Mediator Complex Subunit 12 Mutations Research Articles

Variants in exon 2 of MED12 gene causes uterine leiomyoma’s through over-expression of MMP-9 of ECM pathway

AimsTo study the impact of Mediator complex subunit 12 (MED12) gene variants on the encoded protein’s function and pathogenic relevance for genesis of uterine leiomyoma’s (ULs). MethodsMutational analysis in exon-2 of MED12 gene was performed by PCR amplification and DNA sequencing in 89 clinically diagnosed ULs tissues. Pathogenicity prediction of variation was performed by computational analysis. The functional effects of missense variation were done by quantity RT-PCR and western blot analysis. Result(s)Out of 89 samples, 40 (44.94%) had missense variation in 14 different CDS position of exon-2 of MED12 gene. Out of 40 missense variation, codon 44 had 25 (62.5%) looking as a hotspot region for mutation for ULs, because CDS position c130 and c131present at codon 44 that have necleotide change G>A, T, C at c130 and c131 have necleotide change G>A and C. We also find somenovel somatic mutations oncodon 36 (T > C), 38 (G>T) of exon-2 and 88 (G>C) of intron-2. No mutations were detected in uterine myometrium samples. Our computational analysis suggests that change in Med12c .131 G>A leads to single substitution of amino acid [Glycine (G) to Aspartate (D)] which has a pathogenic and lethal impact and may cause instability of MED12 protein. Further, analysis of extracellular matrix (ECM) component (MMP-2 & 9, COL4A2 and α-SMA) mRNA and protein expression levels in the set of ULs having MED12 mutation showed significantly higher expression of MMP-9 and α-SMA. Conclusion(s)The findings of present study suggest that missense variation in codon 44 of MED12 gene lead to the genesis of leiomyoma’s through over-expression of MMP-9 of ECM pathway which could be therapeutically targeted for non-surgical management of ULs.

MED12 mutation activates the tryptophan/kynurenine/AHR pathway to promote growth of uterine leiomyomas.

Uterine leiomyomas cause heavy menstrual bleeding, anemia, and pregnancy loss in millions of women worldwide. Driver mutations in the transcriptional mediator complex subunit 12 (MED12) gene in uterine myometrial cells initiate 70% of leiomyomas that grow in a progesterone-dependent manner. We showed a distinct chromatin occupancy landscape of MED12 in mutant MED12 (mut-MED12) versus WT-MED12 leiomyomas. Integration of cistromic and transcriptomics data identified tryptophan 2,3-dioxygenase (TDO2) as the top mut-MED12 target gene that was significantly upregulated in mut-MED12 leiomyomas when compared with adjacent myometrium and WT-MED12 leiomyomas. TDO2 catalyzes the conversion of tryptophan to kynurenine, an aryl hydrocarbon receptor (AHR) ligand that we confirmed to be significantly elevated in mut-MED12 leiomyomas. Treatment of primary mut-MED12 leiomyoma cells with tryptophan or kynurenine stimulated AHR nuclear translocation, increased proliferation, inhibited apoptosis, and induced AHR-target gene expression, whereas blocking the TDO2/kynurenine/AHR pathway by siRNA or pharmacological treatment abolished these effects. Progesterone receptors regulated the expression of AHR and its target genes. In vivo, TDO2 expression positively correlated with the expression of genes crucial for leiomyoma growth. In summary, activation of the TDO2/kynurenine/AHR pathway selectively in mut-MED12 leiomyomas promoted tumor growth and may inform the future development of targeted treatments and precision medicine.

O-190 Establishment of non-invasive prediction models for diagnosis of the subtypes and tissue composition of uterine leiomyomas by machine learning using MRI data

Abstract Study question We investigated whether machine learning models using MRI data can predict the subtypes and tissue composition of uterine leiomyomas. Summary answer Our machine learning models using MRI data were able to predict the subtypes and tissue composition of uterine leiomyomas with high accuracy. What is known already Recently, somatic mutations in the Mediator complex subunit 12 (MED12) gene were found to be a biomarker of uterine leiomyomas, which is detected in about 70% of uterine leiomyomas. Uterine leiomyomas are classified into two subtypes with or without the MED12 mutation. These subtypes differ in the ratio of smooth muscle cells and fibroblasts and in the amount of collagen fibers. In addition, sensitivities to female hormones differ between smooth muscle cells and fibroblasts. Thus, the effect of therapeutic drugs (GnRH analogs and selective progesterone receptor modulators) may differ depending on the subtypes and tissue composition of uterine leiomyomas. Study design, size, duration We analyzed 90 uterine leiomyoma nodules (MED12 mutation-positive and negative = 62 and 28) obtained from 51 women who underwent surgery at our hospital between 2020 and 2022. Seventy-one uterine leiomyomas (MED12 mutation-positive and negative = 49 and 22) were assigned to the primary dataset to establish the prediction models. Nineteen uterine leiomyomas (MED12 mutation-positive and negative = 13 and 6) were assigned to the test dataset to validate the prediction model utility. Participants/materials, setting, methods For each leiomyoma, the tumor signal intensity was quantified by five MRI sequences (T2WI, ADC, T1map, T2*BOLD, MTC) for evaluating the collagen amount. After surgery, genotyping of MED12 was examined and Trichrome staining was performed to quantify the collagen amount. Using these results, we established the prediction models based on machine learning by applying support vector classification and logistic regression for subtype prediction, and support vector regression and Ridge regression for tissue composition prediction. Main results and the role of chance The signal intensity of all five MRI sequences differed significantly between the subtypes. The cross-validation within the primary dataset showed that support vector classification and logistic regression models using five MRI sequences and MED12 genotyping data were highly predictive of the subtypes (AUC: 0.984 and 0.995, respectively). The validation using the test dataset showed that both models were able to predict the subtypes for all uterine leiomyomas (AUC: 1.000, both). This result showed higher accuracy than each MRI sequence’s cut-off value alone. On the other hand, four MRI sequence values (other than ADC) showed a significant correlation with the collagen amount. However, to improve accuracy, we added the ADC data and the result of the subtype predicted by the preceding models to the predictors for the collagen amount. Support vector regression and Ridge regression models using five MRI sequences and Trichrome staining data were shown to be highly predictive of the collagen amount in cross-validation within the primary dataset (R2: 0.570 and 0.525, respectively) and in validation using the test dataset (R2: 0.648 and 0.675, respectively). Moreover, the prediction models using only T2WI and ADC, which are taken in general clinical practice, were as accurate as using five MRI sequences. Limitations, reasons for caution In this study, we used limited data by using one specific MRI machine in a single center to establish the prediction models. We have not verified that similar results would be obtained under different conditions. Therefore, further training and evaluation in larger cohorts are required for clinical use. Wider implications of the findings The prediction models established in this study may be useful in clinical support such as predicting the effect of drug therapy and selecting therapeutic drugs, such as GnRH analogs and selective progesterone receptor modulators. Their utilities will be confirmed by validation using our prediction models in studies involving drug therapy. Trial registration number not applicable

Prevalence and clinical significance of co-existing mutations in MED12 and FH in uterine fibroids of Australian women.

Uterine fibroids are exceedingly common benign tumours of the female reproductive system and cause severe symptoms, including acute pain, bleeding, and infertility. Fibroids are frequently associated with genetic alterations affecting mediator complex subunit 12 (MED12), fumarate hydratase (FH), high mobility group AT-hook 2 (HMGA2) and collagen, type IV alpha 5 and alpha 6 (COL4A5-COL4A6). Recently, we reported MED12 exon 2 mutations in 39 out of 65 uterine fibroids (60%) from 14 Australian patients. The aim of this study was to evaluate the status of FH mutations in MED12 mutation-positive and mutation-negative uterine fibroids. FH mutation screening of altogether 65 uterine fibroids and corresponding adjacent normal myometrium (n = 14) was carried out by Sanger sequencing. Three out of 14 patients displayed somatic mutations in FH exon 1 in addition to harbouring MED12 mutation in uterine fibroids. This study is the first to report that the mutations in MED12 and FH co-exist in uterine fibroids of Australian women.

THE DYSREGULATION OF TRYPTOPHAN METABOLISM IN LEIOMYOMAS

To determine the expression of enzymes in tryptophan (Trp) catabolism and the influence of race and MED12 (Mediator Complex Subunit 12) mutation on their expression in fibroids and matched myometrium.

A mediator complex subunit 12 gain-of-function mutation induces partial leiomyoma cell properties in human uterine smooth muscle cells

To clarify whether a mediator complex subunit 12 (MED12) gain-of-function mutation induces leiomyoma cell properties in human uterine smooth muscle cells (USMCs). Experimental study. Academic research laboratory. Women undergoing hysterectomy for leiomyoma. CRISPR/Cas9-mediated genome editing to introduce an MED12 gain-of-function mutation (G44D) into human USMCs. Cell proliferation, collagen production, and invivo tumorigenicity of USMCs with vs. without the MED12 mutation. Uterine smooth muscle cells isolated from the uterine myometrium of a 44-year-old patient were subjected to lentiviral vector-mediated gene transduction of the fluorescent protein Venus, followed by long-term passage. Uterine smooth muscle cells with a normal female karyotype, high cell proliferative activity, and Venus expression, but without stem/progenitor cell populations, were obtained and designated as USMC44. Using CRISPR/Cas9-mediated genome editing, mtUSMC44 (MED12, 131G>A, p.G44D) and mock USMC44 without MED12 mutation (wtUSMC44) were established from USMC44. wtUSMC44 and mtUSMC44 showed similar cell proliferation activity, even in the presence of estradiol and progesterone (EP) together with transforming growth factor-beta 3 (TGFB3). In addition, wtUSMC44 and mtUSMC44 generated similar tiny smooth muscle-like tissue constructs when xenotransplanted beneath the kidney capsule in immunodeficient mice treated with EP alone or TGFB3. In contrast, mtUSMC44 produced more collagen type I than wtUSMC invitro, and this production was likely enhanced by EP and TGFB3. The results suggest that the MED12 gain-of-function mutation is involved in collagen production. Although approximately 70% of leiomyomas have MED12 mutations, additional factors and/or events other than MED12 and/or myometrialstem/progenitor cells may be required for fully inducing leiomyoma cell properties, including transformation, in USMCs.

Aberrant R-loop-induced replication stress in MED12-mutant uterine fibroids

Uterine fibroid (UF) driver mutations in Mediator complex subunit 12 (MED12) trigger genomic instability and tumor development through unknown mechanisms. Herein, we show that MED12 mutations trigger aberrant R-loop-induced replication stress, suggesting a possible route to genomic instability and a novel therapeutic vulnerability in this dominant UF subclass. Immunohistochemical analyses of patient-matched tissue samples revealed that MED12 mutation-positive UFs, compared to MED12 mutation-negative UFs and myometrium, exhibited significantly higher levels of R-loops and activated markers of Ataxia Telangiectasia and Rad3-related (ATR) kinase-dependent replication stress signaling in situ. Single molecule DNA fiber analysis revealed that primary cells from MED12 mutation-positive UFs, compared to those from patient-matched MED12 mutation-negative UFs and myometrium, exhibited defects in replication fork dynamics, including reduced fork speeds, increased and decreased numbers of stalled and restarted forks, respectively, and increased asymmetrical bidirectional forks. Notably, these phenotypes were recapitulated and functionally linked in cultured uterine smooth muscle cells following chemical inhibition of Mediator-associated CDK8/19 kinase activity that is known to be disrupted by UF driver mutations in MED12. Thus, Mediator kinase inhibition triggered enhanced R-loop formation and replication stress leading to an S-phase cell cycle delay, phenotypes that were rescued by overexpression of the R-loop resolving enzyme RNaseH. Altogether, these findings reveal MED12-mutant UFs to be uniquely characterized by aberrant R-loop induced replication stress, suggesting a possible basis for genomic instability and new avenues for therapeutic intervention that involve the replication stress phenotype in this dominant UF subtype.

Long Noncoding RNA MIAT Modulates the Extracellular Matrix Deposition in Leiomyomas by Sponging MiR-29 Family.

The objective of this study was to determine the expression and functional role of a long noncoding RNA (lncRNA) MIAT (myocardial infarction-associated transcript) in leiomyoma pathogenesis. Leiomyoma compared with myometrium (n = 66) expressed significantly more MIAT that was independent of race/ethnicity and menstrual cycle phase but dependent on MED12 (mediator complex subunit 12) mutation status. Leiomyomas bearing the MED12 mutation expressed higher levels of MIAT and lower levels of microRNA 29 family (miR-29a, -b, and -c) compared with MED12 wild-type leiomyomas. Using luciferase reporter activity and RNA immunoprecipitation analysis, MIAT was shown to sponge the miR-29 family. In a 3-dimensional spheroid culture system, transient transfection of MIAT siRNA in leiomyoma smooth muscle cell (LSMC) spheroids resulted in upregulation of miR-29 family and downregulation of miR-29 targets, collagen type I (COL1A1), collagen type III (COL3A1), and TGF-β3 (transforming growth factor β-3). Treatment of LSMC spheroids with TGF-β3 induced COL1A1, COL3A1, and MIAT levels, but repressed miR-29 family expression. Knockdown of MIAT in LSMC spheroids blocked the effects of TGF-β3 on the induction of COL1A1 and COL3A1 expression. Collectively, these results underscore the physiological significance of MIAT in extracellular matrix accumulation in leiomyoma.

Frequency of MED12 Mutation in Relation to Tumor and Patient's Clinical Characteristics: a Meta-analysis.

Mediator complex subunit 12 (MED12) is the most frequently mutated gene in uterine leiomyomas (ULs)-with a frequency of up to 85%-suggesting that it plays key roles in the pathogenesis of ULs. However, there is no established relationship between genetic alteration and other risk factors of UL pathogenesis such as the patient's age, weight, and race. In this meta-analysis, we established an association between these risk factors and the frequency of MED12 mutation. We also established the relationship between MED12 mutation with the number and size of tumors in a patient. A systematic literature search was performed for studies published by May 2020 and performed a meta-analysis according to PRISMA guidelines. Twenty-five studies were included in the analysis, representing 3151 tissue samples. MED12 mutations were more common in Black (74.5%) as compared to White (65.8%) and Asian (53.2%) patients. There was no significant relationship between the patient's age and the frequency of mutations (OR 0.73, 95% CI 0.38 to 1.41). MED12 mutations were common in patients barring small-sized (OR 1.46, 95% CI 1.09 to 1.95) multiple (OR 0.39, 95% CI 0.17 to 0.92) tumors. For the patient's weight, studies were few and the outcome was not statistically significant. This meta-analysis provides valuable information on the relationship between the patient's clinical characteristics and frequency of MED12 mutation among patients barring ULs, which is relevant for understanding the pathogenesis of ULs.Protocol registration: The protocol was registered in PROSPERO with registration number CRD42019123439.

Droplet-digital PCR reveals frequent mutations in TERT promoter region in breast fibroadenomas and phyllodes tumours, irrespective of the presence of MED12 mutations

BackgroundBreast fibroadenoma (FA) and phyllodes tumour (PT) often have variations of gene mediator complex subunit 12 (MED12) and mutations in the telomerase reverse transcriptase promoter region (TERTp). TERTp mutation is usually tested by Sanger sequencing. In this study, we compared Sanger sequencing and droplet-digital PCR (ddPCR) to measure TERTp mutations in FA and PT samples.MethodsFA and PT samples were collected from 82 patients who underwent surgery at our institution from 2005 to 2016. MED12 mutations for all cases and TERTp mutations for 17 tumours were detected by Sanger sequencing. ddPCR was performed to analyse TERTp mutation in all cases.ResultsA total of 75 samples were eligible for analysis. Sanger sequencing detected MED12 mutations in 19/44 FA (42%) and 21/31 PT (68%). Among 17 Sanger sequencing-tested samples, 2/17 (12%) were TERTp mutation-positive. In ddPCR analyses, a significantly greater percentage of PT (19/31, 61%) was TERTp mutation-positive than was FA (13/44, 30%; P = 0.0046). The mutation positivity of TERTp and MED12 did not correlate, in either FA or PT.ConclusionsddPCR was more sensitive for detecting TERTp mutation than Sanger sequencing, being able to elucidate tumorigenesis in FA and PT.

Vitamin D as an effective treatment in human uterine leiomyomas independent of mediator complex subunit 12 mutation

To study whether vitamin D (VitD) inhibits cell proliferation and Wnt/β-catenin and transforming growth factor-β (TGFβ) signaling pathways in uterine leiomyomas independent of mediator complex subunit 12 (MED12) mutation status. Prospective study comparing leiomyoma vs. myometrial tissues and human uterine leiomyoma primary (HULP) cells treated with or without VitD and analyzed by MED12 mutation status. Hospital and university laboratories. Women with uterine leiomyoma without any treatment (n = 37). Uterine leiomyoma and myometrium samples were collected from women undergoing surgery because of symptomatic leiomyoma pathology. Analysis of Wnt/β-catenin and TGFβ pathways and proliferation by quantitative real-time polymerase chain reaction in leiomyoma and myometrial tissue as well as in VitD-treated HULP cells analyzed by Sanger sequencing. Sequencing data showed that 46% of leiomyomas presented MED12 mutation, whereas no mutations were detected in adjacent myometrium. Expression of Wnt/β-catenin and TGFβ pathway genes was significantly increased in MED12-mutated leiomyomas compared to matched myometrium; no significant differences were found in wild-type (WT) leiomyomas. In HULP cells, VitD significantly decreased PCNA expression of both MED12-mutated and WT groups. VitD treatment decreased WNT4 and β-catenin expression in both groups compared to controls, with significance for WNT4 expression in MED12-mutated samples. Similarly, VitD significantly inhibited TGFβ3 expression in cells from both groups. MMP9 expression also decreased. Despite molecular differences between MED12-mutated and WT leiomyomas, VitD inhibited Wnt/β-catenin and TGFβ pathways in HULP cells, suggesting VitD as an effective treatment to reduce proliferation and extracellular matrix formation in different molecular subtypes of uterine leiomyomas.

Systematic molecular and clinical analysis of uterine leiomyomas from fertile-aged women undergoing myomectomy.

What are the distributions and associated clinical characteristics of mediator complex subunit 12 (MED12), high mobility group AT-hook 2 (HMGA2) and fumarate hydratase (FH) aberrations in uterine leiomyomas from fertile-aged myomectomy patients? These driver mutations account for the majority (83%) of tumours in fertile-aged patients. Alterations affecting MED12, HMGA2 and FH account for 80-90% of uterine leiomyomas from middle-aged hysterectomy patients, while the molecular background of tumours from young myomectomy patients has not been systematically studied. A retrospective series of 361 archival uterine leiomyoma samples from 234 women aged ≤45 years undergoing myomectomy in 2009-2014 was examined. Associations between the molecular data and detailed clinical information of the patients and tumours were analysed. DNA was extracted from formalin-fixed paraffin-embedded samples and MED12 exons 1 and 2 were sequenced to identify mutations. Level of HMGA2 expression was evaluated by immunohistochemistry. Biallelic FH inactivation was analysed with 2-succinylcysteine staining, which is an indirect method of assessing FH deficiency. All patients' medical histories were reviewed, and clinical information of patients and tumours was combined with molecular data. The median age at operation was 34 years. The majority (58%) of patients were operated on for a single leiomyoma. Known driver mutations were identified in 83% of tumours (71% MED12; 9% HMGA2; 3% FH). In solitary leiomyomas, the MED12 mutation frequency was only 43%, and 29% were wild-type for all driver alterations. MED12 mutations were associated with multiple tumours, smaller tumour size and subserosal location. Although comprehensive, the study is retrospective in nature and all samples have been collected for routine diagnostic purposes. The use of paraffin-embedded samples and immunohistochemistry may have led to an underestimation of mutations. Due to the limited sample size and rarity of especially FH-deficient leiomyomas, the data are partly descriptive. The contribution of driver mutations in leiomyomas from young myomectomy patients is comparable to tumours obtained from hysterectomies of mostly middle-aged women. Our results support the earlier findings that MED12 mutations are associated with multiple tumours, smaller tumour size and subserosal location. The study emphasizes the distinct molecular background of solitary leiomyomas, and more research is needed to clarify the underlying causes of the notable proportion of wild-type leiomyomas. The study was supported by the Academy of Finland (307773), the Sigrid Jusélius Foundation, the Cancer Foundation Finland and the iCAN Digital Precision Cancer Medicine Flagship. The authors declare no conflicts of interest. N/A.

Introduction of Somatic Mutation in MED12 Induces Wnt4/β-Catenin and Disrupts Autophagy in Human Uterine Myometrial Cell.

Uterine fibroids (UFs) or leiomyoma are frequently associated with somatic mutations in the mediator complex subunit 12 (MED12) gene; however, the function of these mutations in human UF biology is yet to be determined. Herein, we determined the functional role of the most common MED12 somatic mutation in the modulation of oncogenic Wnt4/β-catenin and mammalian target of rapamycin (mTOR) signaling pathways. Using an immortalized human uterine myometrial smooth muscle cell line (UtSM), we constitutively overexpressed either MED12-Wild Type or the most common MED12 somatic mutation (c.131G>A), and the effects of this MED12 mutation were compared between these cell lines. This immortalized cell line was used as a model because it expresses wild type MED12 protein and do not possess MED12 somatic mutations. By comparing the effect between MED12-WT and MED12-mutant (mut) stable cell populations, we observed increased levels of protein expression of Wnt4 and β-catenin in MED12-mut cells as compared with MED12-WT cells. MED12-mut cells also expressed increased levels of mTOR protein and oncogenic cyclin D1 which are hallmarks of cell growth and tumorigenicity. This somatic mutation in MED12 showed an effect on cell-cycle progression by induction of S-phase cells. MED12-mut cells also showed inhibition of autophagy as compared with MED12-WT cells. Together, these findings indicate that the MED12 somatic mutation has the potentials for myometrial cell transformation by dysregulating oncogenic Wnt4/β-catenin and its downstream mTOR signaling which might be associated with autophagy abrogation, cell proliferation, and tumorigenicity.

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

Transcriptional dysregulation induced by disease-defining genetic alterations of proteins in transcriptional machinery is a key feature of cancers. Mediator complex subunit 12 (MED12) is the central architectural subunit in the kinase module of Mediator, a large transcriptional regulatory complex that controls essential steps of transcription. Emerging evidence links deregulated MED12 to human cancers. MED12 is frequently mutated in benign tumors and cancers. Although the missense mutations of MED12 in benign tumors disrupt the kinase activity of Mediator, MED12 mutations in cancers could eliminate the interaction between Mediator complex and RNA polymerase II, leading to severe transcriptional misregulation. Aberrant expression of MED12 is associated with the prognosis of various types of human cancers. Loss of MED12 function has been associated with the development of resistance to chemotherapeutics. Moreover, MED12 is modified by posttranscriptional regulations. Arginine methylation of MED12 has been shown to regulate MED12-mediated transcriptional regulation and response to chemotherapeutics in human cancer cell lines. In this mini-review, the authors provide an overview of the roles of MED12 in the development of benign and malignant tumors as well as its roles in chemoresistance. The studies of MED12 exemplify that aberrant transcriptional programming is a therapeutic vulnerability for certain types of cancer.

Proteomic Analysis Identifies Tenascin-C Expression Is Upregulated in Uterine Fibroids.

Uterine leiomyomas (fibroids) are the most common gynecological tumors, which are enriched in the extracellular matrix (ECM). Fibroids are leading cause of abnormal uterine bleeding and hysterectomy. One of the major questions yet to be answered is the overproduction of specific ECM components in human uterine fibroids, particularly in relation to mutations in the driver gene mediator complex subunit 12 ( MED12). Surgical specimens from 14 patients with uterine leiomyoma having fibroids and corresponding adjacent normal myometrium (ANM) were utilized to analyze genetic and proteomic expression patterns in the tissue samples. MED12 mutations in the fibroids were screened by Sanger sequencing. iTRAQ was used to label the peptides in small-, medium-, and large-sized fibroid samples of annotated MED12 mutation from the same patient. The mixtures of the peptides were fractionated by hydrophilic interaction liquid chromatography (HILIC) and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) to identify the differential expression proteins. Using isobaric tagged-based quantitative mass spectrometry on 3 selected patients, ECM-related protein tenascin-C (TNC) was observed significantly upregulated (>1.5-fold) with a confidence corresponding to false discovery rate (FDR) <1% in small-, medium-, and large-sized fibroid samples regardless of MED12 mutation status. The TNC was validated on additional patient samples using Western blotting (WB) and immunohistochemistry (IHC) and confirmed significant overexpression of this protein in fibroids compared to matched ANM. Proteomic analyses have identified the increased ECM protein expression, TNC, as a hallmark of uterine fibroids regardless of MED12 mutations. Further functional studies focusing on the upregulated ECM proteins in leiomyogenesis will lead to the identification of novel ECM drug targets for fibroid treatment.

MicroRNAs involved in the HMGA2 deregulation and its co-occurrence with MED12 mutation in uterine leiomyoma.

Can the mediator complex subunit 12 (MED12) mutation and high mobility group AT-hook 2 (HMGA2) overexpression co-occurrence be explained by the alternative mechanism of HMGA2 dysregulation in uterine leiomyomas (UL)? The co-occurrence of MED12 mutation and HMGA2 overexpression, and a negative correlation of five validated or predicted microRNAs that target HMGA2 were reported. The recent stratification of UL, according to recurrent and mutually exclusive genomic alterations affecting HMGA2, MED12, fumarate hydratase (FH) and collagen type IV alpha 5-alpha 6 (COL4A5-COL4A6) pointed out the involvement of distinct molecular pathways. However, the mechanisms of regulation involving these drivers are poorly explored. A total of 78 UL and 34 adjacent normal myometrium (NM) tissues was collected from 56 patients who underwent hysterectomies at a single institution. The patients were treated at the Department of Gynecology and Obstetrics, School of Medicine, Sao Paulo State University, Botucatu, SP, Brazil, from October 1995 to February 2004. Gene expression profiling was evaluated from fresh frozen tissues and compared with MED12 mutations at exon 2. In addition, RT-qPCR was applied to evaluate the expression levels of HMGA2 and their predictive miRNA regulators: hsa-let-7a, miR-26a, miR-26b, mir-93 and mir-106b. An unsupervised hierarchical clustering analysis revealed two main clusters with one of them (26 of 42 UL) showing an enrichment of MED12 mutated cases (18 of 26 UL). Increased expression levels of HMGA2 were observed in both clusters, including cases with MED12 mutation (cluster 1:18 UL). A significant HMGA2 overexpression (P < 0.001) in UL in comparison with NM was found. Five miRNAs predicted to regulate HMGA2 were significantly downregulated (P < 0.001) and negatively correlated to HMGA2 expression levels (P < 0.05) in UL. An in vivo functional study was not performed to validate the microRNAs and HMGA2 interaction due to technical limitations. HMGA2 overexpression was detected in a significant number of MED12 mutated ULs, suggesting that these alterations coexist. Furthermore, five miRNAs were described as potential regulators of HMGA2 expression in UL. Data available in the Gene Expression Omnibus GSE42939. This study was supported by grants from Fundação de Amparo a Pesquisa do Estado de São Paulo (# 2008/58835-2) and Conselho Nacional de Pesquisa (# 485032/2007-4), Brazil. The authors declared having no conflicts of interest.

Abstract 4611: Uterine leiomyoma driver events in uterine adenomyomas

Abstract Uterine adenomyosis is a condition in which ectopic endometrial glands are present in myometrial stroma surrounded by smooth muscle cell hyperplasia. Foci of adenomyosis growing as a tumor like mass are called adenomyomas. Uterine adenomyomas are common tumors and they share symptoms, including pelvic pain and abnormal bleeding, with uterine leiomyomas. The two tumor types are challenging to distinguish from one another and the diagnosis is usually confirmed only after surgery by pathological evaluation. The molecular background of uterine adenomyomas is not currently well known. In uterine leiomyomas, somatic mediator complex subunit 12 (MED12) mutations, high mobility group AT-hook (HMGA2) protein overexpression, and fumarate hydratase (FH) inactivation are well established as major mutually exclusive driver events covering 80-90% of the tumors. Here, we have analyzed the presence of these changes in a set of 21 uterine adenomyomas. Representative areas of formalin-fixed paraffin embedded archival uterine adenomyoma tissue samples were used to construct a tissue microarray. The HMGA2 overexpression and the FH inactivation were assessed using immunohistochemistry with anti HMGA and 2SC antibodies, respectively. DNA was extracted from the tumor samples to determine the MED12 mutation status by direct sequencing of exons 1 and 2 of the gene. MED12 c.131GA, p.G44D mutation was found in two adenomyoma samples out of 21 (9.5%). Strong positive staining of 2SC indicating FH inactivation was present in one sample which also showed reduced FH protein expression when validated with an independent method using anti-FH immunostaining. Sequencing revealed a frameshift mutation c.911delC, p.P304fs in exon 7 leading to a premature stop codon 25 codons later. The mutation was also found in a separate uterine leiomyoma of the same patient and both tumor samples mostly presented the mutant allele indicating loss of heterozygosity of the wild type allele. This, together with the patient's medical history of previous uterine leiomyomas, indicates the germline origin of the mutation and thus a hereditary leiomyomatosis and renal cell cancer (HLRCC) syndrome. No changes in HMGA2 expression were detected with all samples presenting normal expression levels. In conclusion, MED12 mutations are present in a subset of uterine adenomyomas. The mutation frequency of 9.5% that was observed here in our adenomyoma sample series is considerably lower than that of 70% in uterine leiomyomas. Our results also suggest that adenomyomas may be linked to HLRCC in which they have not been previously reported. The driver events behind uterine adenomyomas remain mostly unknown and further large-scale studies are warranted to clarify the spectrum of underlying mutations and molecular background of these common tumors. Citation Format: Tuomas A. Heikkinen, Anna Äyräväinen, Janne Hänninen, Terhi Ahvenainen, Ralf Bützow, Annukka Pasanen, Pia Vahteristo. Uterine leiomyoma driver events in uterine adenomyomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4611.

Abstract 1159: Development of uterine leiomyoma 3D in vitro models for high-throughput drug and chemical compound screenings: Towards personalized medicine

Abstract Uterine leiomyomas (ULs) are extremely common smooth muscle tumors, occurring in an estimated 77% of women of reproductive age. Although benign, ULs form a major burden to women's health and are the leading cause of hysterectomy worldwide. Recent findings show that ULs can be classified into at least three distinct molecular subclasses, each with a characteristic genetic driver aberration and global gene expression profile: MED12 (mediator complex subunit 12) mutation-positive, HMGA2 (high mobility group AT-hook 2)-overexpressing, FH (fumarate hydratase)-deficient. However, ULs are currently treated without taking into account the possible different subclasses. The aim of this project is to develop and characterize 3D in vitro models of ULs. The models will be used to examine responses to existing treatments in different subclasses of UL and to identify subclass-specific novel lead compounds for drugs. The work is based on the hypothesis that different molecular UL subclasses have distinct sensitivity and resistance patterns to existing treatment options. Therefore, ULs should not be treated as a single entity. The study material consists of UL tissue belonging to different subclasses and myometrium, collected from hysterectomy patients during surgery in Helsinki University Hospital, Finland. Freshly operated samples are processed to initiate primary cell cultures. The primary cells are seeded on Ultra-Low Attachment (ULA) multiwell plates and grown to form 3D spheroids. In case of MED12-mutated tumors, the spheroids are screened for MED12 mutations by Sanger sequencing. MED12-wild type samples and spheroids are analyzed for overexpression of HMGA2 and FH-deficiency. Immunofluorescence staining is performed to characterize different cell types in the spheroid structure. The presence of live/dead cells in spheroids is monitored during the drug treatments in 384-well plates with high-content confocal microscopy. The approach will first focus on the most common subclasses (MED12-mutated and HMGA2-overexpressing tumors) and most common treatment options. Here, we have set up novel patient-derived cell models for ULs. We have characterized myometrium and UL-derived spheroids and show that they retain the characteristics and genetic driver aberration of the original samples. Furthermore, we show they are composed mostly of live cells. Subsequent in vitro high-throughput drug screening will lead to characterization of drug sensitivities and identification of novel lead compounds for drugs. This information is important for rational drug design, where the effects of candidate drugs can be evaluated in light of the molecular class of the respective tumors. This work will lead to new knowledge towards precision medicine, which has potential for scientific breakthrough, since it would avoid exposing the patients to ineffective treatment options. Citation Format: Simona Bramante, Vilja Pietiäinen, Lassi Paavolainen, Annukka Pasanen, Netta Mäkinen, Hanna-Riikka Heinonen, Pirjo Ikonen, Oskari Heikinheimo, Jari Sjöberg, Ralf Bützow, Lauri Aaltonen. Development of uterine leiomyoma 3D in vitro models for high-throughput drug and chemical compound screenings: Towards personalized medicine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 1159.

Insulin‐like growth factor II messenger RNA‐binding protein‐3 is an independent prognostic factor in uterine leiomyosarcoma

The aim of this study was to identify the prognostic factors of uterine leiomyosarcoma (ULMS). We reviewed 60 cases of surgically resected ULMSs and investigated conventional clinicopathological factors, together with the expression of insulin-like growth factor II messenger RNA-binding protein-3 (IMP3), hormone receptors and cell cycle regulatory markers by immunohistochemistry. Mediator complex subunit 12 (MED12) mutation analysis was also performed. Univariate analyses revealed that advanced stage (P < 0.0001), older age (P = 0.0244) and IMP3 expression (P = 0.0011) were significant predictors of a poor outcome. Multivariate analysis revealed advanced stage (P < 0.0001) and IMP3 (P = 0.0373) as independent predictors of a poor prognosis. Expressions of cell cycle markers and hormone receptors, and MED12 mutations (12% in ULMSs) were not identified as prognostic markers in this study. IMP3 expression in ULMS could be a marker of a poor prognosis.

Uterine Fibroids in Senegal: Polymorphism of MED12 Gene and Correlation With Epidemiological Factors

Background: Mediator complex subunit 12 (MED12) is a part of the mediator complex, which is believed to regulate transcription. Our recent study showed that MED12 is mutated at high frequency and with different mutation frequencies in Senegalese women with uterine fibroids. However, the status of mutations has not been correlated to the epidemiological factors that are implicated in uterine fibroids. Methods: This study aimed to analyze status of MED12 mutations in Senegalese population. MED12 was sequenced in tumoral tissues and blood samples of 54 Senegalese women with uterine fibroids. Clinical and pathological data were obtained from the patient’s records and other parameters were recorded. Mutation Surveyor software version 5.0.1, DnaSP version 5.10, MEGA version 7.0.26 and Arlequin version 3.5.1.3 were used to determine the level of mutations and genetics parameters. To estimate the genetic variation according to the epidemiological parameters, the index of genetic differentiation (Fst) and the genetic structure like analysis of molecular variance (AMOVA) were determined with Arlequin software version 3.5.1.3. The significance level (P-value) was 0.05. Results: Our results showed that MED12 is mutated at 88.89% (48/54) only in tumor tissues. The variants frequencies were not similar to those found in the Finnish populations. The Chi2 test indicates a statistically significant difference for the variants c.130G>C, c.130G>A, c.131G>A and for the Intronic Variant Site (p <0.05). This variable expression of the MED12 gene is further confirmed by the amino acid frequency between blood and tumor tissue with a statistically significant difference for alanine, glutamic acid, lysine, methionine, threonine, valine, tryptophan and tyrosine (p <0.05). In addition, the selection test indicates that codon 44 is under positive selection (p=0.0243) in cases of uterine fibroids. Genetic diversity according to risk factors such as parity and diet was observed in uterine fibroids in Senegal (p <0.05). Conclusion: Significant genetic diversity has been noted in uterine fibroids in Senegal. The codon 44 being under positive selection could be considered as a biomarker in uterine fibroids. Depending on the epidemiological parameters studied, parity and diet seem to be the risk factors most implicated in uterine fibroids in Senegal.