Jaana Tolvanen

MED12, the Mediator Complex Subunit 12 Gene, Is Mutated at High Frequency in Uterine Leiomyomas

Uterine fibroids frequently harbor mutations in a specific gene that has been implicated in transcriptional regulation. Uterine leiomyomas, or fibroids, are benign tumors that affect millions of women worldwide and that can cause considerable morbidity. To study the genetic basis of this tumor type, we examined 18 uterine leiomyomas derived from 17 different patients by exome sequencing and identified tumor-specific mutations in the mediator complex subunit 12 (MED12) gene in 10. Through analysis of 207 additional tumors, we determined that MED12 is altered in 70% (159 of 225) of tumors from a total of 80 patients. The Mediator complex is a 26-subunit transcriptional regulator that bridges DNA regulatory sequences to the RNA polymerase II initiation complex. All mutations resided in exon 2, suggesting that aberrant function of this region of MED12 contributes to tumorigenesis.

Strong family history of uterine leiomyomatosis warrants fumarate hydratase mutation screening

Hereditary leiomyomatosis and renal cell cancer (HLRCC) is a tumor predisposition syndrome characterized by cutaneous and uterine leiomyomas and renal cell cancer. HLRCC is caused by heterozygous germline mutations in the fumarate hydratase (FH) gene. A Finnish family with nine closely related women with uterine leiomyomas was detected by an alert gynecologist. No cutaneous or renal cell tumors were reported in the family when it was referred to genetic analyses. Samples were available from seven patients, and a novel germline FH mutation was detected in five of them. Mutation carriers were symptomatic, had multiple tumors and were diagnosed at an early age. This study emphasizes the importance of considering FH mutation screening when gynecologists encounter families with multiple severe uterine leiomyoma cases. Due to possibility of phenocopies more than one patient should be tested. Early mutation detection allows regular screening of the mutation carriers and enables early detection of possible highly aggressive renal tumors. It may also affect family planning as multiple myomas at early age may significantly reduce fertility.

MED12 mutations and FH inactivation are mutually exclusive in uterine leiomyomas

Background:Uterine leiomyomas from hereditary leiomyomatosis and renal cell cancer (HLRCC) patients are driven by fumarate hydratase (FH) inactivation or occasionally by mediator complex subunit 12 (MED12) mutations. The aim of this study was to analyse whether MED12 mutations and FH inactivation are mutually exclusive and to determine the contribution of MED12 mutations on HLRCC patients’ myomagenesis.Methods:MED12 exons 1 and 2 mutation screening and 2SC immunohistochemistry indicative for FH deficiency was performed on a comprehensive series of HLRCC patients’ (122 specimens) and sporadic (66 specimens) tumours. Gene expression analysis was performed using Affymetrix GeneChip Human Exon Arrays (Affymetrix, Santa Clara, CA, USA).Results:Nine tumours from HLRCC patients harboured a somatic MED12 mutation and were negative for 2SC immunohistochemistry. All remaining successfully analysed lesions (107/116) were deficient for FH. Of sporadic tumours, 35/64 were MED12 mutation positive and none displayed a FH defect. In global gene expression analysis FH-deficient tumours clustered together, whereas HLRCC patients’ MED12 mutation-positive tumours clustered together with sporadic MED12 mutation-positive tumours.Conclusions:Somatic MED12 mutations and biallelic FH inactivation are mutually exclusive in both HLRCC syndrome-associated and sporadic uterine leiomyomas. The great majority of HLRCC patients’ uterine leiomyomas are caused by FH inactivation, but incidental tumours driven by somatic MED12 mutations also occur. These MED12 mutation-positive tumours display similar expressional profiles with their sporadic counterparts and are clearly separate from FH-deficient tumours.

Multiple clinical characteristics separate MED12-mutation-positive and -negative uterine leiomyomas

Up to 86% of uterine leiomyomas harbour somatic mutations in mediator complex subunit 12 (MED12). These mutations have been associated with conventional histology, smaller tumour size, and larger number of tumours within the uterus. Prior studies, with limited sample sizes, have failed to detect associations between other clinical features and MED12 mutations. Here, we prospectively collected 763 uterine leiomyomas and the corresponding normal myometrial tissue from 244 hysterectomy patients, recorded tumour characteristics, collected clinical data from medical records, and screened the tissue samples for MED12 mutations to assess potential associations between clinical variables and mutation status. Out of 763 leiomyomas, 599 (79%) harboured a MED12 mutation. In the analysis of tumour characteristics, positive MED12-mutation status was significantly associated with smaller tumour size, conventional histology, and subserous location, relative to intramural. In the analysis of clinical variables, the number of MED12-mutation-positive tumours showed an inverse association with parity, and the number of mutation-negative tumours showed a positive association with a history of pelvic inflammatory disease. This study confirmed the previously reported differences and discovered novel differentiating features for MED12-mutation-positive and -negative leiomyomas. These findings emphasise the relevance of specific driver mutations in genesis and presentation of uterine leiomyomas.

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

Somatic mutations in exon 2 of the RNA polymerase II transcriptional Mediator subunit MED12 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 MED12 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.

ESR1, WT1, WNT4, ATM and TERT loci are major contributors to uterine leiomyoma predisposition

Uterine leiomyomas (ULs) are benign tumors that are a major burden to women’s health. A genome-wide association study on 5,417 UL cases and 331,791 controls was performed, followed by replication of the genomic risk in two cohorts. Effects of the identified risk alleles were evaluated in view of molecular and clinical features. Five loci displayed a genome-wide significant association; the previously reported TNRC6B, and four novel loci ESR1 (ERα), WT1, WNT4, and ATM. The sixth hit TERT is also a conceivable target. The combined polygenic risk contributed by these loci was associated with MED12 mutation-positive tumors. The findings link genes for uterine development and genetic stability to leiomyomagenesis. While the fundamental role of sex hormones in UL aetiology has been clear, this work reveals a connection to estrogen receptor alpha on genetic level and suggests that determinants of UL growth associated with estrogen exposure have an inherited component.

Abstract 4800: Identification of candidate predisposition genes for familial uterine leiomyomas

Uterine leiomyomas are benign smooth muscle tumors that cause considerable morbidity. They are among the most common human tumors and are the leading indication for hysterectomy. Genetic factors play an important role in the development of these hormone-dependent tumors. First-degree relatives of uterine leiomyoma patients are at increased risk for developing leiomyomas. Twin studies have shown that an inherited predisposition is likely to contribute to the formation of these lesions. Furthermore, the clinical and molecular features of familial leiomyomas differ from those of sporadic cases. Thus far, the only known high risk predisposition defect is dominantly inherited loss of function mutations in fumarate hydratase (FH) that underlie a tumor predisposition syndrome hereditary leiomyomatosis and renal cell cancer (HLRCC). The aim of this study is to identify novel candidate high risk susceptibility genes for uterine leiomyomas. The study material consists of five FH mutation-negative families, which have at least four individuals diagnosed with uterine leiomyomas, originating from Northern Finland. Genomic DNA extracted from peripheral blood lymphocytes from one affected individual from each family has been exome sequenced. Exome sequencing data have been analyzed for rare non-synonymous variants with allele frequencies less than 0.1% in 4 019 controls. All protein-truncating variants as well as missense and splice site variants affecting the same gene in at least two individuals that are predicted to be protein-damaging have been included for further analysis. Preliminarily 25 candidate susceptibility genes have been shortlisted. Relevant variant calls will be verified by direct sequencing, followed by segregation analysis and assessment of loss of heterozygosity in the corresponding tumor samples. Identification of novel genes predisposing to familial uterine leiomyomas may provide a better understanding into the tumorigenic mechanisms and could lead to new prevention approaches and personalized medical treatments in the future. Citation Format: Hanna-Riikka Heinonen, Outi Uimari, Jaana Tolvanen, Markku Ryynanen, Lauri A. Aaltonen, Pia Vahteristo. Identification of candidate predisposition genes for familial uterine leiomyomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4800. doi:10.1158/1538-7445.AM2015-4800

Abstract 3514: MED12 and FH mutations in HLRCC associated uterine leiomyomas

Uterine leiomyomas (fibroids) are benign tumors which originate from the smooth muscle cells of the myometrium. Almost 70% of women are affected by these tumors by the age of 50. Even though uterine leiomyomas are benign, they can cause difficult symptoms and are the most common indication for hysterectomy. Most uterine leiomyomas are sporadic and up to 70% of these lesions harbor very specific mutations in MED12 (mediator complex subunit 12). All MED12 mutations observed in uterine leiomyomas have been missense or insertion deletion mutations retaining the open reading frame. Uterine leiomyomas occur also in the context of Hereditary Leiomyomatosis and Renal Cell Cancer syndrome (HLRCC, OMIM # 150800). In addition to uterine leiomyomas, the syndrome predisposes individuals to cutaneous leiomyomas and renal cell cancer. HLRCC is an autosomal dominant syndrome caused by heterozygous germline mutations in FH (fumarate hydratase). The gene is classified as a tumor suppressor and according to Knudson9s two-hit hypothesis, syndrome related tumors display biallelic inactivation of FH. Recently, we reported 3/34 uterine leiomyomas from HLRCC patients to harbor somatic mutations in MED12. Loss of heterozygosity (LOH) at the FH locus was assessed from the tumors, and interestingly none of the MED12 mutation positive tumors showed biallelic inactivation of FH through LOH. Exome sequencing of seemingly sporadic uterine leiomyomas revealed also one patient to carry a truncating germline mutation in FH. The FH and MED12 loci from the uterine leiomyomas of this patient were further studied with direct sequencing and LOH assessment. Tumors had different MED12 mutations and none of them harbored a somatic mutation or displayed LOH as a second hit at FH locus. The aim of this study is to clarify the role of MED12 in HLRCC syndrome associated uterine leiomyomas and to analyze whether MED12 mutations and biallelic inactivation of FH are truly mutually exclusive. A tissue microarray including uterine leiomyomas from HLRCC patients will be constructed and FH inactivation will be analyzed with immunohistochemical stainings. Expression analyses of the tumors harboring somatic MED12 mutation and FH mutation in germline will show whether these tumors cluster together with MED12 mutation positive tumors, tumors with biallelic FH inactivation, or if they form a separate cluster. Results of these analyses will reveal if there are two distinct molecular mechanisms behind the development of uterine leiomyomas in HLRCC patients; one dependent on the biallelic inactivation of FH and the other on the altered function of MED12. Citation Format: Kati Kampjarvi, Netta Makinen, Miika Mehine, Jaana Tolvanen, Tuomas Heikkinen, Ralf Butzow, Lauri A. Aaltonen, Pia Vahteristo. MED12 and FH mutations in HLRCC associated uterine leiomyomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3514. doi:10.1158/1538-7445.AM2014-3514