探討ALT癌症當中的診斷與治療

Abstract

端粒(telomere)為位於染色體末端的重複序列，於細胞分裂過程中逐漸縮短，最終導致細胞老化與凋亡。癌細胞透過啟動端粒延長機制達到永生化，其中約85%的癌症依賴端粒酶維持端粒長度，而約15%則是透過端粒替代延長機制(alternative lengthening of telomeres, ALT)維持端粒長度。ALT癌細胞主要藉由同源重組方式延長端粒，其基因組高度不穩定，並且常伴隨大量端粒重複序列形成的染色體外環狀 DNA(extrachromosomal telomeric repeats, ECTRs)。目前ALT的診斷需依靠多項分子特徵進行綜合判斷，尚未建立明確且標準化的檢測策略，且多為侵入式檢測，因此開發具特異性且非侵入性的診斷方式具有迫切性。ALT盛行於間質性腫瘤或中樞神經系統腫瘤，其預後不佳且對於化學藥物治療具有抗性，使ALT的鑑定與治療成為重要的臨床挑戰。 本篇研究旨在開發一套鑑定ALT的新策略，透過改良C環狀DNA檢測法並結合環狀DNA定序技術，系統性分析染色體外環狀DNA(extrachromosomal circular DNA, eccDNA)。我們的策略是透過限制酶和外切核酸酶選擇性切除及放大環狀DNA，結合Illumina短讀長定序以及Nanopore長讀長定序進行分析，並以U2OS細胞作為ALT陽性對照組及HeLa細胞作為ALT陰性對照組。定序分析顯示，U2OS細胞中的環狀DNA數量顯著高於HeLa，且端粒、次端粒與核糖體DNA相關序列皆呈現 ALT 細胞中特異性富集。跨平台分析顯示，短讀長定序在偵測範圍與基因覆蓋度上較具優勢，而長讀長定序則能解析eccDNA的完整結構與重複序列組成。研究亦發現，癌細胞中普遍存在源自著絲體衛星序列的重複序列型eccDNA，且eccDNA具有攜帶基因的偏好，特別是啟動子區域。此外，超過90% 的eccDNA包含SINE、LINE、LTR等重複序列因子，說明其偏好產生於基因組不穩定之區塊。 此外，本研究有探討ALT的潛在治療策略。相較於傳統化療藥物，ALT細胞對lurbinectedin展現出較高的敏感性。實驗室前人透過化學定序(Chem-seq)結果顯示lurbinectedin傾向結合於端粒帶有G-四聯體結構的區域，並經由免疫螢光染色加以驗證。同時，lurbinectedin促進端粒區域RNA-DNA雜合環結構的累積，進而招募XPF蛋白並誘發DNA斷裂，過度累積DNA損傷最終造成細胞凋亡，達成選擇性殺傷ALT癌細胞之目的。 綜上所述，本研究成功建立一套ALT相關eccDNA定序的鑑定策略，未來將進一步優化並應用於臨床檢體分析，作為具潛力的ALT診斷方法。同時，本研究闡明lurbinectedin透過干擾端粒穩定性誘發DNA損傷的分子機制，對於ALT癌症的治療提供了新的理論依據與應用方向。

Telomeres are repetitive DNA sequences located at the ends of chromosomes that progressively shorten during cell division, ultimately leading to cellular senescence and apoptosis. To achieve replicative immortality, cancer cells activate telomere maintenance mechanisms. Approximately 85% of cancers maintain telomere length through telomerase activity, whereas the remaining ~15% rely on the alternative lengthening of telomeres (ALT) pathway. ALT-positive cancer cells elongate telomeres primarily through homologous recombination (HR), exhibit pronounced genomic instability, and are frequently associated with abundant extrachromosomal telomeric repeats (ECTRs) derived from telomeric DNA. Currently, ALT diagnosis relies on the combined assessment of multiple molecular features, lacks standardized diagnostic criteria, and often requires invasive procedures. Therefore, the development of a specific and non-invasive diagnostic strategy for ALT remains an urgent clinical need. ALT is prevalent in mesenchymal and central nervous system tumors, which are typically associated with poor prognosis and resistance to conventional chemotherapy, making ALT identification and treatment a significant clinical challenge. In this study, we aimed to establish a novel strategy for ALT identification by integrating a modified C-circle assay with comprehensive extrachromosomal circular DNA (eccDNA) sequencing. Our approach selectively enriches circular DNA through restriction enzyme (RE) and exonuclease (Exo) treatments, followed by rolling circle amplification. Both Illumina short-read sequencing and Nanopore long-read sequencing were employed to systematically characterize eccDNA profiles. U2OS cells were used as an ALT-positive model, while HeLa cells served as an ALT-negative control. Circle-seq analyses revealed that U2OS cells harbored significantly higher levels of eccDNA compared to HeLa cells, with marked enrichment of telomeric, subtelomeric, and ribosomal DNA sequences specific to ALT cells. Cross-platform comparisons demonstrated that short-read Circle-seq provided broader detection coverage and higher gene annotation resolution, whereas long-read Circle-seq enabled the reconstruction of full-length eccDNA structures and detailed characterization of repetitive elements. Notably, cancer cells were found to harbor abundant eccDNAs derived from centromeric satellite repeats. In addition, eccDNA exhibits a strong tendency to carry genic regions, particularly promoter elements. More than 90% of eccDNA molecules contain repetitive sequence elements, including SINEs, LINEs, and LTRs, suggesting a preferential origin from genomically unstable regions. In addition, this study explored potential therapeutic strategies targeting ALT. Compared with conventional chemotherapeutic agents, ALT cells displayed increased sensitivity to lurbinectedin. Chem-seq analysis demonstrated that lurbinectedin preferentially binds to telomeric regions enriched in G-quadruplex structures, a finding further validated by immunofluorescence assays. Moreover, lurbinectedin treatment promoted the accumulation of RNA-DNA hybrids (R-loops) at telomeres, leading to the recruitment of the endonuclease XPF and induction of DNA breaks. Excessive DNA damage ultimately resulted in apoptosis, achieving selective cytotoxicity toward ALT-positive cancer cells. In conclusion, we established a robust eccDNA sequencing-based strategy for the identification of ALT-associated molecular features, which holds promise for further optimization and application to clinical specimens as a potential diagnostic approach for ALT. Furthermore, our findings elucidate a molecular mechanism by which lurbinectedin disrupts telomere stability and induces DNA damage, providing a theoretical basis and therapeutic rationale for targeting ALT-positive cancers.