PROJECT SUMMARY/ABSTRACT Uterine fibroids (UFs) are the most important benign neoplastic threat to women’s health worldwide. As no long- term non-invasive treatment option exists for UFs, deeper insight into tumor etiology is key to develop more effective therapies. Accordingly, this proposal is impactful as it suggests a novel etiological basis for the predominant UF subtype and further offers proof of concept for therapeutic intervention in this specific genetic setting. UFs arise from the genetic transformation of a single myometrial stem cell (MM SC) into a tumor initiating cell (UF SC) that seeds monoclonal tumor growth. Notably, recurrent somatic mutations in the RNA polymerase II (RNAPII) Mediator subunit MED12 account for ~70% of UFs, but how these mutations drive cell transformation and tumor formation is unclear. Previously, we showed that MED12 mutations disrupt CycC- CDK8 kinase activity in Mediator, revealing the first and heretofore only known biochemical defect arising from these pathogenic mutations and further implying a new etiological role for CDK8 in UF pathogenesis. This breakthrough discovery was the basis for our original application which spawned major advances that justify studies in this renewal application to clarify the molecular basis and therapeutic implications of Mediator kinase dysfunction in the pathogenesis of MED12-mutant UFs. Herein, we show that MED12 mutations impair CDK8 activity through T-loop destabilization, leading to a profoundly altered phosphoproteome and dysregulation of cell growth and myogenic gene expression programs that dictate MM SC fate. Further, we show that MED12 mutation-induced CDK8 inactivation triggers R-loop-dependent replication stress, suggesting a possible basis for genomic instability and a new therapeutic vulnerability in this dominant UF subclass. Accordingly, we hypothesize that MED12 mutation-induced Mediator kinase disruption drives tumor initiation and progression through aberrant MM SC reprogra