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Abstract：
BACKGROUND: Poly (ADP-ribose) polymerase inhibitor (PARPi) resistance poses a significant challenge in ovarian carcinoma (OC). While the role of DOT1L in cancer and chemoresistance is acknowledged, its specific role in PARPi resistance remains unclear. This study aims to elucidate the molecular mechanism of DOT1L in PARPi resistance in OC patients.
METHODS: This study analyzed the expression of DOT1L in PARPi-resistant cell lines compared to sensitive ones and correlated it with clinical outcomes in OC patients. Comprehensive in vitro and in vivo functional experiments were conducted using cellular and mouse models. Molecular investigations, including RNA sequencing, chromatin immunoprecipitation (ChIP) and Cleavage Under Targets and Tagmentation (CUT&Tag) assays, were employed to unravel the molecular mechanisms of DOT1L-mediated PARPi resistance.
RESULTS: Our investigation revealed a robust correlation between DOT1L expression and clinical PARPi resistance in non-BRCA mutated OC cells. Upregulated DOT1L expression in PARPi-resistant tissues was associated with diminished survival in OC patients. Mechanistically, we identified that PARP1 directly binds to the DOT1L gene promoter, promoting transcription independently of its enzyme activity. PARP1 trapping induced by PARPi treatment amplified this binding, enhancing DOT1L transcription and contributing to drug resistance. Sequencing analysis revealed that DOT1L plays a crucial role in the transcriptional regulation of PLCG2 and ABCB1 via H3K79me2. This established the PARP1-DOT1L-PLCG2/ABCB1 axis as a key contributor to PARPi resistance. Furthermore, we discovered that combining a DOT1L inhibitor with PARPi demonstrated a synergistic effect in both cell line-derived xenograft mouse models (CDXs) and patient-derived organoids (PDOs).
CONCLUSIONS: Our results demonstrate that DOT1L is an independent prognostic marker for OC patients. The PARP1-DOT1L/H3K79me2-PLCG2/ABCB1 axis is identified as a pivotal contributor to PARPi resistance. Targeted inhibition of DOT1L emerges as a promising therapeutic strategy for enhancing PARPi treatment outcomes in OC patients.
METHODS: This study analyzed the expression of DOT1L in PARPi-resistant cell lines compared to sensitive ones and correlated it with clinical outcomes in OC patients. Comprehensive in vitro and in vivo functional experiments were conducted using cellular and mouse models. Molecular investigations, including RNA sequencing, chromatin immunoprecipitation (ChIP) and Cleavage Under Targets and Tagmentation (CUT&Tag) assays, were employed to unravel the molecular mechanisms of DOT1L-mediated PARPi resistance.
RESULTS: Our investigation revealed a robust correlation between DOT1L expression and clinical PARPi resistance in non-BRCA mutated OC cells. Upregulated DOT1L expression in PARPi-resistant tissues was associated with diminished survival in OC patients. Mechanistically, we identified that PARP1 directly binds to the DOT1L gene promoter, promoting transcription independently of its enzyme activity. PARP1 trapping induced by PARPi treatment amplified this binding, enhancing DOT1L transcription and contributing to drug resistance. Sequencing analysis revealed that DOT1L plays a crucial role in the transcriptional regulation of PLCG2 and ABCB1 via H3K79me2. This established the PARP1-DOT1L-PLCG2/ABCB1 axis as a key contributor to PARPi resistance. Furthermore, we discovered that combining a DOT1L inhibitor with PARPi demonstrated a synergistic effect in both cell line-derived xenograft mouse models (CDXs) and patient-derived organoids (PDOs).
CONCLUSIONS: Our results demonstrate that DOT1L is an independent prognostic marker for OC patients. The PARP1-DOT1L/H3K79me2-PLCG2/ABCB1 axis is identified as a pivotal contributor to PARPi resistance. Targeted inhibition of DOT1L emerges as a promising therapeutic strategy for enhancing PARPi treatment outcomes in OC patients.
摘要：
背景：聚（ADP-核糖）聚合酶抑制剂（PARPi）耐药性在卵巢癌（OC）中提出了重大挑战。虽然DOT1L在癌症和化疗耐药中的作用是公认的，其在PARPi耐药中的具体作用尚不清楚。本研究旨在阐明DOT1L在OC患者PARPi耐药中的分子机制。
方法：本研究分析了DOT1L在PARPi耐药细胞系中的表达与敏感细胞系的比较，并将其与OC患者的临床结局相关联。使用细胞和小鼠模型进行全面的体外和体内功能实验。分子调查，包括RNA测序，染色质免疫沉淀（ChIP）和切割下的目标和标签（CUT和标签）测定，用于阐明DOT1L介导的PARPi抗性的分子机制。
结果：我们的研究显示，在非BRCA突变的OC细胞中，DOT1L表达与临床PARPi耐药之间存在密切的相关性。PARPi耐药组织中DOT1L表达上调与OC患者生存率降低相关。机械上,我们确定PARP1直接与DOT1L基因启动子结合，独立于其酶活性促进转录。PARPi处理诱导的PARP1捕获放大了这种结合，增强DOT1L转录并促进耐药性。测序分析表明，DOT1L通过H3K79me2在PLCG2和ABCB1的转录调控中起着至关重要的作用。这确立了PARP1-DOT1L-PLCG2/ABCB1轴作为PARPi抗性的关键贡献者。此外，我们发现,DOT1L抑制剂与PARPi的联合应用在细胞系来源的异种移植小鼠模型(CDXs)和患者来源的类器官(PDO)中均显示出协同作用.
结论：我们的结果表明DOT1L是OC患者的独立预后指标。PARP1-DOT1L/H3K79me2-PLCG2/ABCB1轴被认为是PARPi抗性的关键贡献者。DOT1L的靶向抑制成为增强OC患者PARPi治疗结果的有希望的治疗策略。
方法：本研究分析了DOT1L在PARPi耐药细胞系中的表达与敏感细胞系的比较，并将其与OC患者的临床结局相关联。使用细胞和小鼠模型进行全面的体外和体内功能实验。分子调查，包括RNA测序，染色质免疫沉淀（ChIP）和切割下的目标和标签（CUT和标签）测定，用于阐明DOT1L介导的PARPi抗性的分子机制。
结果：我们的研究显示，在非BRCA突变的OC细胞中，DOT1L表达与临床PARPi耐药之间存在密切的相关性。PARPi耐药组织中DOT1L表达上调与OC患者生存率降低相关。机械上,我们确定PARP1直接与DOT1L基因启动子结合，独立于其酶活性促进转录。PARPi处理诱导的PARP1捕获放大了这种结合，增强DOT1L转录并促进耐药性。测序分析表明，DOT1L通过H3K79me2在PLCG2和ABCB1的转录调控中起着至关重要的作用。这确立了PARP1-DOT1L-PLCG2/ABCB1轴作为PARPi抗性的关键贡献者。此外，我们发现,DOT1L抑制剂与PARPi的联合应用在细胞系来源的异种移植小鼠模型(CDXs)和患者来源的类器官(PDO)中均显示出协同作用.
结论：我们的结果表明DOT1L是OC患者的独立预后指标。PARP1-DOT1L/H3K79me2-PLCG2/ABCB1轴被认为是PARPi抗性的关键贡献者。DOT1L的靶向抑制成为增强OC患者PARPi治疗结果的有希望的治疗策略。
