Rhabdomyosarcoma (RMS) is a pediatric malignancy of skeletal muscle lineage. The aggressive alveolar subtype is characterized by t(2;13) or t(1;13) translocations encoding for PAX3- or PAX7-FOXO1 ...chimeric transcription factors, respectively, and are referred to as fusion positive RMS (FP-RMS). The fusion gene alters the myogenic program and maintains the proliferative state while blocking terminal differentiation. Here, we investigated the contributions of chromatin regulatory complexes to FP-RMS tumor maintenance. We define the mSWI/SNF functional repertoire in FP-RMS. We find that SMARCA4 (encoding BRG1) is overexpressed in this malignancy compared to skeletal muscle and is essential for cell proliferation. Proteomic studies suggest proximity between PAX3-FOXO1 and BAF complexes, which is further supported by genome-wide binding profiles revealing enhancer colocalization of BAF with core regulatory transcription factors. Further, mSWI/SNF complexes localize to sites of de novo histone acetylation. Phenotypically, interference with mSWI/SNF complex function induces transcriptional activation of the skeletal muscle differentiation program associated with MYCN enhancer invasion at myogenic target genes, which is recapitulated by BRG1 targeting compounds. We conclude that inhibition of BRG1 overcomes the differentiation blockade of FP-RMS cells and may provide a therapeutic strategy for this lethal childhood tumor.
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.
A vast number of cancer genes are transcription factors that drive tumorigenesis as oncogenic fusion proteins. Although the direct targeting of transcription factors remains challenging, therapies ...aimed at oncogenic fusion proteins are attractive as potential treatments for cancer. There is particular interest in targeting the oncogenic PAX3-FOXO1 fusion transcription factor, which induces alveolar rhabdomyosarcoma (aRMS), an aggressive cancer of skeletal muscle cells for which patient outcomes remain dismal. In this work, we have defined the interactome of PAX3-FOXO1 and screened 60 candidate interactors using siRNA-mediated depletion to identify candidates that affect fusion protein activity in aRMS cells. We report that chromodomain helicase DNA binding protein 4 (CHD4), an ATP-dependent chromatin remodeler, acts as crucial coregulator of PAX3-FOXO1 activity. CHD4 interacts with PAX3-FOXO1 via short DNA fragments. Together, they bind to regulatory regions of PAX3-FOXO1 target genes. Gene expression analysis suggested that CHD4 coregulatory activity is essential for a subset of PAX3-FOXO1 target genes. Depletion of CHD4 reduced cell viability of fusion-positive but not of fusion-negative RMS in vitro, which resembled loss of PAX3-FOXO1. It also caused specific regression of fusion-positive xenograft tumors in vivo. Therefore, this work identifies CHD4 as an epigenetic coregulator of PAX3-FOXO1 activity, providing rational evidence for CHD4 as a potential therapeutic target in aRMS.
Collateral lethality occurs when loss of a gene/protein renders cancer cells dependent on its remaining paralog. Combining genome-scale CRISPR/Cas9 loss-of-function screens with RNA sequencing in ...over 900 cancer cell lines, we found that cancers of nervous system lineage, including adult and pediatric gliomas and neuroblastomas, required the nuclear kinase vaccinia-related kinase 1 (VRK1) for their survival in vivo. VRK1 dependency was inversely correlated with expression of its paralog VRK2. VRK2 knockout sensitized cells to VRK1 loss, and conversely, VRK2 overexpression increased cell fitness in the setting of VRK1 loss. DNA methylation of the VRK2 promoter was associated with low VRK2 expression in human neuroblastomas and adult and pediatric gliomas. Mechanistically, depletion of VRK1 reduced barrier-to-autointegration factor phosphorylation during mitosis, resulting in DNA damage and apoptosis. Together, these studies identify VRK1 as a synthetic lethal target in VRK2 promoter-methylated adult and pediatric gliomas and neuroblastomas.
Rhabdomyosarcoma (RMS) is a group of pediatric cancers with features of developing skeletal muscle. The cellular hierarchy and mechanisms leading to developmental arrest remain elusive. Here, we ...combined single-cell RNA sequencing, mass cytometry, and high-content imaging to resolve intratumoral heterogeneity of patient-derived primary RMS cultures. We show that the aggressive alveolar RMS (aRMS) subtype contains plastic muscle stem-like cells and cycling progenitors that drive tumor growth, and a subpopulation of differentiated cells that lost its proliferative potential and correlates with better outcomes. While chemotherapy eliminates cycling progenitors, it enriches aRMS for muscle stem-like cells. We screened for drugs hijacking aRMS toward clinically favorable subpopulations and identified a combination of RAF and MEK inhibitors that potently induces myogenic differentiation and inhibits tumor growth. Overall, our work provides insights into the developmental states underlying aRMS aggressiveness, chemoresistance, and progression and identifies the RAS pathway as a promising therapeutic target.
Abstract
Fusion-positive rhabdomyosarcoma (FP-RMS) is a pediatric tumor driven by an oncogenic fusion protein, PAX3-FOXO1, which acts as a transcription factor. Conventional chemotherapy is effective ...for low risk patients who have a 5-year overall survival greater than 65%, while high risk patients, including those with metastatic disease, have less than 40% survival. Consequently, we hypothesize that targeting the fusion protein or its collaborators in transcription regulation will provide novel therapies for this aggressive subtype of RMS. To identify new druggable PAX3-FOXO1 interactors, we performed a combined proteomic and genetic screen which led to the discovery of the NuRD complex (Nucleosome Remodelling and Deacetylase) as a major PAX3-FOXO1 co-regulator. The NuRD complex is unique among the chromatin remodelling complexes due to its dual enzymatic activity. It can act by histone deacetylation through HDAC1/2 (histone deacetylases) or influence nucleosome positioning through CHD4 (chromodomain-DNA-binding protein 4). Intriguingly, it has been associated with both activating and repressive activities in gene expression and its role in cancer development is not fully understood yet. We found that in FP-RMS, silencing of CHD4 affected the expression of approximately 50% of PAX3-FOXO1 regulated target genes. These were mainly genes which are usually upregulated, suggesting an activating role for NuRD. Consistent with CHD4 activation activity, ChIP-seq experiments demonstrated that CHD4 and HDAC2 co-localize with the fusion protein in cis-regulatory sites of a subset of its target genes. Interestingly, gene expression analysis showed that both CHD4 and HDAC2 are highly expressed in tumor tissue and myoblasts when compared to normal skeletal muscle, inferring a potential role of the NuRD complex in maintaining the undifferentiated phenotype observed in FP-RMS. Importantly, CHD4 silencing had no effect on myoblasts proliferation whereas a profound growth reduction was seen in FP-RMS cell lines, suggesting a unique tumour dependency on this chromatin remodeler. In addition, depletion of CHD4 caused a complete regression of xenograft tumours in mice.In summary, we have identified the NuRD complex as an essential positive co-regulator of PAX3-FOXO1 transcriptional activity. Our data propose a critival role of one of the NuRD core component CHD4 in FP-RMS cell viability, making CHD4 an attractive new target for therapy. To our knowledge, CHD4 is the first chromatin remodeler identified to associate with PAX3-FOXO1 transcriptional activity, thus highlighting the relevance of epigenetic regulation in FP-RMS tumour development. Collectively, our findings suggest CHD4 as a potential novel therapeutic target in this childhood malignancy.Ongoing work is currently underway to identify first-in-class small molecules to inhibit CHD4 protein.
Citation Format: Joana G. Marques, Berkley Gryder, Maria Boehm, Marco Wachtel, Young Song, Hsien-Chao Chou, Rajesh Patidar, Hongling Liao, Javed Khan, Beat W. Schaefer. Chromatin remodeling as a potential new strategy for fusion positive rhabdomyosarcoma therapy. abstract. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4457.
Abstract
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.
Abstract
The alveolar subtype of rhabdomyosarcoma (aRMS) is a highly malignant soft tissue tumor, which commonly occurs in childhood and is associated with the skeletal muscle lineage. aRMS is ...characterized by the expression of the fusion transcription factor PAX3-FOXO1, acting as the main oncogenic driver. Despite the expression of master regulatory factors (MRFs) of skeletal muscle differentiation, aRMS cells are blocked from executing the terminal differentiation program. Since PAX3-FOXO1 plays a role in repressing differentiation, in part by induction of epigenetic changes, we were interested to gain additional knowledge about epigenetic cofactors that mediate this activity of the fusion protein. Here, we identified BRG1, the major catalytic subunit of SWI/SNF complexes, to be essential for tumor cell proliferation in aRMS and the most important regulatory ATPase. We further report that the physiological effects of interference with SWI/SNF complex function involve transcriptional activation of the myogenic differentiation program, accompanied by altered morphology and cell cycle profiles. Proteomic studies suggest that PAX3-FOXO1 is not an integral part of SWI/SNF complexes, but still has close proximity indicating potential functional interaction through chromatin interfaces. Genome-wide ChIP binding profiles in aRMS cells revealed associations of BRG1 with motifs of myogenic factors including MYOG, MYF5, and MYOD, suggesting functional interactions with MRFs. Our data is consistent with a model where BRG1 containing complexes have a repressive function at regulatory elements of downstream differentiation target genes. Ongoing work aims to further characterize BRG1-mediated changes on genome architecture to understand its contribution to differentiation blockade in the background of fusion positive aRMS. We conclude that BRG1 is an essential target to overcome differentiation blockade of aRMS cells. While other SWI/SNF complex members have been proposed previously to be potentially important for RMS oncogenesis, these proteins generally lack druggability. In contrast, efforts to develop BRG1 specific compounds are already underway, including PROTAC compounds. Preclinical studies using these tools will provide novel insights into therapeutic applicability to target SWI/SNF complexes in RMS.
Citation Format: Dominik Laubscher, Benjamin Z. Stanton, Berkley Gryder, Sudipto Das, Thorkell Andresson, Bernd Roschitzki, Witold Wolski, QuyAi Ngo, Joana G. Marques, Marco Wachtel, Christopher Vakoc, Javed Khan, Beat Schäfer. The BRG1 ATPase helps to maintain the de-differentiation phenotype in alveolar rhabdomyosarcoma abstract. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3900.
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
Diffuse midline gliomas are uniformly fatal pediatric central nervous system cancers that are refractory to standard-of-care therapeutic modalities. The primary genetic drivers are a set of recurrent ...amino acid substitutions in genes encoding histone H3 (H3K27M), which are currently undruggable. These H3K27M oncohistones perturb normal chromatin architecture, resulting in an aberrant epigenetic landscape. To interrogate for epigenetic dependencies, we performed a CRISPR screen and show that patient-derived H3K27M-glioma neurospheres are dependent on core components of the mammalian BAF (SWI/SNF) chromatin remodeling complex. The BAF complex maintains glioma stem cells in a cycling, oligodendrocyte precursor cell-like state, in which genetic perturbation of the BAF catalytic subunit SMARCA4 (BRG1), as well as pharmacologic suppression, opposes proliferation, promotes progression of differentiation along the astrocytic lineage, and improves overall survival of patient-derived xenograft models. In summary, we demonstrate that therapeutic inhibition of the BAF complex has translational potential for children with H3K27M gliomas.
Epigenetic dysregulation is at the core of H3K27M-glioma tumorigenesis. Here, we identify the BRG1-BAF complex as a critical regulator of enhancer and transcription factor landscapes, which maintain H3K27M glioma in their progenitor state, precluding glial differentiation, and establish pharmacologic targeting of the BAF complex as a novel treatment strategy for pediatric H3K27M glioma. See related commentary by Beytagh and Weiss, p. 2730. See related article by Mo et al., p. 2906.