EZH2 is required for parathyroid and thymic development through differentiation of the third pharyngeal pouch endoderm.

The Ezh2 gene encodes a histone methyltransferase of the polycomb repressive complex 2 that methylates histone H3 lysine 27. In this study, we investigated whether EZH2 has a role in the development of the pharyngeal apparatus and whether it regulates the expression of the Tbx1 gene, which encodes a key transcription factor required in pharyngeal development. To these ends, we performed genetic in vivo experiments with mouse embryos and used mouse embryonic stem cell (ESC)-based protocols to probe endoderm and cardiogenic mesoderm differentiation. Results showed that EZH2 occupies the Tbx1 gene locus in mouse embryos, and that suppression of EZH2 was associated with reduced expression of Tbx1 in differentiated mouse ESCs. Conditional deletion of Ezh2 in the Tbx1 expression domain, which includes the pharyngeal endoderm, did not cause cardiac defects but revealed that the gene has an important role in the morphogenesis of the third pharyngeal pouch (PP). We found that in conditionally deleted embryos the third PP was hypoplastic, had reduced expression of Tbx1, lacked the expression of Gcm2, a gene that marks the parathyroid domain, but expressed FoxN1, a gene marking the thymic domain. Consistently, the parathyroids did not develop, and the thymus was hypoplastic. Thus, Ezh2 is required for parathyroid and thymic development, probably through a function in the pouch endoderm. This discovery also provides a novel interpretational key for the finding of Ezh2 activating mutations in hyperparathyroidism and parathyroid cancer.

Halting the FGF/FGFR axis leads to antitumor activity in Waldenström macroglobulinemia by silencing MYD88.

The human fibroblast growth factor/fibroblast growth factor receptor (FGF/FGFR) axis deregulation is largely involved in supporting the pathogenesis of hematologic malignancies, including Waldenström macroglobulinemia (WM). WM is still an incurable disease, and patients succumb because of disease progression. Therefore, novel therapeutics designed to specifically target deregulated signaling pathways in WM are required. We aimed to investigate the role of FGF/FGFR system blockade in WM by using a pan-FGF trap molecule (NSC12). Wide-transcriptome profiling confirmed inhibition of FGFR signaling in NSC12-treated WM cells; unveiling a significant inhibition of MYD88 was also confirmed at the protein level. Importantly, the NSC12-dependent silencing of MYD88 was functionally active, as it led to inhibition of MYD88-driven pathways, such as BTK and SYK, as well as the MYD88-downstream target HCK. Of note, both canonical and noncanonical NF-κB cascades were downregulated in WM cells upon NSC12 treatment. Functional sequelae exerted by NSC12 in WM cells were studied, demonstrating significant inhibition of WM cell growth, induction of WM cell apoptosis, halting MAPK, JAK/STAT3, and PI3K-Akt pathways. Importantly, NSC12 exerted an anti-WM effect even in the presence of bone marrow microenvironment, both in vitro and in vivo. Our studies provide the evidence for using NSC12 as a specific FGF/FGFR system inhibitor, thus representing a novel therapeutic strategy in WM.

Epigenetic Aberrations in Multiple Myeloma.

Multiple myeloma (MM) is a plasma cell dyscrasia characterized by proliferation of clonal plasma cells within the bone marrow. Several advances in defining key processes responsible for MM pathogenesis and disease progression have been made; and dysregulation of epigenetics, including DNA methylation and histone modification, has emerged as a crucial regulator of MM pathogenesis. In the present review article, we will focus on the role of epigenetic modifications within the specific context of MM.

TBX1 and Basal Cell Carcinoma: Expression and Interactions with Gli2 and Dvl2 Signaling.

Early events of basal cell carcinoma (BCC) tumorigenesis are triggered by inappropriate activation of SHH signaling, via the loss of Patched1 (Ptch1) or by activating mutations of Smoothened (Smo). TBX1 is a key regulator of pharyngeal development, mainly through expression in multipotent progenitor cells of the cardiopharyngeal lineage. This transcription factor is connected to several major signaling systems, such as FGF, WNT, and SHH, and it has been linked to cell proliferation and to the regulation of cell shape and cell dynamics. Here, we show that TBX1 was expressed in all of the 51 BCC samples that we have tested, while in healthy human skin it was only expressed in the hair follicle. Signal intensity and distribution was heterogeneous among tumor samples. Experiments performed on a cellular model of mouse BCC showed that Tbx1 is downstream to GLI2, a factor in the SHH signaling, and that, in turn, it regulates the expression of Dvl2, which encodes an adaptor protein that is necessary for the transduction of WNT signaling. Consistently, Tbx1 depletion in the cellular model significantly reduced cell migration. These results suggest that TBX1 is part of a core transcription network that promotes BCC tumorigenesis.

Left pulmonary artery in 22q11.2 deletion syndrome. Echocardiographic evaluation in patients without cardiac defects and role of Tbx1 in mice.

INTRODUCTION AND HYPOTHESIS: Patients with 22q11 deletion syndrome (22q11.2DS) present, in about 75% of cases, typical patterns of cardiac defects, with a particular involvement on the ventricular outflow tract and great arteries. However, in this genetic condition the dimensions of the pulmonary arteries (PAs) never were specifically evaluated. We measured both PAs diameter in patients with 22q11.2DS without cardiac defects, comparing these data to a normal control group. Moreover, we measured the PAs diameter in Tbx1 mutant mice. Finally, a cell fate mapping in Tbx1 mutants was used to study the expression of this gene in the morphogenesis of PAs. METHODS: We evaluated 58 patients with 22q11.2DS without cardiac defects. The control group consisted of 54 healthy subjects, matched for age and sex. All cases underwent a complete transthoracic echocardiography. Moreover, we crossed Tbx1+/- mice and harvested fetuses. We examined the cardiovascular phenotype of 8 wild type (WT), 37 heterozygous (Tbx1+/-) and 6 null fetuses (Tbx1-/-). Finally, we crossed Tbx1Cre/+mice with R26RmT-mG Cre reporter mice to study Tbx1 expression in the pulmonary arteries. RESULTS: The echocardiographic study showed that the mean of the LPA/RPA ratio in 22q11.2DS was smaller (0.80 ± 0.12) than in controls (0.97 ± 0.08; p < 0.0001). Mouse studies resulted in similar data as the size of LPA and RPA was not significantly different in WT embryos, but in Tbx1+/- and Tbx1-/- embryos the LPA was significantly smaller than the RPA in both mutants (P = 0.0016 and 0.0043, respectively). We found that Tbx1 is expressed near the origin of the PAs and in their adventitia. CONCLUSIONS: Children with 22q11.2DS without cardiac defects show smaller LPA compared with healthy subjects. Mouse studies suggest that this anomaly is due to haploinsufficiency of Tbx1. These data may be useful in the clinical management of children with 22q11.2DS and should guide further experimental studies as to the mechanisms underlying PAs development.

p53 Suppression partially rescues the mutant phenotype in mouse models of DiGeorge syndrome.

T-box 1 (Tbx1), a gene encoding a T-box transcription factor, is required for embryonic development in humans and mice. Half dosage of this gene in humans causes most of the features of the DiGeorge or Velocardiofacial syndrome phenotypes, including aortic arch and cardiac outflow tract abnormalities. Here we found a strong genetic interaction between Tbx1 and transformation related protein 53 (Trp53). Indeed, genetic ablation of Trp53, or pharmacological inhibition of its protein product p53, rescues significantly the cardiovascular defects of Tbx1 heterozygous and hypomorphic mutants. We found that the Tbx1 and p53 proteins do not interact directly but both occupy a genetic element of Gbx2, which is required for aortic arch and cardiac outflow tract development, and is a known genetic interactor of Tbx1. We found that Gbx2 expression is down-regulated in Tbx1(+/-) embryos and is restored to normal levels in Tbx1(+/-);Trp53(+/-) embryos. In addition, we found that the genetic element that binds both Tbx1 and p53 is highly enriched in H3K27 trimethylation, and upon p53 suppression H3K27me3 levels are reduced, along with Ezh2 enrichment. This finding suggests that the rescue of Gbx2 expression in Tbx1(+/-);Trp53(+/-) embryos is due to reduction of repressive chromatin marks. Overall our data identify unexpected genetic interactions between Tbx1 and Trp53 and provide a proof of principle that developmental defects associated with reduced dosage of Tbx1 can be rescued pharmacologically.

Early thyroid development requires a Tbx1-Fgf8 pathway.

The thyroid develops within the pharyngeal apparatus from endodermally-derived cells. The many derivatives of the pharyngeal apparatus develop at similar times and sometimes from common cell types, explaining why many syndromic disorders express multiple birth defects affecting different structures that share a common pharyngeal origin. Thus, different derivatives may share common genetic networks during their development. Tbx1, the major gene associated with DiGeorge syndrome, is a key player in the global development of the pharyngeal apparatus, being required for virtually all its derivatives, including the thyroid. Here we show that Tbx1 regulates the size of the early thyroid primordium through its expression in the adjacent mesoderm. Because Tbx1 regulates the expression of Fgf8 in the mesoderm, we postulated that Fgf8 mediates critical Tbx1-dependent interactions between mesodermal cells and endodermal thyrocyte progenitors. Indeed, conditional ablation of Fgf8 in Tbx1-expressing cells caused an early thyroid phenotype similar to that of Tbx1 mutant mice. In addition, expression of an Fgf8 cDNA in the Tbx1 domain rescued the early size defect of the thyroid primordium in Tbx1 mutants. Thus, we have established that a Tbx1->Fgf8 pathway in the pharyngeal mesoderm is a key size regulator of mammalian thyroid.