Identification and characterization of the mediator kinase-dependent myometrial stem cell phosphoproteome

Objective: To identify, in myometrial stem/progenitor cells, the presumptive cell of origin for uterine fibroids, substrates of Mediator-associated cyclin dependent kinase 8/19 (CDK8/19), which is known to be disrupted by uterine fibroid driver mutations in Mediator complex subunit 12 (MED12). Design: Experimental study. Setting: Academic research laboratory. Patient(s): Women undergoing hysterectomy for uterine fibroids. Intervention(s): Stable isotopic labeling of amino acids in cell culture (SILAC) coupled with chemical inhibition of CDK8/19 and downstream quantitative phosphoproteomics and transcriptomic analyses in myometrial stem/progenitor cells. Main Outcome Measure(s): High-confidence Mediator kinase substrates identified by SILAC-based quantitative phosphoproteomics were determined using an empirical Bayes analysis and validated orthogonally by in vitro kinase assay featuring reconstituted Mediator kinase modules comprising wild-type or G44D mutant MED12 corresponding to the most frequent uterine fibroid driver mutation in MED12. Mediator kinase-regulated transcripts identified by RNA sequencing were linked to Mediator kinase substrates by computational analyses. Result(s): A total of 296 unique phosphosites in 166 proteins were significantly decreased (≥ twofold) upon CDK8/19 inhibition, including 118 phosphosites in 71 nuclear proteins representing high-confidence Mediator kinase substrates linked to RNA polymerase II transcription, RNA processing and transport, chromatin modification, cytoskeletal architecture, and DNA replication and repair. Orthogonal validation confirmed a subset of these proteins, including Cut Like Homeobox 1 (CUX1) and Forkhead Box K1 (FOXK1), to be direct targets of MED12-dependent CDK8 phosphorylation in a manner abrogated by the most common uterine fibroid driver mutation (G44D) in MED12, implicating these substrates in disease pathogenesis. Transcriptome-wide profiling of Mediator kinase-inhibited myometrial stem/progenitor cells revealed alterations in cell cycle and myogenic gene expression programs to which Mediator kinase substrates could be linked directly. Among these, CUX1 is an established transcriptional regulator of the cell cycle whose corresponding gene on chromosome 7q is the locus for a recurrent breakpoint in uterine fibroids, linking MED12 and Mediator kinase with CUX1 for the first time in uterine fibroid pathogenesis. FOXK1, a transcriptional regulator of myogenic stem cell fate, was found to be coordinately enriched along with kinase, but not core, Mediator subunits in myometrial stem/progenitor cells compared with differentiated uterine smooth muscle cells. Conclusion(s): These studies identify a new catalog of pathologically and biologically relevant Mediator kinase substrates implicated in the pathogenesis of MED12 mutation-positive uterine fibroids, and further uncover a biochemical basis to link Mediator kinase activity with CUX1 and FOXK1 in the regulation of myometrial stem/progenitor cell fate.