Oncogenic exon 2 mutations in Mediator subunit MED12 disrupt allosteric activation of cyclin C-CDK8/19

Abstract

Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit occur at high frequency in uterine fibroids (UFs) and breast fibroepithelial tumors as well as recurrently, albeit less frequently, in malignant uterine leimyosarcomas, chronic lymphocytic leukemias, and colorectal cancers. Previously, we reported that UF-linked mutations in MED12 disrupt its ability to activate cyclin C (CycC)-dependent kinase 8 (CDK8) in Mediator, implicating impaired Mediator-associated CDK8 activity in the molecular pathogenesis of these clinically significant lesions. Notably, the CDK8 paralog CDK19 is also expressed in myometrium, and both CDK8 and CDK19 assemble into Mediator in a mutually exclusive manner, suggesting that CDK19 activity may also be germane to the pathogenesis of mutation-induced UFs. However, whether and how UF-linked mutations in MED12 affect CDK19 activation is unknown. Herein, we show that MED12 allosterically activates CDK19 and that UF-linked exon 2 mutations in MED12 disrupt its CDK19 stimulatory activity. Furthermore, we find that within the Mediator kinase module, MED13 directly binds to the MED12 C terminus, thereby suppressing an apparent UF mutation-induced conformational change in MED12 that otherwise disrupts its association with CycC-CDK8/19. Thus, in the presence of MED13, mutant MED12 can bind, but cannot activate, CycC-CDK8/19. These findings indicate that MED12 binding is necessary but not sufficient for CycC-CDK8/19 activation and reveal an additional step in the MED12-dependent activation process, one critically dependent on MED12 residues altered by UF-linked exon 2 mutations. These findings confirm that UF-linked mutations in MED12 disrupt composite Mediator-associated kinase activity and identify CDK8/19 as prospective therapeutic targets in UFs.

Integrated Epigenome, Exome, and Transcriptome Analyses Reveal Molecular Subtypes and Homeotic Transformation in Uterine Fibroids

“…In contrast, biochemical assays demonstrate that MED12 mutations lead to loss of CycC-CDK8/19 binding and function, suggesting that MED12 mutations might be a loss-of-function (LOF) phenotype (Park et al, 2018). Biochemical analysis further revealed quantitative differences within the various MED12 exon 2 mutations with regard to kinase activity, indicating that maybe all MED12 mutants are not equal (Park et al, 2018). In our study, we discovered mutations distinct from the more common mutations in exons 1 and 2 (Mä kinen et al, 2011), a 24-bp deletion at the end of exon 1, and a 44-bp deletion spanning the wild-type splice acceptor in exon 2, resulting in deletion of 8 and 15 amino acids, respectively (Figure 1; Figure S7E).…”

“…A genetically modified mouse model has been developed that shows that expression of the MED12 mutant transgene on either a MED12-null or WT background leads to fibroid formation, suggesting that MED12 mutation could drive their development through a gain-of-function (GOF) or dominant-negative mechanism (Mittal et al, 2015). In contrast, biochemical assays demonstrate that MED12 mutations lead to loss of CycC-CDK8/19 binding and function, suggesting that MED12 mutations might be a loss-of-function (LOF) phenotype (Park et al, 2018). Biochemical analysis further revealed quantitative differences within the various MED12 exon 2 mutations with regard to kinase activity, indicating that maybe all MED12 mutants are not equal (Park et al, 2018).…”

The emerging role of mediator complex subunit 12 in tumorigenesis and response to chemotherapeutics

“…Moreover, MED13 interacts with the C terminus of MED12 and plays an allosteric role in regulating the interaction between a mutant form of MED12 and CycC‐CDK8/CDK19. Thus, when MED13 is present, mutant MED12 can bind, but is unable to activate, CycC‐CDK8/CDK19 . These studies implicate the MED12‐CycC interface as a putative target in CDK8‐driven cancers.…”

“…Thus, when MED13 is present, mutant MED12 can bind, but is unable to activate, CycC-CDK8/CDK19. 5 These studies implicate the MED12-CycC interface as a putative target in CDK8-driven cancers.…”

Growth disparities in uterine leiomyomas associated with MED12 mutation

“…Mutations in MED12 result in the disruption of mediator kinase activity and consequently alter CDK8 function. In the context of uterine leiomyoma, MED12-linked mutations disrupt its direct interaction with components of the CDK8 and results in suppression of E-induced transcription and diminished cell growth (3). Considering that EPO production is regulated by E, the suppression of E-induced transcription, as observed in the study by Asano et al (2) in response to E. These findings provide clarity for why the lack of MED12 mutation is correlated with accelerated growth in certain leiomyomas, in that elevated transcription of EPO is induced by E. Thus, new treatments inhibiting EPO expression might be considered as a therapy to prevent the growth in uterine leiomyomas lacking MED12 mutation.…”