The NuRD complex subunit CHD4 is essential for fusion-positive rhabdomyosarcoma (FP-RMS) survival, but the mechanisms underlying this dependency are not understood. Here, a NuRD-specific CRISPR screen demonstrates that FP-RMS is particularly sensitive to CHD4 amongst the NuRD members. Mechanistically, NuRD complex containing CHD4 localizes to super-enhancers where CHD4 generates a chromatin architecture permissive for the binding of the tumor driver and fusion protein PAX3-FOXO1, allowing downstream transcription of its oncogenic program. Moreover, CHD4 depletion removes HDAC2 from the chromatin, leading to an increase and spread of histone acetylation, and prevents the positioning of RNA Polymerase 2 at promoters impeding transcription initiation. Strikingly, analysis of genome-wide cancer dependency databases identifies CHD4 as a general cancer vulnerability. Our findings describe CHD4, a classically defined repressor, as positive regulator of transcription and super-enhancer accessibility as well as establish this remodeler as an unexpected broad tumor susceptibility and promising drug target for cancer therapy.

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Ewing sarcoma tumorigenesis is tightly linked to epigenetic deregulation. Indeed, the aberrant fusion transcription factor and tumor driver EWS-FLI1 produces extensive rewiring of the cancer cell epigenome. At enhancers containing canonical ETS motifs, the fusion protein represses gene expression but, when binding to GGAA repeats, EWS-FLI1 induces transcription by recruiting activating epigenetic regulators such as the acetyltransferase p300 and the BAF chromatin remodeling complex. The nucleosome remodeling and deacetylase (NuRD) complex is an ATP-dependent multi-subunit complex that modulates chromatin architecture and regulates DNA damage repair, genome stability and gene expression. In Ewing sarcoma, NuRD subunits, such as LSD1, have been suggested to interfere with EWS-FLI1 activity and prevent tumor progression. Hence, we here aimed to comprehensively study the role of NuRD in Ewing sarcoma pathogenesis. First, we performed a NuRD-centered, negative selection, CRISPR/Cas9 screen and identified the chromatin remodeling helicase CHD4 as essential for Ewing sarcoma tumor cell survival. Validation assays using two doxycycline-inducible shRNAs targeting CHD4 demonstrated that silencing of this remodeler in fact drastically reduces tumor cell proliferation and completely prevents colony formation. This cell proliferation impairment was caused by an induction of cell death by apoptosis and not by cell cycle arrest. Surprisingly, this cell death phenotype was not linked to impaired fusion protein activity. CHD4 and NuRD, despite primarily locating to enhancers and super-enhancers in Ewing sarcoma cells, did not preferentially localize to GGAA repeats, unlike EWS-FLI1. Moreover, RNA sequencing assays performed upon CHD4 silencing showed that this ATPase does not regulate the EWS-FLI1 signature. As CHD4 is a chromatin remodeler able to move nucleosomes along the DNA, we performed ATAC sequencing experiments to investigate changes in chromatin status that could explain the dependency of Ewing sarcoma cells on CHD4 for survival. We observed that CHD4 depletion from Ewing sarcoma cells causes a drastic and global chromatin relaxation which renders tumor cells prone to DNA damage and increasingly sensitive to DNA damaging agents. Interestingly, augmented sensitivity to DNA damage was not observed by silencing CHD4 in non-tumorigenic human fibroblasts. Finally, CHD4 depletion also reduced Ewing sarcoma tumor growth in vivo and prolonged mice survival. In conclusion, we demonstrate for the first time that CHD4 is crucial for Ewing sarcoma cell survival and highlight this helicase as a promising therapeutic target for Ewing sarcoma with potential for combination therapy with drugs inducing DNA damage. Importantly, this work has initiated ongoing efforts to develop first-in-class small molecules specifically targeting CHD4 which will be crucial for the future validation of this ATPase as a viable target with clinical application. Citation Format: Joana Graca Marques, Blaz Pavlovic, Quy Ai Ngo, Gloria Pedot, Michaela Roemmele, Larissa Volken, Marco Wachtel, Beat W. Schäfer. The NuRD subunit CHD4 is essential for ewing sarcoma cell survival as it regulates global chromatin architecture [abstract]. In: Proceedings of the AACR Special Conference: Sarcomas; 2022 May 9-12; Montreal, QC, Canada. Philadelphia (PA): AACR; Clin Cancer Res 2022;28(18_Suppl):Abstract nr B010.

Cancer-specific chromosomal aberrations producing chimeric fusion genes are recurrently found in pediatric sarcomas. Fusion positive rhabdomyosarcoma (FP-RMS) and Ewing sarcoma (ES) are two rare but lethal pediatric malignancies driven by such chromosomal translocations. PAX3-FOXO1 and EWS-FLI1 are the most common products of the fusion genes found in FP-RMS and ES, respectively, and they are commonly perceived as the founding genetic abnormality driving the development of these malignancies by changing gene expression. Since direct targeting of transcription factors is still very challenging, acting on the activity of these oncogenic transcription factors at the chromatin level presents a robust alternative for targeted therapy. The Nucleosome Remodeling and Deacetylase (NuRD) complex subunit CHD4 has been previously identified as an interactor of both PAX3-FOXO1 and EWS-FLI1. Hence, we decided here to further characterize the role of this chromatin remodeler in the regulation of fusion protein-mediated gene expression in both FP-RMS and ES. Our NuRD-centered CRISPR/Cas9 screen demonstrated that both these malignancies are especially dependent on CHD4 amongst all other complex members. In fact, CHD4 silencing in both tumors through shRNA knockdown or CRISPR knockout drastically reduces tumor cell proliferation and induces cell death. In vivo, CHD4 knockdown also impaired tumour growth in both FP-RMS and ES. Mechanistically, our RNA-seq assays demonstrated that silencing of the nucleosome remodeller CHD4 alters gene expression in both tumours and our ChIP-seq experiments show that CHD4 binding sites are highly enriched for the binding motif of PAX3-FOXO1 in FP-RMS and EWS-FLI1 in ES. In FP-RMS, we observed that CHD4 particularly regulates super-enhancer accessibility creating a chromatin architecture permissive to the binding of PAX3-FOXO1 and allowing the expression of the fusion gene signature. Similar studies in ES to further investigate CHD4 as a regulator of gene expression are currently ongoing. Finally, our analysis of genome-wide cancer dependency databases identified CHD4 as general novel cancer vulnerability amongst NuRD subunits and other SNF2-like ATPases. In summary, we have unravelled the prominent role of CHD4 in regulation of super-enhancer driven gene expression in FP-RMS and exposed this chromatin remodeler as novel potential drug target for pediatric sarcoma therapy. Our work has motivated us to establish several collaborations with computational and biophysics experts and we are now currently working to identify the first CHD4 specific small molecule inhibitor. Citation Format: Joana G. Marques, Berkley Gryder, Blaz Pavlovic, Yeonjoo Chung, Quy Ngo, Marco Wachtel, Javed Khan, Beat Schäfer. Disrupting chromatin architecture: The NuRD subunit and ATPase CHD4 as a new therapeutic target in pediatric sarcoma [abstract]. In: Abstracts: AACR Special Virtual Conference on Epigenetics and Metabolism; October 15-16, 2020; 2020 Oct 15-16. Philadelphia (PA): AACR; Cancer Res 2020;80(23 Suppl):Abstract nr PO-009.