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

Palombo, Ramona; Verdile, Veronica; Paronetto, Maria Paola

doi:10.3390/cells9020328

“…With regard to the variant we studied and its effects on poison exon inclusion, there is a growing list of therapeutic strategies that target mechanisms related to alternative splicing. An RNA-based therapeutic triggering poison exon inclusion in DHX9 , a gene involved in Ewing sarcoma, has been exploited to enhance the efficacy of chemotherapy in cancer patients [ 41 ]. Additionally, in Duchenne muscular dystrophy and spinal muscular atrophy (SMA), oligonucleotides that promote exon skipping and alternative exon inclusion, respectively, are becoming increasingly applied in clinical settings [ 42 , 43 ].…”

Section: Discussionmentioning

confidence: 99%

Aberrant regulation of a poison exon caused by a non-coding variant in a mouse model of Scn1a-associated epileptic encephalopathy

Voskobiynyk

¹

,

Battu

²

,

Felker

³

et al. 2021

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Dravet syndrome (DS) is a developmental and epileptic encephalopathy that results from mutations in the Nav1.1 sodium channel encoded by SCN1A. Most known DS-causing mutations are in coding regions of SCN1A, but we recently identified several disease-associated SCN1A mutations in intron 20 that are within or near to a cryptic and evolutionarily conserved “poison” exon, 20N, whose inclusion is predicted to lead to transcript degradation. However, it is not clear how these intron 20 variants alter SCN1A expression or DS pathophysiology in an organismal context, nor is it clear how exon 20N is regulated in a tissue-specific and developmental context. We address those questions here by generating an animal model of our index case, NM_006920.4(SCN1A):c.3969+2451G>C, using gene editing to create the orthologous mutation in laboratory mice. Scn1a heterozygous knock-in (+/KI) mice exhibited an ~50% reduction in brain Scn1a mRNA and Nav1.1 protein levels, together with characteristics observed in other DS mouse models, including premature mortality, seizures, and hyperactivity. In brain tissue from adult Scn1a +/+ animals, quantitative RT-PCR assays indicated that ~1% of Scn1a mRNA included exon 20N, while brain tissue from Scn1a +/KI mice exhibited an ~5-fold increase in the extent of exon 20N inclusion. We investigated the extent of exon 20N inclusion in brain during normal fetal development in RNA-seq data and discovered that levels of inclusion were ~70% at E14.5, declining progressively to ~10% postnatally. A similar pattern exists for the homologous sodium channel Nav1.6, encoded by Scn8a. For both genes, there is an inverse relationship between the level of functional transcript and the extent of poison exon inclusion. Taken together, our findings suggest that poison exon usage by Scn1a and Scn8a is a strategy to regulate channel expression during normal brain development, and that mutations recapitulating a fetal-like pattern of splicing cause reduced channel expression and epileptic encephalopathy.

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“…In terms of the intronic variant we studied, there is a growing list of therapeutic strategies that target mechanisms related to alternative splicing. An RNA-based therapeutic triggering poison exon inclusion in DHX9, a gene involved in Ewing sarcoma, has been exploited to enhance the efficacy of chemotherapy in cancer patients [42]. Additionally, targeting RNA splicing of SMN2, haploinsufficiency of which leads to spinal muscular atrophy (SMA), has led to the development of the first FDA-approved drug to treat SMA [43].…”

Section: Discussionmentioning

confidence: 99%

Aberrant regulation of a poison exon caused by a non-coding variant inScn1a-associated epileptic encephalopathy

Voskobiynyk

¹

,

Battu

²

,

Felker

³

et al. 2020

Preprint

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Dravet syndrome (DS) is a developmental and epileptic encephalopathy that results from mutations in the Na v 1.1 sodium channel encoded by SCN1A. Most known DS-causing mutations are in coding regions of SCN1A, but we recently identified several disease-associated SCN1A mutations in intron 20 that are within or near to a cryptic and evolutionarily conserved "poison" exon, 20N, whose inclusion leads to transcript degradation. However, it is not clear how these intron 20 variants alter SCN1A transcript processing or DS pathophysiology in an organismal context, nor is it clear how exon 20N is regulated in a tissue-specific and developmental context. We address those questions here by generating an animal model of our index case, NM_006920.4(SCN1A):c.3969+2451G>C, using gene editing to create the orthologous mutation in laboratory mice. Scn1a heterozygous knock-in (+/KI) mice exhibited an ~50% reduction in brain Scn1a mRNA and Na v 1.1 protein levels, together with characteristics observed in other DS mouse models, including premature mortality, seizures, and hyperactivity. In brain tissue from adult Scn1a +/+ animals, quantitative RT-PCR assays indicated that ~1% of Scn1a mRNA included exon 20N, while brain tissue from Scn1a +/KI mice exhibited an ~5-fold increase in the extent of exon 20N inclusion. We investigated the extent of exon 20N inclusion in brain during normal fetal development in RNA-seq data and discovered that levels of inclusion were ~70% at E14.5, declining progressively to ~10% postnatally. A similar pattern exists for the homologous sodium channel Na v 1.6, encoded by Scn8a. For both genes, there is an inverse relationship between the level of functional transcript and the extent of poison exon inclusion. Taken together, our findings suggest that poison exon usage by Scn1a and Scn8a is a strategy to regulate channel expression during normal brain development, and that mutations recapitulating a fetal-like pattern of splicing cause reduced channel expression and epileptic encephalopathy.Correspondence to Gregory Barsh (gbarsh@hudsonalpha.org | hudsonalpha.org/faculty/greg-barsh | @GregBarsh ) Correspondence to Erik Roberson (eroberson@uabmc.edu | robersonlab.net | @RobersonLabUAB) Author SummaryDravet syndrome (DS) is a neurological disorder affecting approximately 1:15,700 Americans[1]. While most patients have a mutation in the SCN1A gene encoding Na v 1.1 sodium channels, about 20% do not have a mutation identified by exome sequencing. Recently, we identified variants in intron 20N, a noncoding region of SCN1A, in some DS patients [2]. We predicted that these variants alter SCN1A transcript processing, decrease Na v 1.1 function, and lead to DS pathophysiology via inclusion of exon 20N, a "poison" exon that leads to a premature stop codon. In this study, we generated a knock-in mouse model, Scn1a+/KI, of one of these variants, NM_006920.4(SCN1A):c.3969+2451G>C, which resides in a genomic region that is extremely conserved across vertebrate species. We found that Scn1a+/KI mice have reduced levels ...

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“…A different study revealed that SRSF3 and hnRNPM can bind to DHX9 exon 6A to suppress its inclusion and promote DHX9 expression. Downregulation of these proteins inhibits DHX9 expression, suppresses proliferation and sensitizes Ewing sarcoma cells to doxorubicin treatment, revealing the importance of DHX9 alternative splicing in conferring chemosensitivity or chemoresistance [ 108 ].…”

Section: Dhx9 As a Biomarker And Therapeutic Targetmentioning

confidence: 99%

The Enigmatic Helicase DHX9 and Its Association With the Hallmarks of Cancer

Gulliver

¹

,

Hoffmann

²

,

Baillie

³

2020

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Much interest has been expended lately in characterizing the association between DExH-Box helicase 9 (DHX9) dysregulation and malignant development, however, the enigmatic nature of DHX9 has caused conflict as to whether it regularly functions as an oncogene or tumor suppressor. The impact of DHX9 on malignancy appears to be cell-type specific, dependent upon the availability of binding partners and activation of inter-connected signaling pathways. Realization of DHX9’s pivotal role in the development of several hallmarks of cancer has boosted the enzyme's potential as a cancer biomarker and therapeutic target, opening up novel avenues for exploring DHX9 in precision medicine applications. Our review discusses the ascribed functions of DHX9 in cancer, explores its enigmatic nature and potential as an antineoplastic target.

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

Cited by 24 publications

References 59 publications

Aberrant regulation of a poison exon caused by a non-coding variant in a mouse model of Scn1a-associated epileptic encephalopathy

Aberrant regulation of a poison exon caused by a non-coding variant in a mouse model of Scn1a-associated epileptic encephalopathy

Aberrant regulation of a poison exon caused by a non-coding variant inScn1a-associated epileptic encephalopathy

The Enigmatic Helicase DHX9 and Its Association With the Hallmarks of Cancer

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