Oncogenes accelerate DNA replication, leading to the activation of excessive replication origins. This process triggers replication stress (RS) and genomic instability in cancer cells, positioning RS as a promising therapeutic target. Translesion synthesis (TLS) functions as a DNA damage repair bypass mechanism, compensating for RS and conferring a proliferation advantage to cancer cells. Despite its therapeutic potential, the application of the TLS polymerase REV1 (REV1 DNA directed polymerase (REV1) inhibitor JH‑RE‑06 in colorectal cancer (CRC) remains unexplored. Bioinformatics analysis of clinical samples from The Cancer Genome Atlas (TCGA) database demonstrated marked REV1 upregulation in colorectal tumors compared with normal tissues, which was associated with a poorer prognosis. JH‑RE‑06 effectively suppressed CRC tumorigenesis bothand. Mechanistically, drug rescue experiments and proteomics revealed that cell death triggered by JH‑RE‑06 was associated with elevated oxidative stress and induction of ferroptosis‑associated signaling. Transmission electron microscopy revealed characteristic morphological changes associated with ferroptosis, including a significant reduction in mitochondrial abundance and the presence of autophagic vacuoles containing engulfed mitochondria. Biochemical assays confirmed that JH‑RE‑06 significantly increased intracellular Feand malondialdehyde (MDA) levels while reducing glutathione levels, indicative of ferroptosis. Western blot analysis revealed decreased levels of antioxidant proteins, including superoxide dismutase 2 (SOD2) and glutamate‑cysteine ligase catalytic subunit (GCLC), as well as ferritin. Furthermore, western blot and FerroOrange assays, combined with Autophagy‑related Gene 7 (ATG7) and Nuclear Receptor Coactivator 4 (NCOA4) knockdown experiments, demonstrated that JH‑RE‑06 activated ferroptosis in CRC via NCOA4‑mediated ferritinophagy. Safety evaluation via hematoxylin and eosin staining of major organs in mice showed no notable pathological damage induced by JH‑RE‑06. Taken together, these findings establish REV1 as a potential diagnostic biomarker and therapeutic target in CRC. REV1 inhibitor JH‑RE‑06 promoted NCOA4‑mediated ferritinophagy and induced programmed cell death, thereby highlighting its potential as a safe and effective therapeutic strategy for CRC. in vitro in vivo ²+
