Epstein-Barr virus-associated smooth muscle tumors (EBV-SMT) are rare lesions that occur in immunocompromised patients. Because they have not been fully characterized pathologically or at the molecular level, we have studied 29 tumors from 19 patients, the largest series to date. Cases coded as EBV-SMT were identified in 19 patients from consultation files and from the renal transplant database at Singapore General Hospital. EBV-SMT occurred in adults (mean age 39 years; range, 21-57 years) and predominantly affected males (12 male, 7 female). Causes of immunocompromise were renal transplantation (10), AIDS (8), and steroid therapy (1). Tumors were located in soft tissue (5), lung (5), liver (4), and miscellaneous sites (15). In 13 patients (68%), the tumors were multiple. Infection with EBV was confirmed in all cases by in situ hybridization for EBV early RNAs (EBER). EBV-SMT were typically well-differentiated smooth muscle tumors with little atypia and usually a low level of mitotic activity. Unlike classic leiomyosarcomas, they lacked significant pleomorphism but frequently displayed primitive round cell areas and prominent intratumoral T lymphocytes. No consistent relationship between histologic features and clinical outcome was noted. All expressed actin (29 of 29) and less frequently desmin (14 of 26). Multiple tumors in a given patient were clonally distinct as assessed by the long terminal repeat region of the virus, supporting the view that multifocal tumors arise from multiple infection events rather than from metastasis. Strain typing by analysis of the EBNA-3C gene confirmed the presence of EBV type 2. Two of four tumors assessed were positive for a 30-bp deletion in the LMP1 gene. EBV copy number per cell ranged greatly between patients and between tumors from the same patient. Follow-up information was available in 18 of 19 patients (mean, 25 months; range, 1-105 months). Fifteen patients were alive: 11 with disease and 4 without. Three patients died, 1 due to disease. We conclude that EBV-SMT are histologically distinct from classic soft tissue smooth muscle tumors, are not readily evaluated by means of conventional histologic criteria, and in the case of multifocal tumors are the result of multiple infection events rather than metastasis. EBV-2 can transform smooth muscle cells, independent of the presence of the LMP1 deletion associated with greater virulence.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive pediatric cancer with poor prognosis. As transient and stable modifications to chromatin have emerged as critical mechanisms in oncogenic signaling, efforts to target epigenetic modifiers as a therapeutic strategy have accelerated in recent years. To identify chromatin modifiers that sustain tumor growth, we performed an epigenetic screen and found that inhibition of lysine methyltransferase G9a significantly affected the viability of ARMS cell lines. Targeting expression or activity of G9a reduced cellular proliferation and motility in vitro and tumor growth in vivo. Transcriptome and chromatin immunoprecipitation-sequencing analysis provided mechanistic evidence that the tumor-suppressor PTEN was a direct target gene of G9a. G9a repressed PTEN expression in a methyltransferase activitydependent manner, resulting in increased AKT and RAC1 activity. Re-expression of constitutively active RAC1 in G9adeficient tumor cells restored oncogenic phenotypes, demonstrating its critical functions downstream of G9a. Collectively, our study provides evidence for a G9a-dependent epigenetic program that regulates tumor growth and suggests targeting G9a as a therapeutic strategy in ARMS. Significance: These findings demonstrate that RAC1 is an effector of G9a oncogenic functions and highlight the potential of G9a inhibitors in the treatment of ARMS.

Ewing sarcoma (EWS) is a devastating soft tissue sarcoma affecting predominantly young individuals. Tyrosine kinases (TK) and associated pathways are continuously activated in many malignancies, including EWS; these enzymes provide candidate therapeutic targets. Two high-throughput screens (a siRNA library and a small-molecule inhibitor library) were performed in EWS cells to establish candidate targets. Spleen tyrosine kinase (SYK) phosphorylation was assessed in EWS patients and cell lines. SYK was inhibited by a variety of genetic and pharmacological approaches, and SYK-regulated pathways were investigated by cDNA microarrays. The transcriptional regulation of MALAT1 was examined by ChIP-qPCR, luciferase reporter, and qRT-PCR assays. SYK was identified as a candidate actionable target through both high-throughput screens. SYK was highly phosphorylated in the majority of EWS cells, and SYK inhibition by a variety of genetic and pharmacologic approaches markedly inhibited EWS cells both and Ectopic expression of SYK rescued the cytotoxicity triggered by SYK-depletion associated with the reactivation of both AKT and c-MYC. A long noncoding RNA, MALAT1, was identified to be dependent on SYK-mediated signaling. Moreover, c-MYC, a SYK-promoted gene, bound to the promoter of MALAT1 and transcriptionally activated MALAT1, which further promoted the proliferation of EWS cells. This study identifies a novel signaling involving SYK/c-MYC/MALAT1 as a promising therapeutic target for the treatment of EWS. .

Ewing sarcoma (EWS) is an aggressive bone malignancy that mainly affects children and young adults. The mechanisms by which EWS (EWSR1) fusion genes drive the disease are not fully understood. CRM1 (XPO1) traffics proteins from the nucleus, including tumor suppressors and growth factors, and is overexpressed in many cancers. A small-molecule inhibitor of CRM1, KPT-330, has shown therapeutic promise, but has yet to be investigated in the context of EWS. In this study, we demonstrate that CRM1 is also highly expressed in EWS. shRNA-mediated or pharmacologic inhibition of CRM1 in EWS cells dramatically decreased cell growth while inducing apoptosis, cell-cycle arrest, and protein expression alterations to several cancer-related factors. Interestingly, silencing of CRM1 markedly reduced EWS-FLI1 fusion protein expression at the posttranscriptional level and upregulated the expression of the well-established EWS-FLI1 target gene, insulin-like growth factor binding protein 3 (IGFBP3), which inhibits IGF-1. Accordingly, KPT-330 treatment attenuated IGF-1-induced activation of the IGF-1R/AKT pathway. Furthermore, knockdown of IGFBP3 increased cell growth and rescued the inhibitory effects on IGF-1 signaling triggered by CRM1 inhibition. Finally, treatment of EWS cells with a combination of KPT-330 and the IGF-1R inhibitor, linsitinib, synergistically decreased cell proliferation both in vitro and in vivo. Taken together, these findings provide a strong rationale for investigating the efficacy of combinatorial inhibition of CRM1 and IGF-1R for the treatment of EWS. Cancer Res; 76(9);

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Wnt signaling is down-regulated in embryonal rhabdomyosarcoma (ERMS) and contributes to the block of differentiation. Epigenetic mechanisms leading to its suppression are unknown and could pave the way towards novel therapeutic modalities. We demonstrate that EHMT2 suppresses canonical Wnt signaling by activating expression of the Wnt antagonist DKK1. Inhibition of EHMT2 expression or activity in human ERMS cell lines reduced DKK1 expression and elevated canonical Wnt signaling resulting in myogenic differentiation in vitro and in mouse xenograft models in vivo. Mechanistically, EHMT2 impacted Sp1 and p300 enrichment at the DKK1 promoter. The reduced tumor growth upon EHMT2 deficiency was reversed by recombinant DKK1 or LGK974, which also inhibits Wnt signaling. Consistently, among thirteen drugs targeting chromatin modifiers, EHMT2 inhibitors were highly effective in reducing ERMS cell viability. Our study demonstrates that ERMS cells are vulnerable to EHMT2 inhibitors and suggest that targeting the EHMT2-DKK1-b-catenin node holds promise for differentiation therapy.

Alveolar rhabdomyosarcoma (ARMS) is an aggressive paediatric cancer of skeletal muscle with poor prognosis. A PAX3-FOXO1 fusion protein acts as a driver of malignancy in ARMS by disrupting tightly coupled but mutually exclusive pathways of proliferation and differentiation. While PAX3-FOXO1 is an attractive therapeutic target, no current treatments are designed to block its oncogenic activity. The present work shows that the histone acetyltransferase P/CAF (KAT2B) is overexpressed in primary tumours from ARMS patients. Interestingly, in fusion-positive ARMS cell lines, P/CAF acetylates and stabilizes PAX3-FOXO1 rather than MyoD, a master regulator of muscle differentiation. Silencing P/CAF, or pharmacological inhibition of its acetyltransferase activity, down-regulates PAX3-FOXO1 levels concomitant with reduced proliferation and tumour burden in xenograft mouse models. Our studies identify a P/CAF-PAX3-FOXO1 signalling node that promotes oncogenesis and may contribute to MyoD dysfunction in ARMS. This work exemplifies the therapeutic potential of targeting chromatin-modifying enzymes to inhibit fusion oncoproteins that are a frequent event in sarcomas. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley& Sons, Ltd.