Gastrointestinal stromal tumours (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract. The vast majority of GISTs are driven by oncogenic activation of KIT, PDGFRA or, less commonly, BRAF. Loss of succinate dehydrogenase complex activity has been identified in subsets of KIT/PDGFRA/BRAF‐mutation negative tumours, yet a significant fraction of GISTs are devoid of any of such alterations. To address the pathobiology of these 'quadruple‐negative' GISTs, we sought to explore the possible involvement of fusion genes. To this end we performed transcriptome sequencing on five KIT/PDGFRA/BRAF‐mutation negative, SDH‐proficient tumours. Intriguingly, the analysis unveiled the presence of an ETV6–NTRK3 gene fusion. The screening by FISH of 26 additional cases, including KIT/PDGFRA‐mutated GISTs, failed to detect other ETV6 rearrangements beside the index case. This was a 'quadruple‐negative' GIST located in the rectum, an uncommon primary site for GIST development (∼4% of all GISTs). The fusion transcript identified encompasses exon 4 of ETV6 and exon 14 of NTRK3 and therefore differs from the canonical ETV6–NTRK3 chimera of infantile fibrosarcomas. However, it retains the ability to induce IRS1 phosphorylation, activate the IGF1R downstream signalling pathway and to be targeted by IGF1R and ALK inhibitors. Thus, the ETV6–NTRK3 fusion might identify a subset of GISTs with peculiar clinicopathological characteristics which could be eligible for such therapies. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Purpose: The majority of gastrointestinal stromal tumors (GIST) are driven by KIT, PDGFRA, or, less commonly, BRAF mutations, and SDH gene inactivation is involved in a limited fraction of gastric lesions. However, about 10% of GISTs are devoid of any of such alterations and are poorly responsive to standard treatments. This study aims to shed light on the molecular drivers of quadruple-negative GISTs. Experimental Design: Twenty-two sporadic quadruple-negative GISTs with no prior association with Neurofibromatosis Type 1 syndrome were molecularly profiled for a panel of genes belonging to tyrosine kinase pathways or previously implicated in GISTs. For comparison purposes, 24 GISTs carrying KIT, PDGFRA, or SDH gene mutations were also analyzed. Molecular findings were correlated to clinicopathologic features. Results: Most quadruple-negative GISTs featured intestinal localization, with a female predilection. About 60% (13/22) of quadruple-negative tumors carried NF1 pathogenic mutations, often associated with biallelic inactivation. The analysis of normal tissues, available in 11 cases, indicated the constitutional nature of the NF1 mutation in 7 of 11 cases, unveiling an unrecognized Neurofibromatosis Type 1 syndromic condition. Multifocality and a multinodular pattern of growth were common findings in NF1-mutated quadruple-negative GISTs. Conclusions: NF1 gene mutations are frequent in quadruple-negative GISTs and are often constitutional, indicating that a significant fraction of patients with apparently sporadic quadruple-negative GISTs are affected by unrecognized Neurofibromatosis Type 1 syndrome. Hence, a diagnosis of quadruple-negative GIST, especially if multifocal or with a multinodular growth pattern and a nongastric location, should alert the clinician to a possible Neurofibromatosis Type 1 syndromic condition. Clin Cancer Res; 23(1); 273–82. ©2016 AACR.

The contribution of MEF2 TFs to the tumorigenic process is still mysterious. Here we clarify that MEF2 can support both pro-oncogenic or tumor suppressive activities depending on the interaction with co-activators or co-repressors partners. Through these interactions MEF2 supervise histone modifications associated with gene activation/repression, such as H3K4 methylation and H3K27 acetylation. Critical switches for the generation of a MEF2 repressive environment are class IIa HDACs. In leiomyosarcomas (LMS), this two-faced trait of MEF2 is relevant for tumor aggressiveness. Class IIa HDACs are overexpressed in 22% of LMS, where high levels of MEF2, HDAC4 and HDAC9 inversely correlate with overall survival. The knock out of HDAC9 suppresses the transformed phenotype of LMS cells, by restoring the transcriptional proficiency of some MEF2-target loci. HDAC9 coordinates also the demethylation of H3K4me3 at the promoters of MEF2-target genes. Moreover, we show that class IIa HDACs do not bind all the regulative elements bound by MEF2. Hence, in a cell MEF2-target genes actively transcribed and strongly repressed can coexist. However, these repressed MEF2-targets are poised in terms of chromatin signature. Overall our results candidate class IIa HDACs and HDAC9 in particular, as druggable targets for a therapeutic intervention in LMS. The tumorigenic process is characterized by profound alterations of the transcriptional landscape, aimed to sustain uncontrolled cell growth, resistance to apoptosis and metastasis. The contribution of MEF2, a pleiotropic family of transcription factors, to these changes is controversial, since both pro-oncogenic and tumor-suppressive activities have been reported. To clarify this paradox, we studied the role of MEF2 in an aggressive type of soft-tissue sarcomas, the leiomyosarcomas (LMS). We found that in LMS cells MEF2 become oncogenes when in complex with class IIa HDACs. We have identified different sub-classes of MEF2-target genes and observed that HDAC9 converts MEF2 into transcriptional repressors on some, but not all, MEF2-regulated loci. This conversion correlates with the acquisition by MEF2 of oncogenic properties. We have also elucidated some epigenetic re-arrangements supervised by MEF2. In summary, our studies suggest that the paradoxical actions of MEF2 in cancer can be explained by their dual role as activators/repressors of transcription and open new possibilities for therapeutic interventions.

Extraskeletal myxoid chondrosarcoma (EMC) is a rare sarcoma histotype with uncertain differentiation. EMC is hallmarked by the rearrangement of the NR4A3 gene, which in most cases fuses with EWSR1 or TAF15. TAF15‐translocated EMC seem to feature a more aggressive course compared to EWSR1‐positive EMCs, but whether the type of NR4A3 chimera impinges upon EMC biology is still largely undefined. To gain insights on this issue, a series of EMC samples (7 EWSR1‐NR4A3 and 5 TAF15‐NR4A3) were transcriptionally profiled. Our study unveiled that the two EMC variants display a distinct transcriptional profile and that the axon guidance pathway is a major discriminant. In particular, class 4–6 semaphorins and axonal guidance cues endowed with pro‐tumorigenic activity were more expressed in TAF15‐NR4A3 tumors; vice versa, class 3 semaphorins, considered to convey growth inhibitory signals, were more abundant in EWSR1‐NR4A3 EMC. Intriguingly, the dichotomy in axon guidance signaling observed in the two tumor variants was recapitulated in in vitro cell models engineered to ectopically express EWSR1‐NR4A3 or TAF15‐NR4A3. Moreover, TAF15‐NR4A3 cells displayed a more pronounced tumorigenic potential, as assessed by anchorage‐independent growth. Overall, our results indicate that the type of NR4A3 chimera dictates an axon guidance switch and impacts on tumor cell biology. These findings may provide a framework for interpretation of the different clinical–pathological features of the two EMC variants and lay down the bases for the development of novel patient stratification criteria and therapeutic approaches. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Background: Colorectal cancer is one of the most frequent and deadly tumors. Among the key regulators of CRC growth and progression, the microenvironment has emerged as a crucial player and as a possible route for the development of new therapeutic opportunities. More specifically, the extracellular matrix acts directly on cancer cells and indirectly affecting the behavior of stromal and inflammatory cells, as well as the bioavailability of growth factors. Among the ECM molecules, EMILIN-2 is frequently down-regulated by methylation in CRC and the purpose of this study was to verify the impact of EMILIN-2 loss in CRC development and its possible value as a prognostic biomarker. Methods: The AOM/DSS CRC protocol was applied to Emilin-2 null and wild type mice. Tumor development was monitored by endoscopy, the molecular analyses performed by IHC, IF and WB and the immune subpopulations characterized by flow cytometry. Ex vivo cultures of monocyte/macrophages from the murine models were used to verify the molecular pathways. Publicly available datasets were exploited to determine the CRC patients’ expression profile; Spearman’s correlation analyses and Cox regression were applied to evaluate the association with the inflammatory response; the clinical outcome was predicted by Kaplan-Meier survival curves. Pearson correlation analyses were also applied to a cohort of patients enrolled in our Institute. Results: In preclinical settings, loss of EMILIN-2 associated with an increased number of tumor lesions upon AOM/DSS treatment. In addition, in the early stages of the disease, the Emilin-2 knockout mice displayed a myeloid-derived suppressor cells-rich infiltrate. Instead, in the late stages, lack of EMILIN-2 associated with a decreased number of M1 macrophages, resulting in a higher percentage of the tumor-promoting M2 macrophages. Mechanistically, EMILIN-2 triggered the activation of the Toll-like Receptor 4/MyD88/NF-κB pathway, instrumental for the polarization of macrophages towards the M1 phenotype. Accordingly, dataset and immunofluorescence analyses indicated that low EMILIN-2 expression levels correlated with an increased M2/M1 ratio and with poor CRC patients’ prognosis. Conclusions: These novel results indicate that EMILIN-2 is a key regulator of the tumor-associated inflammatory environment and may represent a promising prognostic biomarker for CRC patients.