Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma

Damon A. Hofman; Jorge Ruiz-Orera; Ian Yannuzzi; Rakesh Murugesan; Adam Brown; Karl R. Clauser; Alexandra L. Condurat; Jip T. van Dinter; Sem A. G. Engels; Amy Goodale; Jasper van der Lugt; Tanaz Abid; Li Wang; Kevin N. Zhou; Jayne Vogelzang; Keith L. Ligon; Timothy N. Phoenix; Jennifer A. Roth; David E. Root; Norbert Hubner; Todd R. Golub; Pratiti Bandopadhayay; Sebastiaan van Heesch; John R. Prensner

doi:https://doi.org/10.1016/j.molcel.2023.12.003

Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma

Damon A. Hofman, Jorge Ruiz-Orera, Ian Yannuzzi, Rakesh Murugesan, Adam Brown, Karl R. Clauser, Alexandra L. Condurat, Jip T. van Dinter, Sem A. G. Engels, Amy Goodale, Jasper van der Lugt, Tanaz Abid, Li Wang, Kevin N. Zhou, Jayne Vogelzang, Keith L. Ligon, Timothy N. Phoenix, Jennifer A. Roth, David E. Root, Norbert HubnerTodd R. Golub, Pratiti Bandopadhayay, Sebastiaan van Heesch, John R. Prensner

Hematology laboratory (VUmc)

Research output: Contribution to journal › Article › Academic › peer-review

Abstract

A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC-family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets.

Original language	English
Pages (from-to)	261-276.e18
Journal	Molecular Cell
Volume	84
Issue number	2
DOIs	https://doi.org/10.1016/j.molcel.2023.12.003
Publication status	Published - 18 Jan 2024

Keywords

CRISPR
Ribo-seq
cancer
gene dependency
lncRNAs
medulloblastoma
non-canonical ORFs
translational regulation
uORF

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https://doi.org/10.1016/j.molcel.2023.12.003

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Hofman, D. A., Ruiz-Orera, J., Yannuzzi, I., Murugesan, R., Brown, A., Clauser, K. R., Condurat, A. L., van Dinter, J. T., Engels, S. A. G., Goodale, A., van der Lugt, J., Abid, T., Wang, L., Zhou, K. N., Vogelzang, J., Ligon, K. L., Phoenix, T. N., Roth, J. A., Root, D. E., ... Prensner, J. R. (2024). Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma. Molecular Cell, 84(2), 261-276.e18. https://doi.org/10.1016/j.molcel.2023.12.003

@article{799a218cb1764f649c91f276c2d6c717,

title = "Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma",

abstract = "A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC-family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets.",

keywords = "CRISPR, Ribo-seq, cancer, gene dependency, lncRNAs, medulloblastoma, non-canonical ORFs, translational regulation, uORF",

author = "Hofman, {Damon A.} and Jorge Ruiz-Orera and Ian Yannuzzi and Rakesh Murugesan and Adam Brown and Clauser, {Karl R.} and Condurat, {Alexandra L.} and {van Dinter}, {Jip T.} and Engels, {Sem A. G.} and Amy Goodale and {van der Lugt}, Jasper and Tanaz Abid and Li Wang and Zhou, {Kevin N.} and Jayne Vogelzang and Ligon, {Keith L.} and Phoenix, {Timothy N.} and Roth, {Jennifer A.} and Root, {David E.} and Norbert Hubner and Golub, {Todd R.} and Pratiti Bandopadhayay and {van Heesch}, Sebastiaan and Prensner, {John R.}",

note = "Funding Information: We thank Greg Newby and David Liu from the Broad Institute for their gracious insights with base editing experiments and providing the ABE8e-NRCH base editor. We thank Edmond Chan (Columbia University) for insights into chromatin immunoprecipitation experiments. We thank Ross Tomiano at the Taplin Mass Spectrometry Facility and Julian Mintseris at the Thermo-Fisher Center for Multiplexed Proteomics at the Harvard Medical School for assistance with mass spectrometry experiments. We thank Maura Berkeley and Zachary Herbert at the Dana-Farber Cancer Institute Molecular Biology Core Facility for assistance with next-generation sequencing. We thank the Dana-Farber Cancer Institute Center for Patient Derived Models for cell line support. We thank the Boston Children{\textquoteright}s Hospital biobank and the DFHCC Neurooncology Program Tissue and Data Bank for biobanking support. We thank the PRISM team at the Broad Institute for assistance with PRISM cell line screening and PRISM cell library sample preparation. We thank Joelle Straehla for sharing Med2112-mCherry-Luc and Med411-GFP-Luc cells. We thank Quang-De Nguyen, Amy Cameron, and Murry Morrow at the Lurie Family Animal Imaging Center at the Dana-Farber Cancer Institute . J.R.P. acknowledges funding from the National Institutes of Health – National Cancer Institute ( K08-CA263552-01A1 ), the Alex{\textquoteright}s Lemonade Stand Foundation Young Investigator Award (# 21-23983 ), the St. Baldrick{\textquoteright}s Foundation Scholar Award (# 931638 ), the DIPG/DMG Research Funding Alliance , the Cure ATRT Now Fund , the Musella Foundation for Brain Tumor Research , and a Collaborative Pediatric Cancer Research Awards Program/Kids Join the Fight award (# 22FN23 ). T.R.G. acknowledges funding from the National Cancer Institute (1 R35 CA242457–01 ). K.L.L. and J.V. acknowledge support from the Pediatric Brain Tumor Foundation , the National Brain Tumor Society , and 3000 Miles to the Cure . S.v.H. acknowledges funding from Fonds Cancers ( FOCA, Belgium ), Stichting Reggeborgh (the Netherlands), and Bergh in het Zadel (the Netherlands). N.H. acknowledges funding from the European Union Horizon 2020 Research and Innovation Program ( AdG788970 ), the Deutsche Forschungsgemeinschaft ( SFB-1470 – B03 ), the Chan Zuckerberg Foundation ( 2019-202666 ), the Leducq Foundation ( 16CVD03 ), the British Heart Foundation , and the Deutsches Zentrum f{\"u}r Herz-Kreislauf-Forschung ( BHF/DZHK: SP/19/1/34461 ). P.B. acknowledges funding from the Isabel V. Marxuach Fund for Medulloblastoma Research and the Jared Branfman Sunflowers for Life Fund for Pediatric Brain and Spinal Cancer Research . Funding Information: K.L.L. reports the following interests: equity in Travera; research funds from Bristol Myers Squibb, SEngine Precision Medicine, Multiple Myeloma Research Foundation, and Eli Lilly and Company; and being a consultant or on the scientific advisory board for Bristol Myers Squibb, Travera, and IntegraGen. P.B. receives grant funding from Novartis Institute of Biomedical Research, and has received grant funding from Deerfield Therapeutics, both for unrelated projects. P.B. has also served on a paid advisory board for qed Therapeutics, unrelated to this work. D.E.R. receives research funding from members of the Functional Genomics Consortium (AbbVie, BMS, Jannsen, Merck, and Vir) and is a director of Addgene, Inc. Publisher Copyright: {\textcopyright} 2023 The Author(s)",

year = "2024",

month = jan,

day = "18",

doi = "https://doi.org/10.1016/j.molcel.2023.12.003",

language = "English",

volume = "84",

pages = "261--276.e18",

journal = "Molecular Cell",

issn = "1097-2765",

publisher = "Cell Press",

number = "2",

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Hofman, DA, Ruiz-Orera, J, Yannuzzi, I, Murugesan, R, Brown, A, Clauser, KR, Condurat, AL, van Dinter, JT, Engels, SAG, Goodale, A, van der Lugt, J, Abid, T, Wang, L, Zhou, KN, Vogelzang, J, Ligon, KL, Phoenix, TN, Roth, JA, Root, DE, Hubner, N, Golub, TR, Bandopadhayay, P, van Heesch, S & Prensner, JR 2024, 'Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma', Molecular Cell, vol. 84, no. 2, pp. 261-276.e18. https://doi.org/10.1016/j.molcel.2023.12.003

TY - JOUR

T1 - Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma

AU - Hofman, Damon A.

AU - Ruiz-Orera, Jorge

AU - Yannuzzi, Ian

AU - Murugesan, Rakesh

AU - Brown, Adam

AU - Clauser, Karl R.

AU - Condurat, Alexandra L.

AU - van Dinter, Jip T.

AU - Engels, Sem A. G.

AU - Goodale, Amy

AU - van der Lugt, Jasper

AU - Abid, Tanaz

AU - Wang, Li

AU - Zhou, Kevin N.

AU - Vogelzang, Jayne

AU - Ligon, Keith L.

AU - Phoenix, Timothy N.

AU - Roth, Jennifer A.

AU - Root, David E.

AU - Hubner, Norbert

AU - Golub, Todd R.

AU - Bandopadhayay, Pratiti

AU - van Heesch, Sebastiaan

AU - Prensner, John R.

N1 - Funding Information: We thank Greg Newby and David Liu from the Broad Institute for their gracious insights with base editing experiments and providing the ABE8e-NRCH base editor. We thank Edmond Chan (Columbia University) for insights into chromatin immunoprecipitation experiments. We thank Ross Tomiano at the Taplin Mass Spectrometry Facility and Julian Mintseris at the Thermo-Fisher Center for Multiplexed Proteomics at the Harvard Medical School for assistance with mass spectrometry experiments. We thank Maura Berkeley and Zachary Herbert at the Dana-Farber Cancer Institute Molecular Biology Core Facility for assistance with next-generation sequencing. We thank the Dana-Farber Cancer Institute Center for Patient Derived Models for cell line support. We thank the Boston Children’s Hospital biobank and the DFHCC Neurooncology Program Tissue and Data Bank for biobanking support. We thank the PRISM team at the Broad Institute for assistance with PRISM cell line screening and PRISM cell library sample preparation. We thank Joelle Straehla for sharing Med2112-mCherry-Luc and Med411-GFP-Luc cells. We thank Quang-De Nguyen, Amy Cameron, and Murry Morrow at the Lurie Family Animal Imaging Center at the Dana-Farber Cancer Institute . J.R.P. acknowledges funding from the National Institutes of Health – National Cancer Institute ( K08-CA263552-01A1 ), the Alex’s Lemonade Stand Foundation Young Investigator Award (# 21-23983 ), the St. Baldrick’s Foundation Scholar Award (# 931638 ), the DIPG/DMG Research Funding Alliance , the Cure ATRT Now Fund , the Musella Foundation for Brain Tumor Research , and a Collaborative Pediatric Cancer Research Awards Program/Kids Join the Fight award (# 22FN23 ). T.R.G. acknowledges funding from the National Cancer Institute (1 R35 CA242457–01 ). K.L.L. and J.V. acknowledge support from the Pediatric Brain Tumor Foundation , the National Brain Tumor Society , and 3000 Miles to the Cure . S.v.H. acknowledges funding from Fonds Cancers ( FOCA, Belgium ), Stichting Reggeborgh (the Netherlands), and Bergh in het Zadel (the Netherlands). N.H. acknowledges funding from the European Union Horizon 2020 Research and Innovation Program ( AdG788970 ), the Deutsche Forschungsgemeinschaft ( SFB-1470 – B03 ), the Chan Zuckerberg Foundation ( 2019-202666 ), the Leducq Foundation ( 16CVD03 ), the British Heart Foundation , and the Deutsches Zentrum für Herz-Kreislauf-Forschung ( BHF/DZHK: SP/19/1/34461 ). P.B. acknowledges funding from the Isabel V. Marxuach Fund for Medulloblastoma Research and the Jared Branfman Sunflowers for Life Fund for Pediatric Brain and Spinal Cancer Research . Funding Information: K.L.L. reports the following interests: equity in Travera; research funds from Bristol Myers Squibb, SEngine Precision Medicine, Multiple Myeloma Research Foundation, and Eli Lilly and Company; and being a consultant or on the scientific advisory board for Bristol Myers Squibb, Travera, and IntegraGen. P.B. receives grant funding from Novartis Institute of Biomedical Research, and has received grant funding from Deerfield Therapeutics, both for unrelated projects. P.B. has also served on a paid advisory board for qed Therapeutics, unrelated to this work. D.E.R. receives research funding from members of the Functional Genomics Consortium (AbbVie, BMS, Jannsen, Merck, and Vir) and is a director of Addgene, Inc. Publisher Copyright: © 2023 The Author(s)

PY - 2024/1/18

Y1 - 2024/1/18

N2 - A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC-family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets.

AB - A hallmark of high-risk childhood medulloblastoma is the dysregulation of RNA translation. Currently, it is unknown whether medulloblastoma dysregulates the translation of putatively oncogenic non-canonical open reading frames (ORFs). To address this question, we performed ribosome profiling of 32 medulloblastoma tissues and cell lines and observed widespread non-canonical ORF translation. We then developed a stepwise approach using multiple CRISPR-Cas9 screens to elucidate non-canonical ORFs and putative microproteins implicated in medulloblastoma cell survival. We determined that multiple lncRNA-ORFs and upstream ORFs (uORFs) exhibited selective functionality independent of main coding sequences. A microprotein encoded by one of these ORFs, ASNSD1-uORF or ASDURF, was upregulated, associated with MYC-family oncogenes, and promoted medulloblastoma cell survival through engagement with the prefoldin-like chaperone complex. Our findings underscore the fundamental importance of non-canonical ORF translation in medulloblastoma and provide a rationale to include these ORFs in future studies seeking to define new cancer targets.

KW - CRISPR

KW - Ribo-seq

KW - cancer

KW - gene dependency

KW - lncRNAs

KW - medulloblastoma

KW - non-canonical ORFs

KW - translational regulation

KW - uORF

UR - http://www.scopus.com/inward/record.url?scp=85182357581&partnerID=8YFLogxK

U2 - https://doi.org/10.1016/j.molcel.2023.12.003

DO - https://doi.org/10.1016/j.molcel.2023.12.003

M3 - Article

C2 - 38176414

SN - 1097-2765

VL - 84

SP - 261-276.e18

JO - Molecular Cell

JF - Molecular Cell

IS - 2

ER -

Translation of non-canonical open reading frames as a cancer cell survival mechanism in childhood medulloblastoma

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Keywords

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