Highlights d C19MC alterations and chr2 gains comprise the most frequent genetic events in ETMRs d C19MC-TTYH1 gene fusion and MYCN DNA interactions create super-enhancers d Super-enhancers and multiple feedback loops fortify a C19MC-LIN28A-MYCN circuitry d BET domain inhibitors abrogate C19MC-LIN28A-MYCN circuit to induce ETMR cell death
Purpose: Malignant rhabdoid tumor (MRT) and atypical teratoid rhabdoid tumors (ATRT) are rare aggressive undifferentiated tumors primarily affecting the kidney and CNS of infants and young children. MRT are almost exclusively characterized by homozygous deletion or inactivation of the chromatin remodeling gene SMARCB1. SMARCB1 protein loss leads to direct impairment of chromatin remodeling and we have previously reported a role for this protein in histone acetylation. This provided the rationale for investigating the therapeutic potential of histone deactylase inhibitors (HDACi) in MRT.Experimental Design: Whereas previously HDACis have been used at doses and schedules that induce cytotoxicity, in the current studies we have tested the hypothesis, both in vitro and in vivo, that sustained treatment of human MRT with lowdose HDACi can lead to sustained cell growth arrest and differentiation.
Hypermethylated-in-Cancer 1 (Hic1) is a tumor suppressor gene frequently inactivated by epigenetic silencing and loss-of-heterozygosity in a broad range of cancers. Loss of HIC1, a sequence-specific zinc finger transcriptional repressor, results in deregulation of genes that promote a malignant phenotype in a lineage-specific manner. In particular, upregulation of the HIC1 target gene SIRT1, a histone deacetylase, can promote tumor growth by inactivating TP53. An alternate line of evidence suggests that HIC1 can promote the repair of DNA double strand breaks through an interaction with MTA1, a component of the nucleosome remodeling and deacetylase (NuRD) complex. Using a conditional knockout mouse model of tumor initiation, we now show that inactivation of Hic1 results in cell cycle arrest, premature senescence, chromosomal instability and spontaneous transformation in vitro. This phenocopies the effects of deleting Brca1, a component of the homologous recombination DNA repair pathway, in mouse embryonic fibroblasts. These effects did not appear to be mediated by deregulation of Hic1 target gene expression or loss of Tp53 function, and rather support a role for Hic1 in maintaining genome integrity during sustained replicative stress. Loss of Hic1 function also cooperated with activation of oncogenic KRas in the adult airway epithelium of mice, resulting in the formation of highly pleomorphic adenocarcinomas with a micropapillary phenotype in vivo. These results suggest that loss of Hic1 expression in the early stages of tumor formation may contribute to malignant transformation through the acquisition of chromosomal instability.
BackgroundDesmoplastic small round cell tumor (DSRCT) is characterized by the presence of a fusion protein EWS/WT1, arising from the t (11;22) (p13;q12) translocation. Here we examine the oncogenic properties of two splice variants of EWS/WT1, EWS/WT1-KTS and EWS/WT1 + KTS.MethodsWe over-expressed both EWS/WT1 variants in murine embryonic fibroblasts (MEFs) of wild-type, p53+/- and p53-/- backgrounds and measured effects on cell-proliferation, anchorage-independent growth, clonogenicity after serum withdrawal, and sensitivity to cytotoxic drugs and gamma irradiation in comparison to control cells. We examined gene expression profiles in cells expressing EWS/WT1. Finally we validated our key findings in a small series of DSRCT.ResultsNeither isoform of EWS/WT1 was sufficient to transform wild-type MEFs however the oncogenic potential of both was unmasked by p53 loss. Expression of EWS/WT1 in MEFs lacking at least one allele of p53 enhanced cell-proliferation, clonogenic survival and anchorage-independent growth. EWS/WT1 expression in wild-type MEFs conferred resistance to cell-cycle arrest after irradiation and daunorubicin induced apoptosis. We show DSRCT commonly have nuclear localization of p53, and copy-number amplification of MDM2/MDMX. Expression of either isoform of EWS/WT1 induced characteristic mRNA expression profiles. Gene-set enrichment analysis demonstrated enrichment of WNT pathway signatures in MEFs expressing EWS/WT1 + KTS. Wnt-activation was validated in cell lines with over-expression of EWS/WT1 and in DSRCT.ConclusionIn conclusion, we show both isoforms of EWS/WT1 have oncogenic potential in MEFs with loss of p53. In addition we provide the first link between EWS/WT1 and Wnt-pathway signaling. These data provide novel insights into the function of the EWS/WT1 fusion protein which characterize DSRCT.
Atypical teratoid rhabdoid tumour (ATRT) is a rare but highly aggressive undifferentiated solid tumour arising in the central nervous system and predominantly affecting infants and young children. ATRT is exclusively characterized by the inactivation of SMARCB1, a member of the SWI/SNF chromatin remodelling complex that is essential for the regulation of large sets of genes required for normal development and differentiation. Histone deacetylase inhibitors (HDACi) are a promising anticancer therapy and are able to mimic the normal acetylation functions of SMARCB1 in SMARCB1-deficient cells and drive multilineage differentiation in extracranial rhabdoid tumours. However, the potential efficacy of HDACi in ATRT is unknown. Here, we show that human ATRT cells are highly responsive to the HDACi panobinostat and that sustained treatment leads to growth arrest, increased cell senescence, decreased clonogenicity and induction of a neurogenesis gene-expression profile. Furthermore, in an orthotopic ATRT xenograft model, continuous panobinostat treatment inhibits tumour growth, increases survival and drives neuronal differentiation as shown by the expression of the neuronal marker, TUJ1. Collectively, this preclinical study supports the therapeutic potential of panobinostat-mediated differentiation therapy for ATRT.
Malignant Rhabdoid Tumor (MRT) is a rare pediatric cancer of the kidney and CNS that is resistant to current treatment protocols. MRT is genetically characterized by homozygous inactivation of SMARCB1, a critical subunit of the SWI/SNF chromatin-remodeling complex. Next-generation sequencing data suggests that inactivation of SMARCB1 is the primary driver mutation, implicating epigenetic deregulation in the pathogenesis of MRT. Recently, we showed that sustained treatment of MRT cell lines with low-dose Panobinostat (LBH589), inhibited tumor growth by driving multi-lineage differentiation in vitro and in vivo. Furthermore, re-expression of physiological levels of SMARCB1 in G401 MRT cells phenocopied the low-dose LBH589 treatment and led to growth inhibition, senescence and terminal differentiation in vitro and in vivo. Enhancer of Zeste homolog 2 (EZH2), a core subunit of the Polycomb Repressive Complex 2 (PRC2), confers transcriptional silencing via the addition of methyl groups to Lysine 27 of Histone 3 (H3K27me3), and is a transcriptional target of SMARCB1. EZH2 expression and H3K27me3 were drastically reduced following sustained low-dose LBH589 treatment and re-expression of SMARCB1 in G401 MRT cells. Sustained siRNA knockdown of EZH2 in G401 cells resulted in reduced cell growth and changes in mRNA expression similar to those observed following low-dose LBH589 treatment and SMARCB1 re-expression. Treatment of MRT cells with the EZH2-catalytic domain inhibitor, GSK-126, had no effect on EZH2 expression and only partially reduced H3K27me3 and cell growth at doses 1nM-10μM suggesting important non-catalytic EZH2 function. However, MRT cells treated in combination with low-dose LBH589 and GSK-126, lost EZH2 and H3K27me3 expression and exhibited significantly reduced cell growth in vitro compared to single agent controls, revealing a synergistic relationship. Similar effects were observed in an in vivo xenograft model, with low-dose LBH589 and GSK-126 treatment leading to a marked reduction in tumor growth, not observed with single agent treatment. This data suggests EZH2 is an important mediator of MRT proliferation and differentiation and provides evidence for dual therapeutic targeting of EZH2 with low-dose HDACi in MRT. Citation Format: Dean Popovski, Elizabeth M. Algar, Catherine R. Cochrane, Anette Szczepny, W. Samantha Jayasekara, David M. Ashley, Peter Downie, D. Neil Watkins, Jason E. Cain. Targeted catalytic inhibition of EZH2 synergizes with low-dose HDACi in malignant rhabdoid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3360. doi:10.1158/1538-7445.AM2017-3360
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