EWS–FLI1 modulated alternative splicing of ARID1A reveals novel oncogenic function through the BAF complex

Selvanathan, Saravana P.; Graham, Garrett T.; Grego, Alexander; Baker, Tabari; Hogg, J. Robert; Simpson, Mark; Batish, Mona; Crompton, Brian D.; Stegmaier, Kimberly; Tomazou, Eleni M.; Kovar, Heinrich; Üren, Aykut; Toretsky, Jeffrey A.

doi:10.1093/nar/gkz699

Cited by 34 publications

(52 citation statements)

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“…As outlined before, a recent study revealed a direct interaction of EWSR1-FLI1 with the BAF complex via its subunit ARID1A [80]. The authors could show that ARID1A was differentially spliced and accumulated in its long isoform ARID1A-L in the presence of EWSR1-FLI1.…”

Section: Targeting Protein-protein Interactions Of Fusion Oncoproteinsmentioning

confidence: 72%

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Targeting the undruggable: exploiting neomorphic features of fusion oncoproteins in childhood sarcomas for innovative therapies

Knott

Hölting²,

Ohmura³

et al. 2019

Cancer Metastasis Rev

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While sarcomas account for approximately 1% of malignant tumors of adults, they are particularly more common in children and adolescents affected by cancer. In contrast to malignancies that occur in later stages of life, childhood tumors, including sarcoma, are characterized by a striking paucity of somatic mutations. However, entity-defining fusion oncogenes acting as the main oncogenic driver mutations are frequently found in pediatric bone and soft-tissue sarcomas such as Ewing sarcoma (EWSR1-FLI1), alveolar rhabdomyosarcoma (PAX3/7-FOXO1), and synovial sarcoma (SS18-SSX1/2/4). Since strong oncogene-dependency has been demonstrated in these entities, direct pharmacological targeting of these fusion oncogenes has been excessively attempted, thus far, with limited success. Despite apparent challenges, our increasing understanding of the neomorphic features of these fusion oncogenes in conjunction with rapid technological advances will likely enable the development of new strategies to therapeutically exploit these neomorphic features and to ultimately turn the "undruggable" into first-line target structures. In this review, we provide a broad overview of the current literature on targeting neomorphic features of fusion oncogenes found in Ewing sarcoma, alveolar rhabdomyosarcoma, and synovial sarcoma, and give a perspective for future developments.

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Section: Targeting Protein-protein Interactions Of Fusion Oncoproteinsmentioning

confidence: 72%

“…One of the subunits of the chromatin remodeling BAF complex, ARID1A, has been recently shown to be a splicing target of EWSR1-FLI1 [80]. ARID1A-L generated by alternative splicing in EwS cells plays an important role in tumor growth and promotes the stability of EWSR1-FLI1.…”

Section: Targeting the Splicing Machinery Involved In Post-transcriptmentioning

confidence: 99%

Targeting the undruggable: exploiting neomorphic features of fusion oncoproteins in childhood sarcomas for innovative therapies

Knott

Hölting²,

Ohmura³

et al. 2019

Cancer Metastasis Rev

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show abstract

“…In this regard, the splicing signature of human cancers is emerging as a powerful tool to distinguish tumor subtypes and stratify patients [32,33]. Notably, although alternative splicing dysregulation has also been reported in Ewing sarcoma [24,[34][35][36][37][38], limited information is available regarding the RBPs responsible for this process and their possible association with prognosis. An important regulator of RNA metabolism with strong implications in Ewing sarcoma malignancy is the DNA/RNA helicase DHX9, which interacts with the oncogene EWS-FLI1 and promotes its transcriptional activity.…”

Section: Discussionmentioning

confidence: 99%

Poison-Exon Inclusion in DHX9 Reduces Its Expression and Sensitizes Ewing Sarcoma Cells to Chemotherapeutic Treatment

Palombo

Verdile

Paronetto

2020

Cells

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Alternative splicing is a combinatorial mechanism by which exons are joined to produce multiple mRNA variants, thus expanding the coding potential and plasticity of eukaryotic genomes. Defects in alternative splicing regulation are associated with several human diseases, including cancer. Ewing sarcoma is an aggressive tumor of bone and soft tissue, mainly affecting adolescents and young adults. DHX9 is a key player in Ewing sarcoma malignancy, and its expression correlates with worse prognosis in patients. In this study, by screening a library of siRNAs, we have identified splicing factors that regulate the alternative inclusion of a poison exon in DHX9 mRNA, leading to its downregulation. In particular, we found that hnRNPM and SRSF3 bind in vivo to this poison exon and suppress its inclusion. Notably, DHX9 expression correlates with that of SRSF3 and hnRNPM in Ewing sarcoma patients. Furthermore, downregulation of SRSF3 or hnRNPM inhibited DHX9 expression and Ewing sarcoma cell proliferation, while sensitizing cells to chemotherapeutic treatment. Hence, our study suggests that inhibition of hnRNPM and SRSF3 expression or activity could be exploited as a therapeutic tool to enhance the efficacy of chemotherapy in Ewing sarcoma.

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“…Like the wild-type FET proteins, EWS-FLI1 binds RNA Pol II, as well as a number of transcription factors and enhancer or repressor complexes [24][25][26][27] . Nevertheless, the mechanism for EWS-FLI1 to broadly modulate transcription remains incomplete.…”

Section: Introductionmentioning

confidence: 99%

Fusion protein EWS-FLI1 is incorporated into a protein granule in cells

Ahmed

Harrell

Schwartz

2020

Preprint

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Ewing sarcoma is driven by fusion proteins containing a low complexity (LC) domain that is intrinsically disordered and a powerful transcription regulator. The most common fusion protein found in Ewing sarcoma, EWS-FLI1, takes its LC domain from the RNA-binding protein EWSR1 (Ewing Sarcoma RNAbinding protein 1) and a DNA-binding domain from the transcription factor FLI1 (Friend Leukemia Virus Integration 1). The LC domain in EWS-FLI1 can bind RNA polymerase II (RNA Pol II) and can selfassemble through a process known as phase separation. The ability of EWSR1 and related RNA-binding proteins to assemble into ribonucleoprotein granules in cells has been vigorously studied and while many questions remain, the role of phase separation in EWS-FLI1 activity is less understood. We investigated the overlapping functions of EWSR1 and EWS-FLI1 in controlling gene expression and tumorigenic cell growth in Ewing sarcoma, which suggested these proteins function closely together. We then studied the nature of interactions between EWS-FLI1, EWSR1, and RNA Pol II. We observed EWSR1 and RNA Pol II to be present in protein granules in cells. We then identified protein granules in cells associated with the fusion protein, EWS-FLI1. The tyrosine residues in the LC domain are required for both the abilities of EWS-FLI1 to bind its partners, EWSR1 and RNA Pol II, and to incorporate into protein granules. These data suggest that interactions between EWS-FLI1, RNA Pol II, and EWSR1 in Ewing sarcoma can occur in the context of a molecular scaffold found within protein granules in the cell. 6 RNA SequencingA673 cells were transfected with 50 nM siRNA as described above. Cells were collected 72 hours posttransfection and total RNA was extracted using TRIzol reagent (ThermoFisher, Cat. #15596026). 1 µg of total RNA was prepared for sequencing using NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB, #E7490) to generate sequencing libraries according to manufacturer's instructions. Soft Agar Colony Formation AssayA673, SK-N-MC, and HEK293T/17 cells transfected with 50 nM siRNA were harvested 24 hours posttransfection. HEK293T/17 cells transfected with 50 nM siRNA and 2 µg of plasmid DNA were harvested 24 hours post-transfection. A673 and SK-N-MC cells were seeded at density of 1.0 x 10 5 cells. Cells were resuspended in 0.35% agarose in growth media and plated onto a bed of solidified 0.6% agarose in growth media. HEK293T/17 cells were seeded at a density of 20k to 30k cells. Cells were resuspended in 0.4% agarose in growth media onto a bed of solidified 0.6% agarose in growth media. A673 and SK-N-MC cells were grown at 37°C and 5% CO2 for 3-4 weeks, imaged, and then colonies were counted using ImageJ software. HEK293T/17 cells were grown at 37°C and 5% CO2 for 1-2 weeks, imaged, and then colonies were counted. Colonies with stained with 0.05% methylene blue. Cell Growth AssayA673 cells were reverse transfected at a density of 4.0 x 10 5 cells in 6-well dishes. Cells were collected by trypsinization and counted on a hemocytometer at 24, 48,...

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EWS–FLI1 modulated alternative splicing of ARID1A reveals novel oncogenic function through the BAF complex

Cited by 34 publications

References 50 publications

Targeting the undruggable: exploiting neomorphic features of fusion oncoproteins in childhood sarcomas for innovative therapies

Targeting the undruggable: exploiting neomorphic features of fusion oncoproteins in childhood sarcomas for innovative therapies

Poison-Exon Inclusion in DHX9 Reduces Its Expression and Sensitizes Ewing Sarcoma Cells to Chemotherapeutic Treatment

Fusion protein EWS-FLI1 is incorporated into a protein granule in cells

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