Rhabdomyosarcoma (RMS) is an aggressive soft-tissue sarcoma with skeletal muscle characteristics in children and adolescents. Among its subtypes, fusion-positive RMS (FP-RMS) is defined by a chromosomal translocation that encodes a fusion oncoprotein, most commonly PAX3::FOXO1 or PAX7::FOXO1. FP-RMS has a weaker response to conventional radiation and chemotherapy, with a 5-year survival rate of only 20–30% in patients with metastatic disease. Natural killer (NK) cell–based therapy has emerged as a promising strategy in cancer immunotherapy, offering a potentially safer, "off-the-shelf" alternative to T-cell therapies as it does not mediate graft-versus-host disease (GVHD). NK cells can recognize and eliminate malignant cells, including FP-RMS, without prior sensitization. However, the long-term success of NK therapies will largely depend on their durability. A key challenge in the field is that it is unclear whether RMS cells would adapt and eventually resist NK cell attack, which could ultimately undermine therapeutic effectiveness.To address this question, we investigated how RMS cells respond to primary human NK cells using cytotoxicity assays and flow cytometry. We found that prolonged NK cell exposure induces an immunoedited state in RMS cells—a process by which tumor cells adapt under immune pressure, leading to the selection of variants with reduced immunogenicity and increased resistance to immune attack. We hypothesize that this immunoedited state represents a stable epigenetic program associated with enhanced resistance to subsequent NK-mediated killing. Phenotypically, this adaptive response includes upregulation of HLA-ABC and HLA-E, which engage inhibitory NK receptors that dampen NK cell cytotoxicity. Concurrent upregulation of PD-L1 on RMS cells further contributes to immune suppression by inhibiting NK effector functions through the PD-1 axis. Future work will include studies to define the transcriptional and epigenetic programs driving this adaptive response. These findings identify a potential mechanism of RMS immune evasion and highlight potential therapeutic opportunities such as blockade of NKG2A or PD-1 pathways to restore anti-tumor immunity and overcome resistance. Zhiyu Song, Benjamin Stanton, Dean Lee. Immunoediting of fusion-positive rhabdomyosarcoma after NK cell-based immunotherapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B010.

Rhabdomyosarcoma (RMS) is an aggressive pediatric soft tissue cancer, with approximately 350 cases per year in the US. The highly aggressive tumor subtype, Fusion-Positive Rhabdomyosarcoma (FP-RMS) is characterized by chromosomal translocations yielding oncogenic fusion transcription factors (TFs). The most common RMS fusion (PAX3::FOXO1) forms an in-frame chimera of the DNA-binding domain of PAX3 with the activator domain of FOXO1. We hypothesize that understanding the precise mechanisms of PAX3::FOXO1’s chromatin engagement and nucleosome invasion would enable precision therapy to target these mechanisms to ultimately improve patient outcomes. Our studies are providing evidence that PAX3::FOXO1 has pioneer activity, including nucleosome engagement, and an ability to recognize multiple chromatin states including both open and repressive chromatin regions. We are investigating the biochemical regulatory functions of PAX3::FOXO1 binding to specific DNA motifs embedded within chromatin or nucleosomes, developing synthetic epigenetics to investigate motifs from our genomics-scale studies. We are interested in the preservation of naturally occurring post-translational modifications from the mammalian cellular context, and have sought to build a highly versatile system for investigation of TF/nucleosome targeting from cell extracts. In our studies, we are finding new evidence for PAX3::FOXO1’s targeting of homeodomain and paired domain DNA motifs in free DNA and also in a nucleosome context. We are validating these findings through parallel approaches including coupling electrophoretic mobility shift assays (EMSAs) with westerns and antibody-targeting of complexes within EMSA experiments. We are finding that PAX3::FOXO1’s binding to high-complexity motifs, conjoining paired domain and homeodomain sequences, occurs during nucleosome invasion. Through iterative comparisons, custom unmodified mono-nucleosomes containing three major classes of DNA binding motifs reveal PAX3::FOXO1’s binding to these nucleosomes without a strong motif preference within the categories examined in our series. These findings support a mechanistic hypothesis where PAX3::FOXO1 may invade nucleosomes, altering their local positioning prior to generation of chromatin accessibility. Additional investigations are underway to unravel these mechanisms further and probe the contexts for motif specificity or generality in nucleosome engagement, and to understand conformational changes in the target nucleosomes. The mechanisms we are uncovering will contribute to the search for new therapeutic vulnerabilities as our community progresses toward targeted therapies for FP-RMS. Chamithi Karunanayake, Hayden Statmore, Ehsan Akbari, Alexi Tallan, Frederic Barr, Michael G. Poirier, Benjamin Stanton. Biochemical mechanisms of PAX3::FOXO1 chromatin invasion in Rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B016.

Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer caused by a chromosomal translocation. The most common translocations occur between PAX3 or PAX7 and FOXO1, which generates a chimeric transcription factor with gain-of-function activities. These fusion proteins contain the PAX3/7 DNA-binding domains (paired and homeobox) and the FOXO1 transactivation domain. For over thirty years, these fusions have been established as the defining genetic drivers of this disease; however, they remain challenging to target pharmacologically. As such, improvements to the standard of care, which include surgery, radiation, and general chemotherapy, have focused on optimizing chemotherapy dosage and are rarely curative. Therapeutic development has, in part, been hindered by unclear in vivo regulatory mechanisms of PAX3/7::FOXO1 across cellular contexts and tumorigenic stages. Previously, our groups showed that PAX3::FOXO1, the most common fusion, is a pioneer transcription factor that reshapes chromatin architecture and can transform zebrafish cells to recapitulate human tumors. Recently, we aimed to understand how PAX3::FOXO1 initially interfaces with and modulates chromatin in vivo. We developed a zebrafish mRNA injection model and expressed human PAX3::FOXO1 during early vertebrate development. PAX3::FOXO1 utilized its homeobox domain and partial homeobox motif recognition for initial genomic binding, a new binding mode that varies from in vitro models. Moreover, PAX3::FOXO1 bound and activated inaccessible chromatin in vivo, two hallmarks of pioneer transcription factor activity. Chromatin and transcriptional profiling revealed that PAX3::FOXO1 binding resulted in activation of rhabdomyosarcoma and neural developmental gene signatures. Despite the common association of rhabdomyosarcoma with incomplete myogenesis, neural pathways and populations are present in primary patient tumors. The neural transcription factor HES3 is one example that we previously characterized as a PAX3::FOXO1 cooperating gene. The neural population is also enriched during chemotherapy treatment in patient-derived xenografts. We are continuing to utilize our zebrafish modeling and parallel approaches to understand the functional role and regulation of these neural pathways. Currently, we are investigating how HES3 modulates this activation, given that it developmentally promotes neural stem renewal, is an established target of PAX3::FOXO1, and correlates with worse patient outcomes. Neural signatures are seen in both PAX3::FOXO1 and PAX7::FOXO1 tumors, therefore we are leveraging in vivo comparative analyses between these fusions to assess if differences in neural activation can explain their divergent clinical outcomes. Altogether, this work highlights neural pathway activation as a core activity of PAX3::FOXO1 and suggests that these programs could play a critical role across multiple stages of tumorigenesis. Our long-term goal is to delineate how PAX3/7::FOXO1 cooperate with neural factors across cellular contexts to identify novel therapeutic vulnerabilities. Jack Kucinski, Alexi Tallan, Matthew Kent, Andrew Vontell, Katherine Silvius, Cenny Taslim, Benjamin Stanton, Genevieve Kendall. In vivo activation of neural gene signatures in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Fusion-Positive Cancer: From Discovery to Therapy; 2026 Jan 13-15; Philadelphia PA. Philadelphia (PA): AACR; Cancer Res 2026;86(1_Suppl):Abstract nr B021.

Quantitative comparison of ChIP-seq profiling between experimental conditions or samples remains technically challenging for the epigenetics field. Here, we report a strategy combining the use of well-defined cellular spike-in ratios of orthologous species' chromatin and a bioinformatic analysis pipeline to facilitate highly quantitative comparisons of 2D chromatin sequencing across experimental conditions. We find that the PerCell methodology results in efficient and consistent levels of spike-in vs. experimental genomic reads. We demonstrate use of the method and pipeline to enable quantitative, internally normalized chromatin sequencing on zebrafish embryos and human cancer cells. Overall, we propose the PerCell method to enable cross-species comparative epigenomics and promote uniformity of data analyses and sharing across labs.

Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer molecularly characterized by arrested myogenesis. The defining genetic driver, PAX3::FOXO1, encodes a chimeric gain-of-function transcription factor. An incomplete understanding of the in vivo chromatin regulatory mechanisms of PAX3::FOXO1 has hindered therapeutic development. Here, we establish a PAX3::FOXO1 zebrafish injection model and a semi-automated ChIP-seq normalization strategy to evaluate how PAX3::FOXO1 initially interfaces with and modulates chromatin in a developmental context. We find that PAX3::FOXO1 interacts with inaccessible chromatin through partial/homeobox motif recognition consistent with pioneering activity. However, PAX3::FOXO1-genome binding through a composite paired box/homeobox motif alters chromatin accessibility and redistributes H3K27ac to activate neural transcriptional programs. We uncover neural signatures that are highly representative of clinical rhabdomyosarcoma gene expression programs that are enriched following chemotherapy. Overall, we identify partial/homeobox motif recognition as a key mode for PAX3::FOXO1 pioneer function and identify neural signatures as a potentially critical PAX3::FOXO1 tumor initiation event.

Abstract Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer with features of arrested skeletal muscle development. PAX3::FOXO1 is the most common and lethal fusion-oncogene of this disease and arises from a translocation between the DNA binding domains of PAX3 with the transactivation domain of FOXO1. This chimeric transcription factor is required for tumor initiation and has pioneering activity allowing it the unique potential to bind to inaccessible chromatin to make regions amenable to transcription. Despite PAX3::FOXO1’s identification over thirty years ago, and well-characterized importance in this disease, we still lack targeted therapies that significantly improve patient outcomes. Furthermore, it has been technically challenging to investigate the initial in vivo activities of PAX3::FOXO1 and how it could establish a tumorigenic cell fate. Here, we established a PAX3::FOXO1 mRNA zebrafish injection model and quantitative spike-in ChIP-seq analytical pipeline to evaluate how PAX3::FOXO1 initially interfaces with chromatin in a developmental context. In our approach, we inject human PAX3::FOXO1 mRNA into zebrafish embryos and observe broad protein expression during early development, peaking at six hours-post fertilization during gastrulation. We then characterized PAX3::FOXO1 activity with 2D chromatin-sequencing and transcriptional profiling. Transcriptionally, we observe the upregulation and enrichment for rhabdomyosarcoma-associated genes and pathways. Further, we find that PAX3::FOXO1 has nucleosomal binding. Using 2D chromatin sequencing, we observe that PAX3::FOXO1 localizes within inaccessible chromatin through partial homeobox motif recognition, consistent with pioneering activity. In contrast, PAX3::FOXO1 binding through its composite motif modifies chromatin accessibility and re-distributes H3K27ac to activate neural transcriptional programs. This includes neural transcriptional signatures found in PAX3::FOXO1 patient tumors, and that are induced in patient-derived xenografts in response to chemotherapy. Our long-term goal is to do comparative analyses across our mRNA injection and zebrafish rhabdomyosarcoma tumor models and patient data to identify conserved PAX3::FOXO1 mechanisms and targets across stages of tumorigenesis. This injection model is versatile, allowing us to functionally evaluate cooperation between PAX3::FOXO1 and other factors of interest. Altogether, we can provide valuable insight into chromatin regulation, which may be applicable across other cancers and development. Citation Format: Jack Kucinski, Alexi Tallan, Cenny Taslim, Meng Weng, Matthew Cannon, Katherine Silvius, Benjamin Z. Stanton, Genevieve C. Kendall. Biological consequences of neural signatures in fusion-positive rhabdomyosarcoma [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Advances in Pediatric Cancer Research; 2024 Sep 5-8; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Res 2024;84(17 Suppl):Abstract nr B010.

We describe exciting recent advances in fusion-driven sarcoma etiology, from an epigenetics perspective. By exploring the current state of the field, we identify and describe the central mechanisms that determine sarcomagenesis. Further, we discuss seminal studies in translational genomics, which enabled epigenetic characterization of fusion-driven sarcomas. Important context for epigenetic mechanisms include, but are not limited to, cell cycle and metabolism, core regulatory circuitry, 3-dimensional chromatin architectural dysregulation, integration with ATP-dependent chromatin remodeling, and translational animal modeling. Paradoxically, while the genetic requirements for oncogenic transformation are highly specific for the fusion partners, the epigenetic mechanisms we as a community have uncovered are categorically very broad. This dichotomy prompts the question of whether the investigation of rare disease epigenomics should prioritize studying individual cell populations, thereby examining whether the mechanisms of chromatin dysregulation are specific to a particular tumor. We review recent advances focusing on rhabdomyosarcoma, synovial sarcoma, alveolar soft part sarcoma, clear cell sarcoma, undifferentiated round cell sarcoma, Ewing sarcoma, myxoid/round liposarcoma, epithelioid hemangioendothelioma and desmoplastic round cell tumor. The growing number of groundbreaking discoveries in the field, motivated us to anticipate further exciting advances in the area of mechanistic epigenomics and direct targeting of fusion transcription factors in the years ahead.

Fusion-positive rhabdomyosarcoma (FP-RMS) is an aggressive pediatric sarcoma driven primarily by the PAX3-FOXO1 fusion oncogene, for which therapies targeting PAX3-FOXO1 are lacking. Here, we screen 62,643 compounds using an engineered cell line that monitors PAX3-FOXO1 transcriptional activity identifying a hitherto uncharacterized compound, P3FI-63. RNA-seq, ATAC-seq, and docking analyses implicate histone lysine demethylases (KDMs) as its targets. Enzymatic assays confirm the inhibition of multiple KDMs with the highest selectivity for KDM3B. Structural similarity search of P3FI-63 identifies P3FI-90 with improved solubility and potency. Biophysical binding of P3FI-90 to KDM3B is demonstrated using NMR and SPR. P3FI-90 suppresses the growth of FP-RMS in vitro and in vivo through downregulating PAX3-FOXO1 activity, and combined knockdown of KDM3B and KDM1A phenocopies P3FI-90 effects. Thus, we report KDM inhibitors P3FI-63 and P3FI-90 with the highest specificity for KDM3B. Their potent suppression of PAX3-FOXO1 activity indicates a possible therapeutic approach for FP-RMS and other transcriptionally addicted cancers.

Correct chromosome organization in the cell nucleus is essential for genome function. However, dynamics and regulations of large-scale chromosomal conformations beyond single compartments at larger than ten megabases in vivo in single cells remain largely unknown. Here we use CRISPR-Sirius, a high-resolution and high-sensitivity real-time imaging technique, to directly visualize distinctions in large-scale chromosomal conformations between live osteosarcoma (OS) cells and osteoblasts, suggesting extensive chromatin reorganization during cell transformation. A surprising discovery is that chromosome 19 long arm is primarily extended in osteoblasts and maintained by H3K27me3. Extended chromosome conformation has been reported in fly and mouse but not in human cells yet. However, in OS cells, chromosome 19 primarily folded into collapsed conformations, which reshape in minutes and are regulated by the chromosome architectural proteins CTCF and cohesin in the presence of H3K27ac. Changes in chromosome conformations by knocking down the cohesin subunit RAD21 resulted in altered gene expression, including proto-oncogenes. Transcription inhibition by a small molecule inhibitor did not have detectable effects on large-scale chromosome conformation, suggesting that local transcription events have limited effects on large-scale chromosomal architecture. Our results provide unique insights into the complex regulatory mechanisms of endogenous large-scale chromosome organization in normal and transformed osteogenic tissues. ### Competing Interest Statement The authors have declared no competing interest.

Abstract Fusion-positive rhabdomyosarcoma is an aggressive pediatric cancer lacking curative therapies, and outcomes for children with this disease have not improved in decades. The chimeric transcription factor PAX3::FOXO1 is the most common and lethal driver of fusion-positive rhabdomyosarcoma, and it consists of the DNA binding domains of PAX3 fused to the transactivation domain of FOXO1. Despite its well-established essentiality for tumorigenesis, how PAX3::FOXO1 initially generates a tumorigenic cell state has been challenging to study due to the lack of in vivo models expressing the fusion-oncogene at accessible early developmental time points. Here, we developed a novel zebrafish mRNA injection model that ubiquitously expresses PAX3::FOXO1 during gastrulation representing all three germ layers. Using this high-throughput model, we used ChIP-seq to identify initial in vivo PAX3::FOXO1 binding sites and targets. PAX3::FOXO1 has pioneering activity, where it can utilize partial-motif recognition to bind to inaccessible chromatin and alter chromatin structure. With biochemical and 2D chromatin sequencing approaches in our model, we find evidence for in vivo PAX3::FOXO1 pioneering activity. PAX3::FOXO1 consists of two DNA binding domains, a paired and homeobox domain, and we demonstrate a new mode of pioneering activity driven by its homeobox domain given a strong enrichment of homeobox-related motifs at PAX3::FOXO1 binding sites. PAX3::FOXO1 activity in developing zebrafish embryos results in an arrested development phenotype and transcriptional signatures enriched for pathways such as an inhibition of segmentation and myogenic development. Critically, we find that PAX3::FOXO1 directly activates neural-related gene targets by increasing chromatin accessibility and re-distribution of the active histone mark H3K27Ac to these loci. These discoveries are particularly striking because rhabdomyosarcoma is traditionally associated with skeletal muscle characteristics. With our findings, we hypothesize neural gene activation is a critical mechanism for fusion-positive rhabdomyosarcoma tumor initiation and could be a critical pathway across various stages of rhabdomyosarcoma tumorigenesis. This hypothesis agrees with our previous discovery of a novel PAX3::FOXO1 cooperating neural transcription factor, HES3, which we find is directly activated by PAX3::FOXO1 in our mRNA injection model. Our long-term goal is to utilize this versatile model to functionally evaluate cooperation between PAX3::FOXO1 and both canonical rhabdomyosarcoma and neural transcription factors. Our approach will employ a cross-species comparative analysis across this injection model, our zebrafish tumor models, other model systems, and patient data. This work will identify conserved targets and mechanisms of PAX3::FOXO1 tumorigenesis that could become new therapeutic vulnerabilities. Citation Format: Jack Kucinski, Cenny Taslim, Alexi Tallan, Matthew Cannon, Katherine Silvius, Benjamin Stanton, Genevieve Kendall. Rhabdomyosarcoma fusion-oncogene alters the chromatin landscape to initially drive a neural signature in vivo [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2861.

CD19 chimeric antigen receptor T-cell therapy (CD19-CAR) has changed the treatment landscape and outcomes for patients with pre-B-cell acute lymphoblastic leukemia (B-ALL). Unfortunately, primary nonresponse (PNR), sustained CD19+ disease, and concurrent expansion of CD19-CAR occur in 20% of the patients and is associated with adverse outcomes. Although some failures may be attributable to CD19 loss, mechanisms of CD19-independent, leukemia-intrinsic resistance to CD19-CAR remain poorly understood. We hypothesize that PNR leukemias are distinct compared with primary sensitive (PS) leukemias and that these differences are present before treatment. We used a multiomic approach to investigate this in 14 patients (7 with PNR and 7 with PS) enrolled in the PLAT-02 trial at Seattle Children's Hospital. Long-read PacBio sequencing helped identify 1 PNR in which 47% of CD19 transcripts had exon 2 skipping, but other samples lacked CD19 transcript abnormalities. Epigenetic profiling discovered DNA hypermethylation at genes targeted by polycomb repressive complex 2 (PRC2) in embryonic stem cells. Similarly, assays of transposase-accessible chromatin-sequencing revealed reduced accessibility at these PRC2 target genes, with a gain in accessibility of regions characteristic of hematopoietic stem cells and multilineage progenitors in PNR. Single-cell RNA sequencing and cytometry by time of flight analyses identified leukemic subpopulations expressing multilineage markers and decreased antigen presentation in PNR. We thus describe the association of a stem cell epigenome with primary resistance to CD19-CAR therapy. Future trials incorporating these biomarkers, with the addition of multispecific CAR T cells targeting against leukemic stem cell or myeloid antigens, and/or combined epigenetic therapy to disrupt this distinct stem cell epigenome may improve outcomes of patients with B-ALL.

We report a comprehensive drug synergy study in acute myeloid leukemia (AML). In this work, we investigate a panel of cell lines spanning both MLL-rearranged and non-rearranged subtypes. The work comprises a resource for the community, with many synergistic drug combinations that could not have been predicted a priori, and open source code for automation and analyses. We base our definitions of drug synergy on the Chou-Talalay method, which is useful for visualizations of synergy experiments in isobolograms, and median-effects plots, among other representations. Our key findings include drug synergies affecting the chromatin state, specifically in the context of regulation of the modification state of histone H3 lysine-27. We report open source high throughput methodology such that multidimensional drug screening can be accomplished with equipment that is accessible to most laboratories. This study will enable preclinical investigation of new drug combinations in a lethal blood cancer, with data analysis and automation workflows freely available to the community.

Materials and Methods used for experiments in Supplemental Figures and Supplemental References

Supplementary Figures S1-S9

ADVERTISEMENT RETURN TO ISSUEViewpointNEXTFunctional Epigenomics: Pioneering Changes in Chromatin StructureAlexi TallanAlexi TallanAbigail Wexner Research Institute, Nationwide Children's Hospital, Center for Childhood Cancer and Blood Diseases, Columbus, Ohio 43205, United StatesMolecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, Ohio 43210, United StatesMore by Alexi Tallanhttps://orcid.org/0000-0002-6043-3233, Rachel A. HoffmanRachel A. HoffmanAbigail Wexner Research Institute, Nationwide Children's Hospital, Center for Childhood Cancer and Blood Diseases, Columbus, Ohio 43205, United StatesMore by Rachel A. Hoffmanhttps://orcid.org/0000-0002-0032-2304, and Benjamin Z. Stanton*Benjamin Z. StantonAbigail Wexner Research Institute, Nationwide Children's Hospital, Center for Childhood Cancer and Blood Diseases, Columbus, Ohio 43205, United StatesMolecular, Cellular, and Developmental Biology Program, The Ohio State University, Columbus, Ohio 43210, United StatesDepartment of Pediatrics, The Ohio State University College of Medicine, Columbus, Ohio 43210, United StatesDepartment of Biological Chemistry and Pharmacology, The Ohio State University College of Medicine, Columbus, Ohio 43210, United States*Email: [email protected]More by Benjamin Z. Stantonhttps://orcid.org/0000-0002-2613-2955Cite this: Biochemistry 2023, 62, 6, 1111–1113Publication Date (Web):February 27, 2023Publication History Received28 January 2023Published online27 February 2023Published inissue 21 March 2023https://pubs.acs.org/doi/10.1021/acs.biochem.3c00048https://doi.org/10.1021/acs.biochem.3c00048article-commentaryACS PublicationsCopyright © 2023 American Chemical Society. This publication is available under these Terms of Use. Request reuse permissions This publication is free to access through this site. Learn MoreArticle Views1632Altmetric-Citations-LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail PDF (2 MB) Get e-AlertscloseSUBJECTS:Conformation,Genetics,Genomics,Molecular structure,Targeting Get e-Alerts

Rhabdomyosarcoma (RMS) is a pediatric soft tissue cancer with a lack of precision therapy options for patients. We hypothesized that with a general paucity of known mutations in RMS, chromatin structural driving mechanisms are essential for tumor proliferation. Thus, we carried out high-depth in situ Hi-C in representative cell lines and patient-derived xenografts (PDXs) to define chromatin architecture in each major RMS subtype. We report a comprehensive 3D chromatin structural analysis and characterization of fusion-positive (FP-RMS) and fusion-negative RMS (FN-RMS). We have generated spike-in in situ Hi-C chromatin interaction maps for the most common FP-RMS and FN-RMS cell lines and compared our data with PDX models. In our studies, we uncover common and distinct structural elements in large Mb-scale chromatin compartments, tumor-essential genes within variable topologically associating domains and unique patterns of structural variation. Our high-depth chromatin interactivity maps and comprehensive analyses provide context for gene regulatory events and reveal functional chromatin domains in RMS.