Bian Ke, Gruber Charley C, Byers Emine, Leclerc Paul, Hemann Michael T, Walker Graham C
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139.
Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139.
Proc Natl Acad Sci U S A. 2025 Sep 16;122(37):e2511136122. doi: 10.1073/pnas.2511136122. Epub 2025 Sep 10.
The mutagenic translesion synthesis (TLS) pathway, which is critically dependent on REV1's ability to recruit inserter TLS polymerases and the POLζ extender polymerase, enables cancer cells to bypass DNA lesions while introducing mutations that likely contribute to the development of chemotherapy resistance and secondary malignancies. Targeting this pathway represents a promising therapeutic strategy. Here, we demonstrate that the expression of the C-terminal domain (CTD) of human REV1, a ca. 100 amino acid scaffold essential for TLS polymerase interactions, disrupts REV1/POLζ-dependent TLS in mammalian cells. Inducible expression of REV1-CTD in multiple human and murine cancer cell lines sensitizes cells to DNA-damaging agents such as cisplatin, benzo[a]pyrene diol epoxide, and methyl methanesulfonate, without intrinsic cytotoxicity. REV1-CTD expression increases genomic instability, decreases mutagenesis, and enhances G2 arrest following genotoxic stress. Mutational disruption of the CTD's interaction interfaces abrogates these effects, confirming a dominant-negative mechanism via sequestration of TLS components. In a xenograft mouse model, REV1-CTD expression markedly enhances cisplatin efficacy, significantly reducing tumor burden. These findings establish the REV1-CTD as an effective dominant-negative inhibitor of TLS and support its development as a therapeutic agent delivered to cancer cells to enhance the efficacy of genotoxic chemotherapy.
易错跨损伤合成（TLS）途径严重依赖REV1招募插入型TLS聚合酶和POLζ延伸聚合酶的能力，该途径使癌细胞能够绕过DNA损伤，同时引入可能导致化疗耐药性和继发性恶性肿瘤发生的突变。靶向该途径是一种很有前景的治疗策略。在此，我们证明人类REV1的C末端结构域（CTD）（约100个氨基酸的支架，对TLS聚合酶相互作用至关重要）的表达会破坏哺乳动物细胞中REV1/POLζ依赖性的TLS。在多种人类和小鼠癌细胞系中诱导表达REV1-CTD会使细胞对顺铂、苯并[a]芘二醇环氧化物和甲磺酸甲酯等DNA损伤剂敏感，且本身无细胞毒性。REV1-CTD的表达会增加基因组不稳定性，减少诱变，并增强基因毒性应激后的G2期阻滞。CTD相互作用界面的突变破坏会消除这些效应，证实了通过隔离TLS组分的显性负性机制。在异种移植小鼠模型中，REV1-CTD的表达显著增强顺铂疗效，显著减轻肿瘤负担。这些发现确立了REV1-CTD作为TLS的有效显性负性抑制剂，并支持将其开发为一种递送至癌细胞以增强基因毒性化疗疗效的治疗剂。
