Nuclear-targeted GDOWN1 potently suppresses tumors by inducing premature transcription termination and exporting Pol Ⅱ CTD phosphatases to activate RB.

Dysregulated transcription and aberrant cell cycle progression represent core cancer hallmarks, yet not many transcriptional regulators are known to simultaneously govern both pathways as potent tumor suppressors. GDOWN1 is a Pol II-interacting factor mainly sequestered in the cytoplasm in somatic cells, and its nuclear biological functions remain largely undefined. We previously generated a nuclear-targeted GDOWN1 mutant (nGDOWN1(10 M)) that strongly represses transcription and cell proliferation. Here, we systematically dissect the underlying molecular mechanisms and validate its broad-spectrum anticancer potential. Combining genetic manipulation, multi-omics profiling, and multiple functional assays, we demonstrate that nGDOWN1(10 M) disturbs Pol II CTD phosphorylation homeostasis and triggers promoter-proximal premature termination, causing global repression of nascent transcription. Terminated Pol II complexes undergo cytoplasmic translocation alongside associated the CTD phosphatases PP2A and PP1, leading to massive cytoplasmic accumulation of these phosphatases. Concurrently, TP53 and CDKN1A are targeted by nGDOWN1(10 M) and transcriptionally upregulated. The above dual regulatory circuit synergistically activates RB family tumor suppressors: p53-p21 signaling inhibits cyclin-dependent kinases, while cytoplasmic PP2A/PP1 enhance cellular phosphatase activity to dephosphorylate and stabilize RB and p130. Activated RB and p130 form functional RB and DREAM complexes and inhibit the transcription of their target genes to induce robust cell cycle arrest. Notably, nGDOWN1(10 M) exerts profound antiproliferative effects across cancer cell lines, mouse xenograft models, and patient-derived tumor organoids, independent of p53 status. Collectively, this work uncovers a novel interconnected regulatory cascade triggered by nuclear translocation of GDOWN1: it remodels Pol II phosphorylation landscape and induces global transcriptional repression, which further drives cytoplasmic kinase-phosphatase coordination to activate master cell cycle suppressors and ultimately arrest cell cycle progression. Our results illuminate a coordinated molecular circuit governing two central cancer hallmarks and establish nuclear GDOWN1 as a promising therapeutic candidate for both p53-proficient and -deficient malignancies.