RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells

Rehna Krishnan; Mariah Lapierre; Brandon Gautreau; Kevin C.J. Nixon; Samah El Ghamrasni; Parasvi S. Patel; Jun Hao; V. Talya Yerlici; Kiran Kumar Naidu Guturi; Jonathan St-Germain; Francesca Mateo; Amine Saad; Arash Algouneh; Rebecca Earnshaw; Duan Shili; Alma Seitova; Joshua Miller; Negin Khosraviani; Adam Penn; Brandon Ho; Otto Sanchez; M. Prakash Hande; Jean Yves Masson; Grant W. Brown; Moulay Alaoui-Jamali; John J. Reynolds; Cheryl Arrowsmith; Brian Raught; Miguel A. Pujana; Karim Mekhail; Grant S. Stewart; Anne Hakem; Razqallah Hakem

doi:10.1093/nar/gkad733

RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells

Rehna Krishnan, Mariah Lapierre, Brandon Gautreau, Kevin C.J. Nixon, Samah El Ghamrasni, Parasvi S. Patel, Jun Hao, V. Talya Yerlici, Kiran Kumar Naidu Guturi, Jonathan St-Germain, Francesca Mateo, Amine Saad, Arash Algouneh, Rebecca Earnshaw, Duan Shili, Alma Seitova, Joshua Miller, Negin Khosraviani, Adam Penn, Brandon HoOtto Sanchez, M. Prakash Hande, Jean Yves Masson, Grant W. Brown, Moulay Alaoui-Jamali, John J. Reynolds, Cheryl Arrowsmith, Brian Raught, Miguel A. Pujana, Karim Mekhail, Grant S. Stewart, Anne Hakem^*, Razqallah Hakem^*

^*Corresponding author for this work

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Abstract

Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.

Original language	English
Pages (from-to)	10484-10505
Number of pages	22
Journal	Nucleic Acids Research
Volume	51
Issue number	19
Early online date	11 Sept 2023
DOIs	https://doi.org/10.1093/nar/gkad733
Publication status	Published - 27 Oct 2023

Bibliographical note

Copyright © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License
(https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]

Data Access Statement

The data underlying this article are available in GEO at https://www.ncbi.nlm.nih.gov/geo/, and can be accessed with accession number GSE202723.

Funding

Funding: Canadian Institute of Health Research [FDN-143214 to R.H., FDN-159913 to G.W.B., FDN-388879 to J.Y.M., CIHR-399687 to K.M., MOP119289 to B.R.]; Canadian Cancer Society [705367, 706439 to R.H.]; Worldwide Cancer Research [110215 to R.H.]; Carlos III Institute of Health [PI18/01029 to M.A.P.]; Generalitat de Catalunya SGR [2017-449 to M.A.P.]; CERCA Program (to M.A.P.); Quebec Breast Cancer Foundation (to M.A-J.); Cancer Research UK programme [C17183/A23303 to G.S.S.]; R.K. is supported by the Princess Margaret Cancer Foundation, the Hold’Em for Life Foundation, and the Strategic Training in Transdisciplinary Radiation Science for the 21st Century (STARS21); J.H. is supported by the Princess Margaret Cancer Foundation and CIHR fellowship; J.J.R. is supported by the University of Birmingham; Canada Graduate Scholarships, Ontario Graduate Scholarship (to M.L., A.A. and P.S.P.); STARS21, Terry Fox Foundation, Princess Margaret Cancer Foundation (to P.S.P.); B.H. was supported by an Alexander Graham Bell Canada Graduate Scholarship from the Natural Sciences and Engi- neering Research Council of Canada; C.H. is supported by the Structural Genomics Consortium; S.G.S. is a registered charity [1097737] that receives funds from Bayer AG, Boehringer Ingelheim, Bristol Myers Squibb, Genentech, Genome Canada through Ontario Genomics Institute [OGI-196]; EU/EFPIA/OICR/McGill/KTH/Diamond Innovative Medicines Initiative 2 Joint Undertaking [EU-bOPEN grant 875510]; Janssen, Merck KGaA (aka EMD in Canada and US), Pfizer and Takeda. Funding for open access charge: CIHR.

Funders	Funder number
Roche SpA

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10.1093/nar/gkad733

R Krishnan et al_RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells
Copyright © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]
Final published version, 2.22 MBLicence: CC BY-NC 4.0

Cite this

Krishnan, R., Lapierre, M., Gautreau, B., Nixon, K. C. J., El Ghamrasni, S., Patel, P. S., Hao, J., Yerlici, V. T., Guturi, K. K. N., St-Germain, J., Mateo, F., Saad, A., Algouneh, A., Earnshaw, R., Shili, D., Seitova, A., Miller, J., Khosraviani, N., Penn, A., ... Hakem, R. (2023). RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells. Nucleic Acids Research, 51(19), 10484-10505. https://doi.org/10.1093/nar/gkad733

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title = "RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells",

abstract = "Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.",

author = "Rehna Krishnan and Mariah Lapierre and Brandon Gautreau and Nixon, {Kevin C.J.} and {El Ghamrasni}, Samah and Patel, {Parasvi S.} and Jun Hao and Yerlici, {V. Talya} and Guturi, {Kiran Kumar Naidu} and Jonathan St-Germain and Francesca Mateo and Amine Saad and Arash Algouneh and Rebecca Earnshaw and Duan Shili and Alma Seitova and Joshua Miller and Negin Khosraviani and Adam Penn and Brandon Ho and Otto Sanchez and Hande, {M. Prakash} and Masson, {Jean Yves} and Brown, {Grant W.} and Moulay Alaoui-Jamali and Reynolds, {John J.} and Cheryl Arrowsmith and Brian Raught and Pujana, {Miguel A.} and Karim Mekhail and Stewart, {Grant S.} and Anne Hakem and Razqallah Hakem",

note = "Copyright {\textcopyright} The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]",

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month = oct,

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doi = "10.1093/nar/gkad733",

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Krishnan, R, Lapierre, M, Gautreau, B, Nixon, KCJ, El Ghamrasni, S, Patel, PS, Hao, J, Yerlici, VT, Guturi, KKN, St-Germain, J, Mateo, F, Saad, A, Algouneh, A, Earnshaw, R, Shili, D, Seitova, A, Miller, J, Khosraviani, N, Penn, A, Ho, B, Sanchez, O, Hande, MP, Masson, JY, Brown, GW, Alaoui-Jamali, M, Reynolds, JJ, Arrowsmith, C, Raught, B, Pujana, MA, Mekhail, K, Stewart, GS, Hakem, A & Hakem, R 2023, 'RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells', Nucleic Acids Research, vol. 51, no. 19, pp. 10484-10505. https://doi.org/10.1093/nar/gkad733

TY - JOUR

T1 - RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells

AU - Krishnan, Rehna

AU - Lapierre, Mariah

AU - Gautreau, Brandon

AU - Nixon, Kevin C.J.

AU - El Ghamrasni, Samah

AU - Patel, Parasvi S.

AU - Hao, Jun

AU - Yerlici, V. Talya

AU - Guturi, Kiran Kumar Naidu

AU - St-Germain, Jonathan

AU - Mateo, Francesca

AU - Saad, Amine

AU - Algouneh, Arash

AU - Earnshaw, Rebecca

AU - Shili, Duan

AU - Seitova, Alma

AU - Miller, Joshua

AU - Khosraviani, Negin

AU - Penn, Adam

AU - Ho, Brandon

AU - Sanchez, Otto

AU - Hande, M. Prakash

AU - Masson, Jean Yves

AU - Brown, Grant W.

AU - Alaoui-Jamali, Moulay

AU - Reynolds, John J.

AU - Arrowsmith, Cheryl

AU - Raught, Brian

AU - Pujana, Miguel A.

AU - Mekhail, Karim

AU - Stewart, Grant S.

AU - Hakem, Anne

AU - Hakem, Razqallah

N1 - Copyright © The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact [email protected]

PY - 2023/10/27

Y1 - 2023/10/27

N2 - Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.

AB - Breast cancer linked with BRCA1/2 mutations commonly recur and resist current therapies, including PARP inhibitors. Given the lack of effective targeted therapies for BRCA1-mutant cancers, we sought to identify novel targets to selectively kill these cancers. Here, we report that loss of RNF8 significantly protects Brca1-mutant mice against mammary tumorigenesis. RNF8 deficiency in human BRCA1-mutant breast cancer cells was found to promote R-loop accumulation and replication fork instability, leading to increased DNA damage, senescence, and synthetic lethality. Mechanistically, RNF8 interacts with XRN2, which is crucial for transcription termination and R-loop resolution. We report that RNF8 ubiquitylates XRN2 to facilitate its recruitment to R-loop-prone genomic loci and that RNF8 deficiency in BRCA1-mutant breast cancer cells decreases XRN2 occupancy at R-loop-prone sites, thereby promoting R-loop accumulation, transcription-replication collisions, excessive genomic instability, and cancer cell death. Collectively, our work identifies a synthetic lethal interaction between RNF8 and BRCA1, which is mediated by a pathological accumulation of R-loops.

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U2 - 10.1093/nar/gkad733

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JF - Nucleic Acids Research

IS - 19

ER -

RNF8 ubiquitylation of XRN2 facilitates R-loop resolution and restrains genomic instability in BRCA1 mutant cells

Abstract

Bibliographical note

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Funding

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