Abstract PR010: Understanding extrachromosomal telomere generation in ALT cancers

Abstract

Abstract Introduction: Alternative Lengthening of Telomeres (ALT) is a telomere maintenance mechanism mediated by break-induced replication (BIR), evident in approximately 15% of human cancers. A characteristic feature of ALT cancers is the presence of C-circles, circular single-stranded telomeric DNAs composed of Cytosine-rich (CCCTAA) sequences. These C-circles are unique biomarkers of ALT cells and may be generated from extrachromosomal telomeric single-stranded DNAs (ssDNAs), also unique to ALT cells, but the generation process remains undefined. Methods and results: In order to investigate C-rich ssDNA, we developed a highly sensitive method to detect single stranded telomeric DNA, called 4SET (Strand-Specific Southern-blot for Single-stranded Extrachromosomal Telomeres) assay. Utilizing 4SET, we are able to capture C-rich single stranded DNAs that are near 200 to 1500 nucleotides in size. Both telomeric ssDNAs and C-circles are abundant in cytoplasm and nucleoplasm fractions, which supports the hypothesis that telomeric ssDNA accumulation may indeed precede C-circle formation. We also found that telomeric ssDNAs originate during Okazaki fragment processing during C-rich lagging strand DNA synthesis at telomere ends during telomere elongation in ALT cancers. During lagging strand priming and synthesis, the CST-PP complex (CTC1/STN1/TEN1-PRIMASE-Polymerase alpha) primes the telomeric C-rich strand; then, Polymerase delta and BLM helicase facilitate excessive strand displacement, leading to the accumulation of telomeric ssDNAs in the nucleoplasm and cytoplasm which is potential precursor material for C-circles in ALT cancers. Conclusion: Here, we introduce a highly sensitive 4SET method for studying telomeric ssDNA, which we used to propose a new model for the generation of telomeric C-rich ssDNAs and C-circles during ALT-mediated telomere elongation. Our work contributes to a growing body of knowledge pertaining to the idiosyncrasies of the ALT mechanism based on investigation of its unique biomarkers. Citation Format: Junyeop Lee, Jina Lee, Eric J. Sohn, Angelo Taglialatela, Roderick J. O’Sullivan, Alberto Ciccia, Jaewon Min. Understanding extrachromosomal telomere generation in ALT cancers [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: DNA Damage Repair: From Basic Science to Future Clinical Application; 2024 Jan 9-11; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2024;84(1 Suppl):Abstract nr PR010.