Lurbinectedin 標靶 ALT 癌細胞中的端粒及探討TERRA與人類老化的關聯性

Abstract

端粒維持機制在癌症進展與細胞老化中扮演關鍵角色。多數癌細胞依賴端粒酶以維持端粒長度，然而約有10-15%的癌症，特別是肉瘤，則使用另一種機制，稱為非典型端粒延長模式（alternative lengthening of telomeres, ALT）。ALT癌細胞常對傳統化學治療藥物產生抗藥性，因此尋找新的治療策略至關重要。本研究利用生物素標記的 lurbinectedin 結合chem-seq技術，探討其在 ALT 陽性癌細胞中的基因組結合分佈。我們的結果顯示，lurbinectedin 偏好結合於端粒區域及 G-四股螺旋結構（G-quadruplex, G4），此現象經由motif分析及 GC 含量富集進一步確認。此外，lurbinectedin 亦顯示在基因啟動子（transcription start sites, TSS）有顯著結合，暗示其可能具備更廣泛的調控功能。本研究揭示 lurbinectedin 於 ALT 癌細胞中標靶端粒與 G4 結構的潛力，提供未來治療策略的新方向。 另一部分的研究著重於一種長鏈分編碼RNA，稱之為（telomeric repeat-containing RNA, TERRA），此RNA包含端粒重複序列，源自亞端粒區域，參與端粒功能及基因組穩定性的調控。然而，TERRA 表達與人類老化之間的關係仍未被充分探討。本研究應用 RNA 納米孔定序（Nanopore RNA direct sequencing）及次世代定序(Next generation sequencing ) 結合生物資訊分析（TERRA-QUANT），量化TERRA 表達量，並進一步使用 poly(A)+/non poly(A) TERRA 捕獲定序以解析染色體端特定的多腺苷酸化偏好。分析結果顯示，TERRA 在血液、腦部及纖維母細胞中隨年齡顯著增加。此外，早老症（Hutchinson-Gilford progeria syndrome, HGPS）患者的纖維母細胞則表現出異常的 TERRA 表達型態。此結果指出，TERRA 可能為老化及相關疾病的重要分子標誌。

Telomere maintenance mechanisms play crucial roles in cancer progression and cellular aging. While most cancers rely on telomerase to sustain telomere length, approximately 10-15% of cancers, particularly sarcomas, utilize the alternative lengthening of telomeres (ALT) pathway. ALT cancers often exhibit resistance to conventional chemotherapeutic agents, highlighting the need for alternative treatment strategies. In this study, we employed chem-seq with biotinylated lurbinectedin to investigate its genome-wide binding profile in ALT-positive cancer cells. Our results reveal that lurbinectedin preferentially binds to telomeric regions and G quadruplex (G4) structures, as confirmed by motif analysis and GC content enrichment. Furthermore, lurbinectedin exhibits notable binding at transcription start sites (TSS), suggesting broader regulatory roles. These findings reveal the mechanism by which lurbinectedin efficiently eliminates ALT cancer cells through targeting telomeres. Second part of my thesis focuses on telomeric repeat-containing RNA (TERRA), a long non-coding RNA transcribed from subtelomeric regions. TERRA serves as a key regulator of telomere function and genome stability, but its relationship to human aging remains poorly understood. To address this, we combine Nanopore RNA direct sequencing and next-generation sequencing to annotate TERRA transcription regions. We established a customized bioinformatics pipeline (TERRA-QUANT) to quantify TERRA levels. Coupling this with poly(A)+ and non poly(A) TERRA capture sequencing allowed us to uncover chromosome specific polyadenylation preferences conserved across cell types. Our analyses also demonstrated that TERRA expression significantly increases with age in blood, brain, and fibroblasts. Notably, abnormal TERRA expression patterns were detected in Hutchinson-Gilford progeria syndrome (HGPS) fibroblasts. These results suggest that TERRA may serve as a molecular marker for aging and age-associated diseases.