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Spliceosomal vulnerability of MYCN-amplified neuroblastoma is contingent on PRMT5-mediated regulation of epitranscriptomic and metabolomic pathways

Jodie Bojko Orcid Logo, Madhu Kollareddy Orcid Logo, Marianna Szemes, Jacob Bellamy Orcid Logo, Evon Poon Orcid Logo, Ahmad Moukachar, Danny Legge, Emma E. Vincent Orcid Logo, Nick Jones Orcid Logo, Sally Malik Orcid Logo, Alexander Greenhough Orcid Logo, Alex Paterson Orcid Logo, Ji Hyun Park, Kelli Gallacher Orcid Logo, Louis Chesler, Karim Malik

Cancer Letters, Volume: 604, Start page: 217263

Swansea University Author: Nick Jones Orcid Logo

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DOI (Published version): 10.1016/j.canlet.2024.217263

Abstract

Approximately 50 % of poor prognosis neuroblastomas arise due to MYCN over-expression. We previously demonstrated that MYCN and PRMT5 proteins interact and PRMT5 knockdown led to apoptosis of MYCN-amplified (MNA) neuroblastoma. Here we evaluate the highly selective first-in-class PRMT5 inhibitor GSK...

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Published in: Cancer Letters
ISSN: 0304-3835
Published: Elsevier BV 2024
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URI: https://cronfa.swan.ac.uk/Record/cronfa67770
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Here we evaluate the highly selective first-in-class PRMT5 inhibitor GSK3203591 and its in vivo analogue GSK3326593 as targeted therapeutics for MNA neuroblastoma. Cell-line analyses show MYCN-dependent growth inhibition and apoptosis, with approximately 200-fold greater sensitivity of MNA neuroblastoma lines. RNA sequencing of three MNA neuroblastoma lines treated with GSK3203591 reveal deregulated MYCN transcriptional programmes and altered mRNA splicing, converging on key regulatory pathways such as DNA damage response, epitranscriptomics and cellular metabolism. Stable isotope labelling experiments in the same cell lines demonstrate that glutamine metabolism is impeded following GSK3203591 treatment, linking with disruption of the MLX/Mondo nutrient sensors via intron retention of MLX mRNA. Interestingly, glutaminase (GLS) protein decreases after GSK3203591 treatment despite unchanged transcript levels. We demonstrate that the RNA methyltransferase METTL3 and cognate reader YTHDF3 proteins are lowered following their mRNAs undergoing GSK3203591-induced splicing alterations, indicating epitranscriptomic regulation of GLS; accordingly, we observe decreases of GLS mRNA m6A methylation following GSK3203591 treatment, and decreased GLS protein following YTHDF3 knockdown. In vivo efficacy of GSK3326593 is confirmed by increased survival of Th-MYCN mice, with drug treatment triggering splicing events and protein decreases consistent with in vitro data. 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spelling 2024-10-21T14:44:13.6519940 v2 67770 2024-09-23 Spliceosomal vulnerability of MYCN-amplified neuroblastoma is contingent on PRMT5-mediated regulation of epitranscriptomic and metabolomic pathways 0fce0f7ddbdbfeb968f4e2f1e3f86744 0000-0003-4846-5117 Nick Jones Nick Jones true false 2024-09-23 MEDS Approximately 50 % of poor prognosis neuroblastomas arise due to MYCN over-expression. We previously demonstrated that MYCN and PRMT5 proteins interact and PRMT5 knockdown led to apoptosis of MYCN-amplified (MNA) neuroblastoma. Here we evaluate the highly selective first-in-class PRMT5 inhibitor GSK3203591 and its in vivo analogue GSK3326593 as targeted therapeutics for MNA neuroblastoma. Cell-line analyses show MYCN-dependent growth inhibition and apoptosis, with approximately 200-fold greater sensitivity of MNA neuroblastoma lines. RNA sequencing of three MNA neuroblastoma lines treated with GSK3203591 reveal deregulated MYCN transcriptional programmes and altered mRNA splicing, converging on key regulatory pathways such as DNA damage response, epitranscriptomics and cellular metabolism. Stable isotope labelling experiments in the same cell lines demonstrate that glutamine metabolism is impeded following GSK3203591 treatment, linking with disruption of the MLX/Mondo nutrient sensors via intron retention of MLX mRNA. Interestingly, glutaminase (GLS) protein decreases after GSK3203591 treatment despite unchanged transcript levels. We demonstrate that the RNA methyltransferase METTL3 and cognate reader YTHDF3 proteins are lowered following their mRNAs undergoing GSK3203591-induced splicing alterations, indicating epitranscriptomic regulation of GLS; accordingly, we observe decreases of GLS mRNA m6A methylation following GSK3203591 treatment, and decreased GLS protein following YTHDF3 knockdown. In vivo efficacy of GSK3326593 is confirmed by increased survival of Th-MYCN mice, with drug treatment triggering splicing events and protein decreases consistent with in vitro data. Together our study demonstrates the PRMT5-dependent spliceosomal vulnerability of MNA neuroblastoma and identifies the epitranscriptome and glutamine metabolism as critical determinants of this sensitivity. Journal Article Cancer Letters 604 217263 Elsevier BV 0304-3835 1 11 2024 2024-11-01 10.1016/j.canlet.2024.217263 COLLEGE NANME Medical School COLLEGE CODE MEDS Swansea University Another institution paid the OA fee he authors would like to thank Cancer Research UK (A12743/A21046), Neuroblastoma UK, the Biotechnology and Biological Sciences Research Council (BB/P008232/1), and Children's Cancer and Leukaemia Group (CCLG) and The Little Princess Trust (LPT) for funding this study. E.P. and L.C. were supported by Cancer Research UK Programme Award A28278 and ICR institutional funding. A.G. is funded by the Wallscourt Foundation. 2024-10-21T14:44:13.6519940 2024-09-23T12:22:44.2913048 Faculty of Medicine, Health and Life Sciences Swansea University Medical School - Biomedical Science Jodie Bojko 0000-0003-2595-8648 1 Madhu Kollareddy 0000-0001-7412-096x 2 Marianna Szemes 3 Jacob Bellamy 0000-0003-2345-3590 4 Evon Poon 0000-0002-0616-2487 5 Ahmad Moukachar 6 Danny Legge 7 Emma E. Vincent 0000-0002-8917-7384 8 Nick Jones 0000-0003-4846-5117 9 Sally Malik 0009-0006-7098-7340 10 Alexander Greenhough 0000-0002-8306-811x 11 Alex Paterson 0000-0003-3025-8347 12 Ji Hyun Park 13 Kelli Gallacher 0000-0001-6551-8968 14 Louis Chesler 15 Karim Malik 16 67770__32661__946ec0d5ccb5477dac6d3d4e3c564ec8.pdf 67770.VoR.pdf 2024-10-21T14:42:37.5178111 Output 13206715 application/pdf Version of Record true Crown Copyright © 2024. This is an open access article under the CC BY license. true eng http://creativecommons.org/licenses/by/4.0/
title Spliceosomal vulnerability of MYCN-amplified neuroblastoma is contingent on PRMT5-mediated regulation of epitranscriptomic and metabolomic pathways
spellingShingle Spliceosomal vulnerability of MYCN-amplified neuroblastoma is contingent on PRMT5-mediated regulation of epitranscriptomic and metabolomic pathways
Nick Jones
title_short Spliceosomal vulnerability of MYCN-amplified neuroblastoma is contingent on PRMT5-mediated regulation of epitranscriptomic and metabolomic pathways
title_full Spliceosomal vulnerability of MYCN-amplified neuroblastoma is contingent on PRMT5-mediated regulation of epitranscriptomic and metabolomic pathways
title_fullStr Spliceosomal vulnerability of MYCN-amplified neuroblastoma is contingent on PRMT5-mediated regulation of epitranscriptomic and metabolomic pathways
title_full_unstemmed Spliceosomal vulnerability of MYCN-amplified neuroblastoma is contingent on PRMT5-mediated regulation of epitranscriptomic and metabolomic pathways
title_sort Spliceosomal vulnerability of MYCN-amplified neuroblastoma is contingent on PRMT5-mediated regulation of epitranscriptomic and metabolomic pathways
author_id_str_mv 0fce0f7ddbdbfeb968f4e2f1e3f86744
author_id_fullname_str_mv 0fce0f7ddbdbfeb968f4e2f1e3f86744_***_Nick Jones
author Nick Jones
author2 Jodie Bojko
Madhu Kollareddy
Marianna Szemes
Jacob Bellamy
Evon Poon
Ahmad Moukachar
Danny Legge
Emma E. Vincent
Nick Jones
Sally Malik
Alexander Greenhough
Alex Paterson
Ji Hyun Park
Kelli Gallacher
Louis Chesler
Karim Malik
format Journal article
container_title Cancer Letters
container_volume 604
container_start_page 217263
publishDate 2024
institution Swansea University
issn 0304-3835
doi_str_mv 10.1016/j.canlet.2024.217263
publisher Elsevier BV
college_str Faculty of Medicine, Health and Life Sciences
hierarchytype
hierarchy_top_id facultyofmedicinehealthandlifesciences
hierarchy_top_title Faculty of Medicine, Health and Life Sciences
hierarchy_parent_id facultyofmedicinehealthandlifesciences
hierarchy_parent_title Faculty of Medicine, Health and Life Sciences
department_str Swansea University Medical School - Biomedical Science{{{_:::_}}}Faculty of Medicine, Health and Life Sciences{{{_:::_}}}Swansea University Medical School - Biomedical Science
document_store_str 1
active_str 0
description Approximately 50 % of poor prognosis neuroblastomas arise due to MYCN over-expression. We previously demonstrated that MYCN and PRMT5 proteins interact and PRMT5 knockdown led to apoptosis of MYCN-amplified (MNA) neuroblastoma. Here we evaluate the highly selective first-in-class PRMT5 inhibitor GSK3203591 and its in vivo analogue GSK3326593 as targeted therapeutics for MNA neuroblastoma. Cell-line analyses show MYCN-dependent growth inhibition and apoptosis, with approximately 200-fold greater sensitivity of MNA neuroblastoma lines. RNA sequencing of three MNA neuroblastoma lines treated with GSK3203591 reveal deregulated MYCN transcriptional programmes and altered mRNA splicing, converging on key regulatory pathways such as DNA damage response, epitranscriptomics and cellular metabolism. Stable isotope labelling experiments in the same cell lines demonstrate that glutamine metabolism is impeded following GSK3203591 treatment, linking with disruption of the MLX/Mondo nutrient sensors via intron retention of MLX mRNA. Interestingly, glutaminase (GLS) protein decreases after GSK3203591 treatment despite unchanged transcript levels. We demonstrate that the RNA methyltransferase METTL3 and cognate reader YTHDF3 proteins are lowered following their mRNAs undergoing GSK3203591-induced splicing alterations, indicating epitranscriptomic regulation of GLS; accordingly, we observe decreases of GLS mRNA m6A methylation following GSK3203591 treatment, and decreased GLS protein following YTHDF3 knockdown. In vivo efficacy of GSK3326593 is confirmed by increased survival of Th-MYCN mice, with drug treatment triggering splicing events and protein decreases consistent with in vitro data. Together our study demonstrates the PRMT5-dependent spliceosomal vulnerability of MNA neuroblastoma and identifies the epitranscriptome and glutamine metabolism as critical determinants of this sensitivity.
published_date 2024-11-01T05:22:48Z
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