PROJECT SUMMARY/ABSTRACT Uterine leiomyomas (LM; fibroids) are monoclonal neoplasms of the myometrium (MM) and represent the most frequent tumors in women worldwide. Although benign, they nonetheless account for significant gynecologic and reproductive dysfunction. As no long-term non-invasive treatment option exists for LM, deeper insight regarding tumor etiology is key to the development of newer targeted therapies. Accordingly, this proposal is impactful as it suggests an etiologic basis for the predominant LM subtype and further offers proof of concept for therapeutic intervention involving new druggable targets in this specific genetic setting. LM arise from the genetic transformation of a single MM stem cell (SC) into a tumor initiating cell (LM SC) that seeds and sustains fibroid growth through asymmetric cell divisions. Heretofore, the dominant drivers of cell transformation have been largely identified. The most prevalent among these, accounting for ~70% of LM, are recurrent somatic mutations in the gene encoding the MED12 subunit of Mediator, a multiprotein signal processor through which regulatory information conveyed by gene- specific transcription factors is transduced to RNA polymerase II (Pol II). However, the impact of these mutations on MED12 function and the molecular basis for their tumorigenic potential remain unknown. Herein, we show that LM-linked mutations in MED12 disrupt its ability to activate Cyclin C (CycC)-dependent kinase 8 (CDK8) in Mediator, leading to reduced site-specific RNA Pol II phosphorylation and global gene dysregulation. We also identify genetic programs uniquely dysregulated in MED12-mutant fibroids, leading us to hypothesize that Mediator kinase disruption as a consequence of MED12 mutations elicits transcriptional reprogramming and altered signaling sufficient to drive MM SC transformation. We further hypothesize that MED12-mutant LM are therapeutically susceptible to reactivation of CDK8 or pharmacologic modulation of uniquely dysregulated signaling pathways. To test these hypotheses we will: (1) Establish the pathogenic role of Mediator kinase disruption in MED12-mutant LM. We will ask if genetic or chemical disruption of CDK8 (or its paralog CDK19) in Mediator can induce fibrotic transformation of MM SCs and, conversely, if WT MED12 can restore CDK8/19 kinase activity and suppress the fibrotic phenotype of MED12-mutant LM SCs; (2) Elucidate the pathogenic mechanism of Mediator kinase disruption in MED12- mutant LM. We will define the biochemical basis by which MED12 mutations disrupt CycC-CDK8/19 kinase activity and employ an integrated genome-scale approach to acquire the unique transcriptomic and epigenomic profiles of MED12 WT and mutant LM SCs; (3) Examine the therapeutic implications of Mediator kinase disruption in MED12-mutant LM. We will ask if reactivation of CDK8/19 or pharmacologic manipulation of signaling pathways uniquely dysregulated in MED12-mutant LM SCs can reverse their fibrotic p...