Triple-negative breast cancer (TNBC) is an aggressive disease characterized by remarkable intratumor heterogeneity (ITH), which poses therapeutic challenges. However, the clinical relevance and key determinant of ITH in TNBC are poorly understood. Here, we comprehensively characterized ITH levels using multi-omics data across our center’s cohort (n = 260), The Cancer Genome Atlas cohort (n = 134), and four immunotherapy-treated cohorts (n = 109). Our results revealed that high ITH was associated with poor patient survival and immunotherapy resistance. Importantly, we identified zinc finger protein 689 (ZNF689) deficiency as a crucial determinant of ITH formation. Mechanistically, the ZNF689–TRIM28 complex was found to directly bind to the promoter of long interspersed element-1 (LINE-1), inducing H3K9me3-mediated transcriptional silencing. ZNF689 deficiency reactivated LINE-1 retrotransposition to exacerbate genomic instability, which fostered ITH. Single-cell RNA sequencing, spatially resolved transcriptomics and flow cytometry analysis confirmed that ZNF689 deficiency-induced ITH inhibited antigen presentation and T-cell activation, conferring immunotherapy resistance. Pharmacological inhibition of LINE-1 significantly reduced ITH, enhanced antitumor immunity, and eventually sensitized ZNF689-deficient tumors to immunotherapy in vivo. Consistently, ZNF689 expression positively correlated with favorable prognosis and immunotherapy response in clinical samples. Altogether, our study uncovers a previously unrecognized mechanism underlying ZNF689 deficiency-induced ITH and suggests LINE-1 inhibition combined with immunotherapy as a novel treatment strategy for TNBC.

三阴性乳腺癌（TNBC）是一种侵袭性疾病，其特征是显着的瘤内异质性（ITH），这给治疗带来了挑战。然而，人们对 TNBC 中 ITH 的临床相关性和关键决定因素知之甚少。在这里，我们使用我们中心队列 ( n  = 260)、癌症基因组图谱队列 ( n  = 134) 和四个免疫治疗队列 ( n  = 109) 的多组学数据全面表征 ITH 水平。我们的结果显示，高 ITH 与患者生存率低和免疫治疗耐药性相关。重要的是，我们发现锌指蛋白 689 (ZNF689) 缺乏是 ITH 形成的关键决定因素。从机制上讲，ZNF689-TRIM28 复合物被发现直接与长散布元件 1 (LINE-1) 的启动子结合，诱导 H3K9me3 介导的转录沉默。ZNF689 缺陷重新激活 LINE-1 逆转录转座，加剧基因组不稳定性，从而促进 ITH。单细胞 RNA 测序、空间分辨转录组学和流式细胞术分析证实，ZNF689 缺陷诱导的 ITH 抑制抗原呈递和 T 细胞激活，从而产生免疫治疗耐药性。LINE-1的药理学抑制显着降低ITH，增强抗肿瘤免疫力，并最终使ZNF689缺陷的肿瘤对体内免疫治疗敏感。一致地，ZNF689 表达与临床样本中良好的预后和免疫治疗反应呈正相关。总而言之，我们的研究揭示了 ZNF689 缺乏引起的 ITH 的一个以前未被认识的机制，并建议 LINE-1 抑制与免疫疗法相结合作为 TNBC 的一种新治疗策略。