The nucleolar deubiquitinase OTUD4 coordinates spatial organization of perinucleolar heterochromatin to restrain tumor immunogenicity

Subnuclear compartmentalization is a foundational determinant of transcriptional homeostasis and cell fate. The nucleolus serves as a scaffold for specialized heterochromatin, yet the mechanisms orchestrating their spatial assembly in malignant contexts remain elusive. Here we identify the nucleolar deubiquitinase OTUD4 as a key regulator of perinucleolar heterochromatin architecture in tumor cells. Casein kinase II (CK2)-mediated phosphorylation activates OTUD4 to specifically remove K63-linked ubiquitin chains from nucleophosmin (NPM1) at lysine 27. This deubiquitination is indispensable for stabilizing the pentameric NPM1 structural scaffold, thereby anchoring H3K9me3-marked heterochromatin to nucleolus. Disruption of the CK2–OTUD4–NPM1 axis in tumor cells triggers heterochromatin detachment, eliciting nucleolar stress and retroelement derepression. This topological collapse activates cytosolic DNA-sensing pathways to potentiate tumor-intrinsic immunogenicity. Clinically, an OTUD4-nucleolus gene signature correlates with adverse survival and immunotherapy resistance in patients with cancer. Collectively, targeting this signal-responsive spatial architecture offers a therapeutic strategy to overcome immune evasion in refractory malignancies. Here the authors describe a nucleolar phosphorylation–deubiquitination cascade that regulates perinucleolar heterochromatin organization, showing that loss of OTUD4 in cancer cells induces nucleolar stress and retroelement derepression, restoring DNA sensor-mediated tumor immunogenicity.