing CENP-B box motifs associated with specific evolutionarily conserved nucleoprotein complexes to coordinate the essential functions of each domain. Centromeres and telomeres typically occupy exclusive nuclear territories. Except in specialized instances during meiosis in Schizosaccharomyces pombe6, the compartmentalization of telomeres and centromeres within defined chromosomal domains is considered essential for genome preservation. Here we present evidence for telomere–centromere contacts, centromeric repeat insertions and the establishment of discrete arrays of CENPA nucleosomes at telomeres in cancer cells that activate the ALT mechanism. Telomere–centromere contacts in ALT The clustering of numerous telomeres within promyelocytic leukaemia (PML) bodies, forming ALT-associated PML bodies (APBs), is unique to cancer cells that activate ALT1. DNA fluorescence in situ hybridization (FISH) with PNA probes for centromeric α-sat repeats revealed a subset of signals overlapping with telomeres and PML protein in U2OS cells, a standard ALT cancer cell line (Fig. 1a,b and Extended Data Fig. 1a). These contacts were absent in HeLa cells expressing telomerase (TERT+) and in IMR90 SV40-LT-expressing fibroblasts, which lack an active telomere lengthening mechanism. However, the acute ALT activation by ASF1A and ASF1B depletion in HeLa cells7 or constitutive ALT activation by disrupting ATRX (α-thalassaemia/intellectual disability X-linked) protein expression in IMR90 SV40-LT cells8 produced similar frequencies of overlapping centromere–telomere– PML foci (Fig. 1a,b and Extended Data Fig. 1a). Structured illumination 1 Department of Pharmacology and Chemical Biology, University of Pittsburgh, School of Medicine, UPMC Hillman Cancer Center, Pittsburgh, PA, USA. 2The Salk Institute for Biological Studies, La Jolla, CA, USA. 3Department of Experimental Pediatric Oncology, University Children’s Hospital of Cologne, Medical Faculty, University of Cologne, Cologne, Germany. 4Center for Molecular Medicine Cologne (CMMC), Medical Faculty, University of Cologne, Cologne, Germany. 5Department of Cell Biology, University of Pittsburgh, School of Medicine, UPMC Hillman Cancer Center, Pittsburgh, PA, USA. 6Center for Biologic Imaging (CBI), University of Pittsburgh, School of Medicine, UPMC Hillman Cancer Center, Pittsburgh, PA, USA. 7Razavi Newman Integrative Genomics and Bioinformatics Core Facility, La Jolla, CA, USA. 8Department of Immunology, University of Pittsburgh, School of Medicine, UPMC Hillman Cancer Center, Pittsburgh, PA, USA. 9 Center for Systems Immunology, University of Pittsburgh, School of Medicine, UPMC Hillman Cancer Center, Pittsburgh, PA, USA. 10Department of Computational and Systems Biology, University of Pittsburgh, School of Medicine, UPMC Hillman Cancer Center, Pittsburgh, PA, USA. 11These authors contributed equally: Megan A. Mahlke, Tobias T. Schmidt. 12These authors jointly supervised this work: Yael Nechemia-Arbely, Roderick J. O’Sullivan. ✉e-mail: ; Nature | www.nature.com | 1 Article siRNA: n = 89 4 IMR-90-E6/E7 non-neoplastic i Location of α-sat motifs in telomeric reads ≤1 bp mismatch TERT + n = 191 n = 173 n = 151 PM L BL M ALT G-292 U2OS SK-N-FI Saos-2 HT-29 Population doublings SK-N-AS 66 69 71 85 96 106 HOS α-Sat HT-1080 CENP-B box CENP-B box–TTAGGG co-localization + Clinical TMM HG002 TERT high MNA IMR90-E6/E7 (PD66.2) TERT-RA ATRX mut. Calu-3 APB HR MNA HOS TMM + HT-1080 Motifs per bin Yes – HR non-MNA TERT Localized >18m + ALT + Stage 4S Ambiguous No HT-29 j −3 1.5 × 10 SK-N-AS Saos-2 + SK-N-FI U2OS G-292 −50000 Subtelomere 0 50000 % tel. molecules with α-Sat motif ≤2 mismatches 1,000 100 10 1 1,000 100 10 1 1,000 100 10 1 1,000 100 10 1 1,000 100 10 1 1,000 100 10 1 1,000 100 10 1 1,000 100 10 1 1,000 100 10 1 1,000 100 10 1 1,000 100 10 1 Location (bp) −100000 HeLa U2OS TERT h 0 HeLa U2OS ALT Normal n = 197 1 0.1 HG002 * * C trl 2 0 ** LD 3 4 Calu-3 2 0 α-Sat motif (AAACTAGACAGAAGCAT) ≤1 bp mismatch 6 6 n = 64 Number of co-localizations per metaphase ** 0.05 siRNA: n = 41 n = 40 TTAGGG CENP-B box DAPI 8 0.10 g P < 0.0001 P < 0.0001 * Number of EdU–Cen–TTAGGG co-localizations per nucleus EdU Merged IMR90 SV40LT 0.15 PO HeLa Ctrl ATRX KO f + U2OS (ALT ) P = 0.0160 Telo-seq reads with α-sat motifs (%) + P = 0.0064 P = 0.0045 Enlarged Enlarged U2OS HeLa (TERT ) BLM 0 Ctrl ASF1 KD e n = 389 n = 170 0.1 PML P < 0.0001 0.3 0.2 POLD3 Ctrl Cen P = 0.0004 0.4 n = 356 Merged 0.5 n = 319 Number of PML–Cen–TTAGGG co-localizations per nucleus Cen P (...truncated)