Molecular basis of MED12 in the pathogenesis of uterine fibroids

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 reprogramming and replication stress-dependent chromosomal
instability. We further propose that clinically relevant ATR axis inhibitors will provide therapeutic benefit in a
preclinical model of MED12-mutant UFs. To test this, we will: (1) Elucidate the biochemical basis by which
MED12 mutations disrupt Mediator kinase activity. Using structural biology and biochemistry, we will determine
the impact of mutant MED12 on CDK8 T-loop stability and conformational dynamics as well as CycC-CDK8
substrate binding and catalytic efficiency; (2) Elucidate the molecular basis by which Mediator kinase disruption
drives UF initiation. We will link Mediator kinase-dependent changes in MM SC self-renewal and differentiation
with genome-wide enhancer reprogramming and altered transcriptional output and further ask if Mediator kinase
disruption can reprogram MM SCs to form UF tumors vivo; (3) Elucidate the molecular basis by which Mediator
kinase disruption drives UF progression. We will investigate RNAPII promoter pausing defects as a basis for
aberrant R-loop accrual, determine if R-loop-induced replication stress triggers mitotic chromosomal breaks, and
evaluate the efficacy of ATR axis inhibitors in a preclinical mouse model of MED12-mutant UFs.

Grant Number: 5R01HD087417-10
NIH Institute/Center: NIH
Principal Investigator: THOMAS BOYER