A phosphorylation switch in PAGE4 drives MED12-mutant fibroid pathogenesis

Recurrent somatic mutations in MED12, found in ∼70% of uterine leiomyomas (ULs), define the dominant molecular subtype of these highly prevalent tumors, yet the downstream effector mechanisms remain poorly understood. Using an integrated multi-omics workflow, encompassing discovery-phase DDA proteomics of matched leiomyoma–myometrium pairs, validation-phase DIA proteomics across genetically stratified cohorts (MED12 p.G44D, RAD51B–HMGA2, IRS4/FH subtypes), phosphoproteomics, immunohistochemistry, and AP-MS interactomics, we identify prostate-associated gene 4 (PAGE4) as a central effector of MED12-mutant UL pathogenesis. PAGE4 emerged as one of the most significantly upregulated proteins in MED12-mutant ULs and harbored the highest-occupancy hyperphosphorylation sites (T51, T85) in the tumor phosphoproteome, a pattern confirmed by immunohistochemistry and orthogonal phosphoproteomics. Kinase-substrate enrichment nominated HIPK2 as the primary upstream kinase, with supporting evidence from the broader CMGC family. Subsequent analysis revealed that phosphorylation acts as a molecular switch, fundamentally restructuring the PAGE4 interactome to favor the Mediator complex and RNA Pol II transcriptional machinery. This rewiring was functionally validated by ChIP-seq and luciferase reporter assays, which demonstrated corresponding shifts in transcription factor occupancy and downstream pathway activity. Collectively, these data establish phospho-PAGE4 as a critical mechanistic node downstream of MED12 mutation, expand PAGE4 biology from prostate cancer to female reproductive tumors, and nominate it as a mechanistic biomarker and candidate therapeutic vulnerability in the most prevalent molecular subtype of uterine leiomyomas.