Abstract 1393: Chromatin remodelers as potential new targets for therapy of pediatric sarcoma
Authors: Marques Joana G. , Gryder Berkley , Wachtel Marco , Khan Javed , Schaefer Beat 
Published: July 2017

Abstract
Fusion-positive rhabdomyosarcoma (FP-RMS) is a pediatric malignancy driven by the fusion transcription factor PAX3-FOXO1, which generates an aberrant gene expression signature leading to cell transformation. Since FP-RMS cells are highly addicted to the fusion protein, it is in focus as target for alternative therapies. Nevertheless, PAX3-FOXO1, as a transcription factor, does not contain structural cavities and has a low druggability. We therefore hypothesize that we can affect this aggressive subtype of RMS by targeting the co-regulators that collaborate with the fusion protein in regulating transcription. Recently, we have identified the NuRD (Nucleosome Remodeling and Deacetylase) complex as a potential partner of PAX3-FOXO1 in gene expression modulation. The NuRD complex is unique among chromatin remodeling complexes due to its dual enzymatic activity (histone deacetylation through HDAC1/2 and nucleosome positioning by CHD4 - chromodomain-DNA-binding protein 4), offering new possible therapeutic targets. Silencing of two core members of NuRD, CHD4 and RBBP4, led to a drastic decrease in FP-RMS cell viability. Additionally, CHD4 depletion caused a complete regression of mouse tumor xenografts, but it did not affect proliferation of myoblasts, fibroblasts or fusion negative RMS cells, despite the fact that these cells also carry high CHD4 expression levels. We further investigated the nucleosome remodeler CHD4 and learnt that it affects the expression of approximately 50% of PAX3-FOXO1 target genes with most of these genes being upregulated, suggesting an activating role for CHD4 in these cases. Consistent with a positive effect of CHD4 on gene expression, ChIP-seq experiments with FP-RMS cell lines demonstrated that NuRD occupies promoter and enhancer regions of highly expressed genes and co-localizes with the fusion protein at regulatory regions of a subset of its target genes. Next, we studied the influence of this nucleosome remodeler on the chromatin status by DNase hypersensitivity assays and determined that the presence of a DNase signal at PAX3-FOXO1 binding sites is concordant with the presence of CHD4. Hence, we suggest a scenario where CHD4 plays an essential role on FP-RMS tumorigenesis by allowing chromatin to acquire an open architecture that enables PAX3-FOXO1 mediated gene expression. In summary, our data propose that CHD4 has a crucial role as a co-regulator of PAX3-FOXO1 driven gene expression. To our knowledge, CHD4 is the first identified chromatin remodeler associated with PAX3-FOXO1 transcriptional activity, thus highlighting the relevance of epigenetic regulation in FP-RMS tumor development and opening chromatin remodelling as a possible new field of action against this tumor, which is driving ongoing work aimed at finding first-in-class small molecules to inhibit CHD4 function.
Citation Format: Joana G. Marques, Berkley Gryder, Marco Wachtel, Javed Khan, Beat Schaefer. Chromatin remodelers as potential new targets for therapy of pediatric sarcoma [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 1393. doi:10.1158/1538-7445.AM2017-1393