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FANCM is Required for the PAX3::FOXO1-Driven Oncogenic Program in Rhabdomyosarcoma [CRISPR Screen]

ABSTRACT: Alveolar rhabdomyosarcoma (ARMS) is a highly aggressive pediatric soft tissue sarcoma driven by the PAX3::FOXO1 fusion protein. Despite multipronged treatment approaches, survival rates for patients with fusion-positive (FP) ARMS remain poor, underscoring the need for novel therapeutic strategies. Here, we employ sequential multimodal CRISPR/Cas9 genetic screens to identify essential genes in FP ARMS, revealing FANCM, a key component of the Fanconi Anemia pathway, as a critical dependency. FANCM loss selectively impairs FP ARMS cell growth in vitro and in vivo, reduces PAX3::FOXO1 levels, and induces myogenic differentiation. Mechanistically, FANCM depletion exacerbates replication stress, particularly at PAX3::FOXO1 target gene loci, which leads to ATR/ATM-mediated DNA damage signaling and selective downregulation of the fusion-driven oncogenic program. CRISPR exon-tiling screens highlighted FANCM's helicase and DNA-binding domains as essential for its FP-specific dependency, linking FANCM-mediated replication fork binding and downstream ATR signaling to FP ARMS survival. This study positions FANCM as a promising therapeutic target in FP ARMS and introduces a novel paradigm for exploiting replication stress vulnerabilities in oncofusion-driven malignancies.

ORGANISM(S): Homo sapiens unidentified

PROVIDER: GSE292882 | GEO | 2026/08/04

REPOSITORIES: GEO

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FANCM is Required for the PAX3::FOXO1-Driven Oncogenic Program in Rhabdomyosarcoma [ChIP-Seq]

Project description:Alveolar rhabdomyosarcoma (ARMS) is a highly aggressive pediatric soft tissue sarcoma driven by the PAX3::FOXO1 fusion protein. Despite multipronged treatment approaches, survival rates for patients with fusion-positive (FP) ARMS remain poor, underscoring the need for novel therapeutic strategies. Here, we employ sequential multimodal CRISPR/Cas9 genetic screens to identify essential genes in FP ARMS, revealing FANCM, a key component of the Fanconi Anemia pathway, as a critical dependency. FANCM loss selectively impairs FP ARMS cell growth in vitro and in vivo, reduces PAX3::FOXO1 levels, and induces myogenic differentiation. Mechanistically, FANCM depletion exacerbates replication stress, particularly at PAX3::FOXO1 target gene loci, which leads to ATR/ATM-mediated DNA damage signaling and selective downregulation of the fusion-driven oncogenic program. CRISPR exon-tiling screens highlighted FANCM's helicase and DNA-binding domains as essential for its FP-specific dependency, linking FANCM-mediated replication fork binding and downstream ATR signaling to FP ARMS survival. This study positions FANCM as a promising therapeutic target in FP ARMS and introduces a novel paradigm for exploiting replication stress vulnerabilities in oncofusion-driven malignancies.

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FANCM is Required for the PAX3::FOXO1-Driven Oncogenic Program in Rhabdomyosarcoma [RNA-Seq]

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Project description:Fusion-positive rhabdomyosarcoma (FP-RMS) is characterized by the presence of tumor-specific chromosomal translocation products, most commonly PAX3::FOXO1, and typically results in lower survival rates compared to fusion-negative RMS cases. PAX3::FOXO1 plays a critical role in FP-RMS oncogenesis in both tumor initiation and maintenance, making it an excellent target for therapeutic intervention in FP-RMS. We created Proteolysis Targeting Chimeras (PROTACs) by combining PAX3::FOXO1 binding small molecules with E3 ligase recruiters for cereblon (CRBN) or S-Phase Kinase Associated Protein 1 (SKP1). PROTACs achieved degradation of endogenous PAX3::FOXO1 protein in FP-RMS cell lines in a concentration-, time-, and proteasome-dependent manner. Moreover, bulk RNA-seq revealed that PAX3::FOXO1-targeting PROTAC treatment impaired endogenous PAX3::FOXO1 target gene expression and induced myogenic differentiation. Importantly, treatment of FP-RMS cells with PAX3::FOXO1-PROTACs synergized with vincristine treatment and demonstrated an impairment in anchorage-independent growth in soft agar. Taken together, we demonstrate the proof of principle of PROTACs targeting the oncogenic fusion protein PAX3::FOXO1 in FP-RMS cells. PROTACs created in this study will not only be useful tools in studying PAX3::FOXO1 biology in laboratory models but could also serve as scaffolds for designing clinical-grade molecules to assess the therapeutic potential of PAX3::FOXO1-targeting PROTACs in FP-RMS patients.

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Project description:Rhabdomyosarcoma (RMS), a malignancy of impaired myogenic differentiation, is the most common soft tissue pediatric cancer. PAX3-FOXO1 oncofusions drive the majority of the clinically more aggressive fusion-positive rhabdomyosarcoma (FP-RMS). Recent studies have established an epigenetic basis for PAX3-FOXO1-driven oncogenic processes. However, details of PAX3-FOXO1 epigenetic mechanisms, including interactions with, and dependence on, other chromatin and transcription factors, are incompletely understood. We previously identified a novel disease-promoting epigenetic axis in RMS, involving the histone demethylase KDM3A and the ETS1 transcription factor, and demonstrated that this epigenetic axis interfaces with PAX3-FOXO1 both phenotypically and transcriptomically, including co-regulation of biological processes and genes important to FP-RMS progression. In this study, we demonstrate that KDM3A and ETS1 colocalize with PAX3-FOXO1 to enhancers of important disease-promoting genes in FP-RMS, including FGF8, IL4R, and MEST, as well as PODXL, which we define herein as a new FP-RMS-promoting gene. We show that ETS1, which is induced by both PAX3-FOXO1 and KDM3A, exists in complex with PAX3-FOXO1, and augments PAX3-FOXO1 chromatin occupancy. We further show that the PAX3-FOXO1/ETS1 complex can be disrupted by the clinically relevant small molecule inhibitor YK-4-279. YK-4-279 displaces PAX3-FOXO1 from chromatin and interferes with PAX3-FOXO1-dependent gene regulation, resulting in potent inhibition of growth and invasive properties in FP-RMS, along with downregulation of FGF8, IL4R, MEST and PODXL expression. We additionally show that, in some FP-RMS, KDM3A also increases PAX3-FOXO1 levels. Together, our studies illuminate mechanisms of action of the KDM3A/ETS1 regulatory module, and reveal novel targetable mechanisms of PAX3-FOXO1 chromatin complex regulation, in FP-RMS.

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BioID HEK293T PAX3-FOXO1 proximity labelling

Project description:PAX3-FOXO1 is a fusion transcription factor that is the main driver of tumorigenesis leading to the development of alveolar rhabdomyosarcoma (aRMS). Since aRMS cells are addicted to PAX3-FOXO1 activity, the fusion protein also represents a major target for therapeutic interference, which is however challenging as transcription factors usually cannot be inhibited directly by small molecules. Hence, characterization of the biology of PAX3-FOXO1 might lead to the discovery of new possibilities for an indirect inhibition of its activity. Here, our goal was to characterize the proteomic neighborhood of PAX3-FOXO1 and to find candidates potentially affecting its activity and tumor cell viability. Towards this aim, we expressed BirA fused versions of PAX3-FOXO1 (N- and C-terminal) in HEK293T cells under presence of biotin. In the control setup, we expressed the BirA enzyme alone. After Streptavidin purification of biotinylated proteins, we performed mass spectrometry and quantified relative abundances compared to control conditions. This enabled us to determine PAX3-FOXO1 proximal proteins, which we investigated further in orthogonal endogenous systems.

2021-09-16 | PXD022187 | Pride

PAX3-FOXO1 suppresses cellular senescence through RASSF4-mediated restraint of the mammalian Hippo/MST1 pathway

Project description:Alveolar rhabdomyosarcoma (aRMS) is an aggressive sarcoma of skeletal muscle characterized by expression of the PAX3-FOXO1 fusion gene. Despite its discovery over almost 20 years ago, PAX3-FOXO1 remains an enigmatic tumor driver. Previously, we reported that PAX3-FOXO1 supports aRMS initiation by enabling bypass of cellular senescence. Here, we show that bypass occurs in part by PAX3-FOXO1-mediated upregulation of RASSF4, a Ras-association domain family (RASSF) member, which then suppresses the evolutionarily conserved mammalian Hippo/Mst1 pathway. RASSF4 loss-of-function activates Hippo/Mst1 and inhibits downstream YAP, causing aRMS cell cycle arrest and senescence. This is the first evidence for an oncogenic role for RASSF4, and a novel mechanism for Hippo signaling suppression in human cancer. Human skeletal muscle myoblasts (HSMMs) were retrovirally transduced with either an empty vector (Vp, pK1) or PAX3-FOXO1 (PFp, pK1-PAX3-FOXO1) and selected on puromycin. Presenescent (presen) cells were harvested before the senescence checkpoint. Since cells expressing PAX3-FOXO1 can bypass the senescence checkpoint, postsenescent (postsen) cells expressing PAX3-FOXO1 were also harvested. the gene expression affected by the introduction of PAX3-FOXO1

2014-05-15 | E-GEOD-40543 | biostudies-arrayexpress

NuRD subunit CHD4 regulates super-enhancer accessibility in Rhabdomyosarcoma and represents a general tumor dependency

Project description:ATP-dependent chromatin remodelers modulate gene expression by regulating genome accessibility and can contribute to tumorigenesis. In fusion-positive rhabdomyosarcoma (FP-RMS), we have previously identified the chromatin remodeler and NuRD subunit CHD4 as an essential gene for tumor survival. Here, we demonstrate that the FP-RMS vulnerability to CHD4 goes beyond its function as a NuRD member. Mechanistically, CHD4 interacts with BRD4 and co-localizes with the tumor driver and fusion protein PAX3-FOXO1 at super-enhancers where it generates a chromatin architecture permissive for the binding of the fusion protein. This allows the positioning of RNA polymerase 2 at promoters and the expression of the oncogenic program of PAX3-FOXO1. Additionally, analysis of genome-wide cancer dependency databases identifies CHD4 amongst the NuRD subunits as general novel cancer vulnerability. Our findings describe, for the first time, CHD4 as a regulator of super-enhancer-mediated gene expression and establish this chromatin remodeler as an unexpected broad tumor susceptibility.

2020-08-08 | GSE155861 | GEO

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