Noncanonical open reading frames encode functional proteins essential for cancer cell survival

John R. Prensner; Oana M. Enache; Victor Luria; Karsten Krug; Karl R. Clauser; Joshua M. Dempster; Amir Karger; Li Wang; Karolina Stumbraite; Vickie M. Wang; Ginevra Botta; Nicholas J. Lyons; Amy Goodale; Zohra Kalani; Briana Fritchman; Adam Brown; Douglas Alan; Thomas Green; Xiaoping Yang; Jacob D. Jaffe; Jennifer A. Roth; Federica Piccioni; Marc W. Kirschner; Zhe Ji; David E. Root; Todd R. Golub

doi:10.1038/s41587-020-00806-2

Noncanonical open reading frames encode functional proteins essential for cancer cell survival

John R. Prensner, Oana M. Enache, Victor Luria, Karsten Krug, Karl R. Clauser, Joshua M. Dempster, Amir Karger, Li Wang, Karolina Stumbraite, Vickie M. Wang, Ginevra Botta, Nicholas J. Lyons, Amy Goodale, Zohra Kalani, Briana Fritchman, Adam Brown, Douglas Alan, Thomas Green, Xiaoping Yang, Jacob D. JaffeJennifer A. Roth, Federica Piccioni, Marc W. Kirschner, Zhe Ji, David E. Root, Todd R. Golub^*

^*Corresponding author for this work

Pharmacology

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

79 Scopus citations

Abstract

Although genomic analyses predict many noncanonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442—renamed glycine-rich extracellular protein-1 (GREP1)—encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.

Original language	English (US)
Pages (from-to)	697-704
Number of pages	8
Journal	Nature biotechnology
Volume	39
Issue number	6
DOIs	https://doi.org/10.1038/s41587-020-00806-2
State	Published - Jun 2021

Funding

We thank D. Bondeson, P. Tsvetkov, S. Corsello, U. Ben-David and T. Ouspenskaia for helpful discussions and critical reading of the manuscript. We thank M. Zhong for technical assistance with cloning and Z. Demere for assistance with CRISPR-sequencing. We thank D. Nusinow and S. Gygi for insights into identification of small peptides in proteomics datasets. We thank R. Tomaino for assistance with mass spectrometry at the Talpin Biological Mass Spectrometry Facility at Harvard Medical School. We thank J. Chen for assistance with the Slncky algorithm. We thank J. Gould for assistance with gene datasets. We thank I. Cheeseman for provision of DOX-inducible HeLa Cas9 cells. J.R.P. was supported by the Harvard K-12 in Central Nervous System tumors (grant 5K12 CA 90354-18). V.L and M.W.K. were supported by the National Institutes of Health (grants R01 HD073104 and RO1 HD091846 to M.W.K.).

ASJC Scopus subject areas

Biotechnology
Bioengineering
Applied Microbiology and Biotechnology
Molecular Medicine
Biomedical Engineering

UN SDGs

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10.1038/s41587-020-00806-2

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Prensner, J. R., Enache, O. M., Luria, V., Krug, K., Clauser, K. R., Dempster, J. M., Karger, A., Wang, L., Stumbraite, K., Wang, V. M., Botta, G., Lyons, N. J., Goodale, A., Kalani, Z., Fritchman, B., Brown, A., Alan, D., Green, T., Yang, X., ... Golub, T. R. (2021). Noncanonical open reading frames encode functional proteins essential for cancer cell survival. Nature biotechnology, 39(6), 697-704. https://doi.org/10.1038/s41587-020-00806-2

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title = "Noncanonical open reading frames encode functional proteins essential for cancer cell survival",

abstract = "Although genomic analyses predict many noncanonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442—renamed glycine-rich extracellular protein-1 (GREP1)—encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.",

author = "Prensner, {John R.} and Enache, {Oana M.} and Victor Luria and Karsten Krug and Clauser, {Karl R.} and Dempster, {Joshua M.} and Amir Karger and Li Wang and Karolina Stumbraite and Wang, {Vickie M.} and Ginevra Botta and Lyons, {Nicholas J.} and Amy Goodale and Zohra Kalani and Briana Fritchman and Adam Brown and Douglas Alan and Thomas Green and Xiaoping Yang and Jaffe, {Jacob D.} and Roth, {Jennifer A.} and Federica Piccioni and Kirschner, {Marc W.} and Zhe Ji and Root, {David E.} and Golub, {Todd R.}",

note = "Publisher Copyright: {\textcopyright} 2021, The Author(s), under exclusive licence to Springer Nature America, Inc.",

year = "2021",

month = jun,

doi = "10.1038/s41587-020-00806-2",

language = "English (US)",

volume = "39",

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Prensner, JR, Enache, OM, Luria, V, Krug, K, Clauser, KR, Dempster, JM, Karger, A, Wang, L, Stumbraite, K, Wang, VM, Botta, G, Lyons, NJ, Goodale, A, Kalani, Z, Fritchman, B, Brown, A, Alan, D, Green, T, Yang, X, Jaffe, JD, Roth, JA, Piccioni, F, Kirschner, MW, Ji, Z, Root, DE & Golub, TR 2021, 'Noncanonical open reading frames encode functional proteins essential for cancer cell survival', Nature biotechnology, vol. 39, no. 6, pp. 697-704. https://doi.org/10.1038/s41587-020-00806-2

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AU - Prensner, John R.

AU - Enache, Oana M.

AU - Luria, Victor

AU - Krug, Karsten

AU - Clauser, Karl R.

AU - Dempster, Joshua M.

AU - Karger, Amir

AU - Wang, Li

AU - Stumbraite, Karolina

AU - Wang, Vickie M.

AU - Botta, Ginevra

AU - Lyons, Nicholas J.

AU - Goodale, Amy

AU - Kalani, Zohra

AU - Fritchman, Briana

AU - Brown, Adam

AU - Alan, Douglas

AU - Green, Thomas

AU - Yang, Xiaoping

AU - Jaffe, Jacob D.

AU - Roth, Jennifer A.

AU - Piccioni, Federica

AU - Kirschner, Marc W.

AU - Ji, Zhe

AU - Root, David E.

AU - Golub, Todd R.

PY - 2021/6

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N2 - Although genomic analyses predict many noncanonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442—renamed glycine-rich extracellular protein-1 (GREP1)—encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.

AB - Although genomic analyses predict many noncanonical open reading frames (ORFs) in the human genome, it is unclear whether they encode biologically active proteins. Here we experimentally interrogated 553 candidates selected from noncanonical ORF datasets. Of these, 57 induced viability defects when knocked out in human cancer cell lines. Following ectopic expression, 257 showed evidence of protein expression and 401 induced gene expression changes. Clustered regularly interspaced short palindromic repeat (CRISPR) tiling and start codon mutagenesis indicated that their biological effects required translation as opposed to RNA-mediated effects. We found that one of these ORFs, G029442—renamed glycine-rich extracellular protein-1 (GREP1)—encodes a secreted protein highly expressed in breast cancer, and its knockout in 263 cancer cell lines showed preferential essentiality in breast cancer-derived lines. The secretome of GREP1-expressing cells has an increased abundance of the oncogenic cytokine GDF15, and GDF15 supplementation mitigated the growth-inhibitory effect of GREP1 knockout. Our experiments suggest that noncanonical ORFs can express biologically active proteins that are potential therapeutic targets.

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Noncanonical open reading frames encode functional proteins essential for cancer cell survival

Abstract

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