Alternative lengthening of telomeres stratifies complex karyotype sarcomas into distinct genomic and transcriptomic states
Complex karyotype sarcomas (CKS) are heterogeneous mesenchymal malignancies that typically lack recurrent actionable oncogenic drivers and remain therapeutically challenging. Loss of ATRX is a recurrent feature of CKS and defines a particularly high-risk subgroup. ATRX loss is also associated with activation of the alternative lengthening of telomeres (ALT) pathway, and ALT-positive sarcomas have been linked to poor clinical outcomes. However, the molecular underpinnings underlying ALT-status-dependent differences in CKS, as well as the therapeutic vulnerabilities associated with ALT, remain poorly defined. By integrating C-circle-based ALT detection across 776 sarcoma samples with multi-modal sequencing of five CKS subtypes, we find that ALT activity is associated with enriched hallmarks of genomic instability. ALT-positive transcriptomes are dominated by a coordinated DNA damage response and mitotic program, in contrast to oncogenic signaling pathways that drive TERT activation in ALT-negative tumors. Long-read sequencing reveals telomere repeat clusters and telomere-mediated healing at structural breakpoints in ALT-positive tumors. These events also occur on extrachromosomal DNA (ecDNA), linking ALT activity to ecDNA biology. Together, our findings position ALT status as an important stratifying feature of CKS and identify ALT-associated transcriptional programs as potential therapeutic targets.