Summary Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 occur at very high frequency (∼70%) in uterine leiomyomas. However, the influence of these mutations on Mediator function and the molecular basis for their tumorigenic potential remain unknown. To clarify the impact of these mutations, we used affinity-purification mass spectrometry to establish the global protein-protein interaction profiles for both wild-type and mutant MED12. We found that uterine leiomyoma-linked mutations in MED12 led to a highly specific decrease in its association with Cyclin C-CDK8/CDK19 and loss of Mediator-associated CDK activity. Mechanistically, this occurs through disruption of a MED12-Cyclin C binding interface that we also show is required for MED12-mediated stimulation of Cyclin C-dependent CDK8 kinase activity. These findings indicate that uterine leiomyoma-linked mutations in MED12 uncouple Cyclin C-CDK8/19 from core Mediator and further identify the MED12/Cyclin C interface as a prospective therapeutic target in CDK8 driven cancers.

Ohdo syndrome comprises a heterogeneous group of disorders characterized by intellectual disability (ID) and typical facial features, including blepharophimosis. Clinically, these blepharophimosis-ID syndromes have been classified in five distinct subgroups, including the Maat-Kievit-Brunner (MKB) type, which, in contrast to the others, is characterized by X-linked inheritance and facial coarsening at older age. We performed exome sequencing in two families, each with two affected males with Ohdo syndrome MKB type. In the two families, MED12 missense mutations (c.3443G>A [p.Arg1148His] or c.3493T>C [p.Ser1165Pro]) segregating with the phenotype were identified. Upon subsequent analysis of an additional cohort of nine simplex male individuals with Ohdo syndrome, one additional de novo missense change (c.5185C>A [p.His1729Asn]) in MED12 was detected. The occurrence of three different hemizygous missense mutations in three unrelated families affected by Ohdo syndrome MKB type shows that mutations in MED12 are the underlying cause of this X-linked form of Ohdo syndrome. Together with the recently described KAT6B mutations resulting in Ohdo syndrome Say/Barber/Biesecker/Young/Simpson type, our findings point to aberrant chromatin modification as being central to the pathogenesis of Ohdo syndrome.

Highlights d Heterozygous missense mutations in SMARCA2 impair neural differentiation d SMARCA2 mutations result in enhancer reprogramming due to SMARCA4 retargeting d SOX3-dependent neural enhancers are lost in SMARCA2 mutant neural progenitor cells d AP-1 factors drive de novo enhancer formation in SMARCA2 mutant cells

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Dysregulation of Wnt/β-catenin signaling promotes colorectal cancer (CRC) and other types of malignancies through unprogrammed changes in gene transcription that drive tumorigenesis. CDK8 is a CRC oncoprotein whose amplification-dependent overexpression identifies a significant subset of CRC patients with poor prognosis. CDK8 kinase activity, along with Cyclin C (CycC), drives tumorigenesis by stimulating β-catenin transcriptional activity. Thus, inhibition of CDK8 kinase function offers a promising therapeutic approach for CDK8-overexpressing CRCs. CycC-CDK8, along with MED12 and MED13, compose a four-subunit “kinase” module within Mediator, a conserved multiprotein interface between gene-specific transcription factors and RNA Polymerase II. We have previously identified a network of physical and functional interactions within the Mediator kinase module critical for oncogenic Wnt/β-catenin signaling. Mechanistically, β-catenin binds directly to MED12 in Mediator, thus activating CycC-dependent CDK8 kinase activity and β-catenin transcriptional activity. More specifically, MED12-dependent CDK8 activation occurs through a direct interaction involving the MED12 N-terminus and a phylogenetically conserved surface groove on CycC. Here, we demonstrate that mutagenic disruption of the MED12/CycC interface in CRC cell lines leads to uncoupling of CycC-CDK8 to MED12 and core Mediator, and concomitant loss of Mediator-associated CDK8 activity. Transcriptome analysis of cells lacking CDK8 kinase function revealed downregulation of Wnt/β-catenin signaling and other oncogenic pathways, consequently impairing CRC cell proliferation and clonogenicity. Our studies therefore identify the MED12/CycC interface as a critical transducer of oncogenic Wnt/β-catenin signaling. By validating MED12/CycC as a potential therapeutic target, our findings have significant implications in current methods of treating CRC as they pave a way for development of novel, targeted inhibitors of Wnt/β-catenin signaling to treat colorectal and other CDK8-driven driven malignancies. Supported by MH085320-05 (TGB), RP140435 (TGB) and T32DE14318 (ADC). Citation Format: Alison Clark, Marieke Oldenbroek, Yao Wang, Jason Spaeth, Fangjian Gao, Victor X. Jin, Thomas Boyer. Mediator kinase module as a transducer of oncogenic Wnt/beta-catenin signaling. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4607.