Transcriptional and post-transcriptional regulation of histone variant H2A.Z during sea urchin development.

Histone variant H2A.Z promotes chromatin accessibility at transcriptional regulatory elements and is developmentally regulated in metazoans. We characterize the transcriptional and post-transcriptional regulation of H2A.Z in the purple sea urchin Strongylocentrotus purpuratus. H2A.Z depletion by antisense translation-blocking morpholino oligonucleotides during early development causes developmental collapse, in agreement with its previously demonstrated general role in transcriptional multipotency. During H2A.Z peak expression in 24-h embryos, endogenous H2A.Z 3' UTR sequences stabilize GFP mRNAs relative to those with SV40 3' UTR sequences, although the 3' UTR of H2A.Z does not determine the spatial distribution of H2A.Z transcripts during embryonic and postembryonic development. We elaborated an H2A.Z::GFP BAC reporter that reproduces embryonic H2A.Z expression. Genome-wide chromatin accessibility analysis using ATAC-seq revealed a cis-regulatory module (CRM) that, when deleted, causes a significant decline of the H2A.Z reporter expression. In addition, the mutation of a Sox transcription factor binding site motif and, more strongly, of a Myb motif cause significant decline of reporter gene expression. Our results suggest that an undetermined Myb-family transcription factor controls the transcriptional regulation of H2A.Z.

Patterns of deregulation of insulin growth factor signalling pathway in paediatric and adult gastrointestinal stromal tumours.

BACKGROUND: Data regarding the patterns and the mechanisms of deregulation of the insulin growth factor (IGF) pathway in adult and paediatric gastrointestinal stromal tumours (GISTs) are limited. METHODS: We investigated the expression profiling of the genes encoding the main components of the IGF signalling pathway in 131 GISTs (106 adults, 21 paediatric and four young adults) and 25 other soft-tissue sarcomas (STS) using an Affymetrix U133A platform. IGF2 was investigated for loss of imprinting (LOI) whereas IGF1R was analysed for copy number aberration and mutation. RESULTS: IGF2 was the most highly overexpressed gene of the IGF pathway in GIST. IGF2 expression was also significantly higher than in other STS. IGF2 expression was correlated to the age onset and mutational status of GIST. Indeed, IGF2 expression was significantly higher in the 'adult' group than in the 'paediatric' and 'young adult' groups. Among adult GIST, IGF2 expression was higher in tumours lacking Homo sapiens v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) or alpha-type platelet-derived growth factor receptor (PDGFRA) mutations in comparison with mutated cases. A trend for a higher expression of IGF2 in resistant GIST in comparison to responsive GIST was also found. Overexpression of IGF2 was not related to LOI. Conversely, the expression of the IGF1R gene was significantly higher in the paediatric group than in the adult group. No copy number gains or mutations of IGF1R were observed. CONCLUSION: The IGF pathway is deregulated in GIST with distinct patterns according to age onset and mutational status. The IGF pathway may represent a therapeutic target in patients with primary or secondary resistance to imatinib.

A novel WWTR1-CAMTA1 gene fusion is a consistent abnormality in epithelioid hemangioendothelioma of different anatomic sites.

The classification of epithelioid vascular tumors remains challenging, as there is considerable morphological overlap between tumor subtypes, across the spectrum from benign to malignant categories. A t(1;3)(p36.3;q25) translocation was reported in two cases of epithelioid hemangioendothelioma (EHE), however, no follow-up studies have been performed to identify the gene fusion or to assess its prevalence in a larger cohort of patients. We undertook a systematic molecular analysis of 17 EHE, characterized by classic morphological and immunophenotypic features, from various anatomical locations and with different malignant potential. For comparison, we analyzed 13 epithelioid hemangiomas, five epithelioid angiosarcomas, and four epithelioid sarcoma-like EHE. A fluorescence in situ hybridization (FISH) positional cloning strategy, spanning the cytogenetically defined regions on chromosomes 1p36.3 and 3q25, confirmed rearrangements in two candidate genes from these loci in all EHE cases tested. None of the other benign or malignant epithelioid vascular tumors examined demonstrated these abnormalities. Subsequent reverse transcription-polymerase chain reaction (RT-PCR) confirmed in three EHE the WWTR1-CAMTA1 fusion product. CAMTA1 and WWTR1 have been previously shown to play important roles in oncogenesis. Our results demonstrate the presence of a WWTR1-CAMTA1 fusion in all EHE tested from bone, soft tissue, and visceral location (liver, lung) in keeping with a unique and specific pathological entity. Thus, FISH or RT-PCR analysis for the presence of WWTR1-CAMTA1 fusion may serve as a useful molecular diagnostic tool in challenging diagnoses.

IGF2 over-expression in solitary fibrous tumours is independent of anatomical location and is related to loss of imprinting.

Solitary fibrous tumour (SFT) is a mesenchymal neoplasm composed of CD34-positive fibroblastic cells. The pathogenesis driving this neoplasm remains unclear, with no recurrent genetic aberrations described to date. Previous reports suggest a role for IGF2 over-expression in the pathogenesis of these tumours, implicated in triggering hypoglycaemia in some patients. The expression profiling of 23 SFTs was investigated using an Affymetrix U133A platform. The transcriptional signature was compared to a set of 34 soft tissue sarcomas spanning seven subtypes. Potential candidate genes were then further investigated for activating mutations or loss of imprinting (LOI). SFT had a distinct expression signature and clustered in a tight genomic cluster, separate from all other sarcoma subtypes. A number of over-expressed receptor tyrosine kinase (RTK) genes were identified in SFT, including DDR1, ERBB2 and FGFR1; however, no mutations were identified by cDNA sequencing. Over-expression of IGF2 was uniformly detected in SFT, regardless of anatomical location, and was related to LOI. In contrast, IGF1 and JUN over-expression was seen in pleural, but not meningeal, locations. SFT shows a distinctive expression signature, with over-expression of DDR1, ERBB2 and FGFR1. Despite of lack of activating mutations in these RTKs, therapy with specific inhibitors targeting these kinases might be considered in advanced/metastatic cases. Our results confirm LOI in several tumours expressing high levels of IGF2, which may explain the observed paraneoplastic hypoglycaemia.

Mechanisms of sunitinib resistance in gastrointestinal stromal tumors harboring KITAY502-3ins mutation: an in vitro mutagenesis screen for drug resistance.

PURPOSE: Although tyrosine kinase inhibitors have improved survival in advanced gastrointestinal stromal tumor (GIST), complete response is rare and most patients eventually fail the first-line treatment with imatinib. Sunitinib malate is the only approved second-line therapy for patients with imatinib-resistant or imatinib-intolerant GIST. The clinical benefit of sunitinib is genotype-dependent in regards to both primary and secondary mutations, with GIST patients harboring the KIT(AY502-3ins) exon 9 mutation being the most sensitive. EXPERIMENTAL DESIGN: As sunitinib resistance is now emerging, our goal was to investigate mechanisms of progression and to test the efficacy of novel tyrosine kinase inhibitor on these resistant mutants in vitro. N-ethyl-N-nitrosourea mutagenesis of Ba/F3 cells expressing the KIT(AY502-3ins) mutant was used to investigate novel patterns of resistant mutations evolving in the presence of sunitinib. RESULTS: Tumors from patients who developed sunitinib resistance after at least 1 year of radiographic response were analyzed, showing similar findings of a primary KIT(AY502-3ins) mutation and a secondary mutation in the KIT activation loop. Ba/F3 cells expressing these sunitinib-resistant double mutants showed sensitivity to both dasatinib and nilotinib. CONCLUSIONS: Sunitinib resistance in GIST shares similar pathogenetic mechanisms identified in imatinib failure, with acquisition of secondary mutations in the activation domain after an extended initial response to the drug. Moreover, in vitro mutagenesis with or without N-ethyl-N-nitrosourea of Ba/F3 cells expressing KIT(AY502-3ins) showed acquisition of secondary mutations restricted to the second kinase domain of KIT. In contrast, in vitro resistance to imatinib produces a broader spectrum of secondary mutations including mutations in both KIT kinase domains.