BackgroundRecent studies have reported mutations in the telomerase reverse transcriptase promoter (TERTp) in meningiomas. We sought to determine the frequency, clonality and clinical significance of telomere gene alterations in a cohort of patients with progressive/higher-grade meningiomas.MethodsWe characterized 64 temporally- and regionally-distinct specimens from 26 WHO grade III meningioma patients. On initial diagnoses, the meningiomas spanned all WHO grades (3 grade I, 13 grade II and 10 grade III). The tumor samples were screened for TERTp and ATRX/DAXX mutations, and TERT rearrangements. Additionally, TERTp was sequenced in a separate cohort of 19 patients with radiation-associated meningiomas. We examined the impact of mutational status on patients’ progression and overall survival.ResultsSomatic TERTp mutations were detected in six patients (6/26 = 23%). Regional intratumoral heterogeneity in TERTp mutation status was noted. In 4 patients, TERTp mutations were detected in recurrent specimens but not in the available specimens of the first surgery. Additionally, a TERT gene fusion (LPCAT1-TERT) was found in one sample. In contrary, none of the investigated samples harbored an ATRX or DAXX mutation. In the cohort of radiation-induced meningiomas, TERTp mutation was detected in two patients (10.5%). Importantly, we found that patients with emergence of TERTp mutations had a substantially shorter OS than their TERTp wild-type counterparts (2.7 years, 95% CI 0.9 – 4.5 years versus 10.8 years, 95% CI 7.8 -12.8 years, p=0.003).ConclusionsIn progressive/higher-grade meningiomas,TERTp mutations are associated with poor survival, supporting a model in which selection of this alteration is a harbinger of aggressive tumor development. In addition, we observe spatial intratumoral heterogeneity of TERTp mutation status, consistent with this model of late emergence in tumor evolution. Thus, early detection of TERTp mutations may define patients with more aggressive meningiomas. Stratification for TERT alterations should be adopted in future clinical trials of progressive/higher-grade meningiomas.

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Progressive meningiomas that have failed surgery and radiation have a poor prognosis and no standard therapy. While meningiomas are more common in females overall, progressive meningiomas are enriched in males. We performed a comprehensive molecular characterization of 169 meningiomas from 53 patients with progressive/high-grade tumors, including matched primary and recurrent samples. Exome sequencing in an initial cohort (n = 24) detected frequent alterations in genes residing on the X chromosome, with somatic intragenic deletions of the dystrophin-encoding and muscular dystrophy-associated DMD gene as the most common alteration (n = 5, 20.8%), along with alterations of other known X-linked cancer-related genes KDM6A (n =2, 8.3%), DDX3X, RBM10 and STAG2 (n = 1, 4.1% each). DMD inactivation (by genomic deletion or loss of protein expression) was ultimately detected in 17/53 progressive meningioma patients (32%). Importantly, patients with tumors harboring DMD inactivation had a shorter overall survival (OS) than their wild-type counterparts [5.1 years (95% CI 1.3-9.0) vs. median not reached (95% CI 2.9-not reached, p = 0.006)]. Given the known poor prognostic association of TERT alterations in these tumors, we also assessed for these events, and found seven patients with TERT promoter mutations and three with TERT rearrangements in this cohort (n = 10, 18.8%), including a recurrent novel RETREG1-TERT rearrangement that was present in two patients. In a multivariate model, DMD inactivation (p = 0.033, HR = 2.6, 95% CI 1.0-6.6) and TERT alterations (p = 0.005, HR = 3.8, 95% CI 1.5-9.9) were mutually independent in predicting unfavorable outcomes. Thus, DMD alterations identify a subset of progressive/high-grade meningiomas with worse outcomes.

GnRH and activin independently and synergistically activate transcription of the FSH beta-subunit gene, the subunit that provides specificity and is the limiting factor in the synthesis of the mature hormone. This synergistic interaction, as determined by two-way ANOVA, is specific for FSHbeta and may, therefore, contribute to differential expression of the two gonadotropin hormones, which is critical for the reproductive cycle. We find that the cross-talk between the GnRH and activin signaling pathways occurs at the level of p38 MAPK, because the synergy is dependent on p38 MAPK activity, which is activated by GnRH, and activin cotreatment augments p38 activation by GnRH. Both the Smad and activator protein-1 binding sites on the FSHbeta promoter are necessary and sufficient for synergy. After cotreatment, Smad 3 proteins are more highly phosphorylated on the activin-receptor signaling-dependent residues on the C terminus than with activin treatment alone, and c-Fos is more highly expressed than with GnRH treatment alone. Inhibition of p38 by either of two different inhibitors or a dominant-negative p38 kinase abrogates synergy on FSHbeta expression, reduces c-Fos induction by GnRH, and prevents the further increase in c-Fos levels that occurs with cotreatment. Additionally, p38 is necessary for maximal Smad 3 C-terminal phosphorylation by activin treatment alone and for the further increase caused by cotreatment. Thus, p38 is the pivotal signaling molecule that integrates GnRH and activin interaction on the FSHbeta promoter through higher induction of c-Fos and elevated Smad phosphorylation.

Despite extensive studies on GnRH regulation of the gonadotropin subunit genes, very little is known about mechanism of induction of intermediary immediate early genes, such as c-Fos, that are direct targets of GnRH signaling and that upon induction, activate transcription of gonadotropin genes. Although c-Fos is induced by a variety of stimuli in other cell types, in the gonadotropes, only GnRH induces c-Fos and through it FSHβ. Thus, understanding the specificity of c-Fos induction by GnRH will provide insight into GnRH regulation of FSHβ gene expression. GnRH induction of c-Fos in LβT2 cells requires the serum response factor (SRF)-binding site, but not the Ets/ELK1 site. This is in contrast to c-Fos induction by growth factors in other cells, which activate c-Fos transcription via phosphorylation of ELK1 and require the ELK1-binding site. The SRF site alone is sufficient for induction by GnRH, whereas induction by 12-tetradecanoylphorbol-13-acetate (TPA) requires both the ELK1 and SRF sites. Although ELK1 site is not required, upon GnRH stimulation, ELK1 interacts with SRF and is recruited to the SRF site. GnRH phosphorylates ELK1 through ERK1/2 and p38 MAPK, which correlates with the signaling pathways necessary for c-Fos and FSHβ induction. GnRH also causes phosphorylation of SRF through calmodulin-dependent kinase II (CamKII), which leads to increased binding to its site. CamKII activation is sufficient for phosphorylation of SRF and for induction of the c-Fos gene through the SRF site. Thus, GnRH uses a combination of growth factor signaling and the CamKII pathway to induce c-Fos to regulate FSHβ gene expression in gonadotrope cells.