Mechanisms of synthetic lethality between BRCA1/2 and 53BP1 deficiencies and DNA polymerase theta targeting

George E. Ronson; Katarzyna Starowicz; Elizabeth J. Anthony; Ann Liza Piberger; Lucy C. Clarke; Alexander J. Garvin; Andrew D. Beggs; Celina M. Whalley; Matthew J. Edmonds; James F. J. Beesley; Joanna R. Morris

doi:10.1038/s41467-023-43677-2

Mechanisms of synthetic lethality between BRCA1/2 and 53BP1 deficiencies and DNA polymerase theta targeting

George E. Ronson, Katarzyna Starowicz, Elizabeth J. Anthony, Ann Liza Piberger, Lucy C. Clarke, Alexander J. Garvin, Andrew D. Beggs, Celina M. Whalley, Matthew J. Edmonds, James F. J. Beesley, Joanna R. Morris^*

^*Corresponding author for this work

Cancer and Genomic Sciences

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Abstract

A synthetic lethal relationship exists between disruption of polymerase theta (Polθ), and loss of either 53BP1 or homologous recombination (HR) proteins, including BRCA1; however, the mechanistic basis of these observations are unclear. Here we reveal two distinct mechanisms of Polθ synthetic lethality, identifying dual influences of 1) whether Polθ is lost or inhibited, and 2) the underlying susceptible genotype. Firstly, we find that the sensitivity of BRCA1/2- and 53BP1-deficient cells to Polθ loss, and 53BP1-deficient cells to Polθ inhibition (ART558) requires RAD52, and appropriate reduction of RAD52 can ameliorate these phenotypes. We show that in the absence of Polθ, RAD52 accumulations suppress ssDNA gap-filling in G2/M and encourage MRE11 nuclease accumulation. In contrast, the survival of BRCA1-deficient cells treated with Polθ inhibitor are not restored by RAD52 suppression, and ssDNA gap-filling is prevented by the chemically inhibited polymerase itself. These data define an additional role for Polθ, reveal the mechanism underlying synthetic lethality between 53BP1, BRCA1/2 and Polθ loss, and indicate genotype-dependent Polθ inhibitor mechanisms.

Original language	English
Article number	7834
Number of pages	16
Journal	Nature Communications
Volume	14
Issue number	1
DOIs	https://doi.org/10.1038/s41467-023-43677-2
Publication status	Published - 29 Nov 2023

Bibliographical note

Acknowledgements
ART558 was a kind gift of Graeme Smith Artios Pharma Ltd. Cambridge, U.K. SV40 1: pBSSVD2005 was a gift from David Ron (Addgene plasmid # 21826; http://n2t.net/addgene:21826; RRID:Addgene_21826). Anti murine BARD1 antibody was a gift from Richard Baer (Columbia). We thank Claudia Lukas (University of Copenhagen) for the RAD52 antibody. 53BP1 animals were a gift from Andre Nussenzweig, NIH (National Institutes of Health), Bethesda. G.R., J.B. and K.S. are funded by Cancer research, UK (C8820/A28283), M.E. and K.S. by Breast Cancer Now, UK (2015MayPR499), E.A. by the Midlands Integrative Biosciences Training Partnership, funded by the Biotechnology and Biological Sciences Research Council, UK. A.J.G. by the Wellcome Trust (206343/Z/17/Z), A.B. and C.W. by Cancer Research UK (C31641/A23923). We thank the microscopy and Imaging services (MISBU) at the University of Birmingham Technology Hub for their assistance and maintenance of equipment. We sincerely apologise to colleagues whose excellent and relevant work was not referenced due to space restrictions.

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10.1038/s41467-023-43677-2Licence: Creative Commons: Attribution (CC BY)

RonsonG2023MechanismsFinal published version, 4.62 MBLicence: Creative Commons: Attribution (CC BY)

1 Active
2 Finished

BRCA1 pathways in cancer predisposition and treatment.
Morris, J. (Principal Investigator), Beesley, J. (Co-Investigator), Starowicz, K. (Co-Investigator), Ronson, G. (Co-Investigator) & Mackay, H. (Co-Investigator)
Cancer Research Uk
1/01/20 → 31/12/25
Project: Research
Regulation of SUMO in DNA double-strand break repair.
Morris, J. (Principal Investigator)
The Wellcome Trust
11/12/17 → 11/01/23
Project: Research
Identifciation of the Second Tumour Suppressor Function of Brca1
Petermann, E. (Co-Investigator) & Morris, J. (Principal Investigator)
BREAST CANCER NOW
1/02/16 → 19/12/19
Project: Research

Cite this

Ronson, G. E., Starowicz, K., Anthony, E. J., Piberger, A. L., Clarke, L. C., Garvin, A. J., Beggs, A. D., Whalley, C. M., Edmonds, M. J., Beesley, J. F. J., & Morris, J. R. (2023). Mechanisms of synthetic lethality between BRCA1/2 and 53BP1 deficiencies and DNA polymerase theta targeting. Nature Communications, 14(1), Article 7834. https://doi.org/10.1038/s41467-023-43677-2

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title = "Mechanisms of synthetic lethality between BRCA1/2 and 53BP1 deficiencies and DNA polymerase theta targeting",

abstract = "A synthetic lethal relationship exists between disruption of polymerase theta (Polθ), and loss of either 53BP1 or homologous recombination (HR) proteins, including BRCA1; however, the mechanistic basis of these observations are unclear. Here we reveal two distinct mechanisms of Polθ synthetic lethality, identifying dual influences of 1) whether Polθ is lost or inhibited, and 2) the underlying susceptible genotype. Firstly, we find that the sensitivity of BRCA1/2- and 53BP1-deficient cells to Polθ loss, and 53BP1-deficient cells to Polθ inhibition (ART558) requires RAD52, and appropriate reduction of RAD52 can ameliorate these phenotypes. We show that in the absence of Polθ, RAD52 accumulations suppress ssDNA gap-filling in G2/M and encourage MRE11 nuclease accumulation. In contrast, the survival of BRCA1-deficient cells treated with Polθ inhibitor are not restored by RAD52 suppression, and ssDNA gap-filling is prevented by the chemically inhibited polymerase itself. These data define an additional role for Polθ, reveal the mechanism underlying synthetic lethality between 53BP1, BRCA1/2 and Polθ loss, and indicate genotype-dependent Polθ inhibitor mechanisms.",

author = "Ronson, {George E.} and Katarzyna Starowicz and Anthony, {Elizabeth J.} and Piberger, {Ann Liza} and Clarke, {Lucy C.} and Garvin, {Alexander J.} and Beggs, {Andrew D.} and Whalley, {Celina M.} and Edmonds, {Matthew J.} and Beesley, {James F. J.} and Morris, {Joanna R.}",

note = "Acknowledgements ART558 was a kind gift of Graeme Smith Artios Pharma Ltd. Cambridge, U.K. SV40 1: pBSSVD2005 was a gift from David Ron (Addgene plasmid # 21826; http://n2t.net/addgene:21826; RRID:Addgene_21826). Anti murine BARD1 antibody was a gift from Richard Baer (Columbia). We thank Claudia Lukas (University of Copenhagen) for the RAD52 antibody. 53BP1 animals were a gift from Andre Nussenzweig, NIH (National Institutes of Health), Bethesda. G.R., J.B. and K.S. are funded by Cancer research, UK (C8820/A28283), M.E. and K.S. by Breast Cancer Now, UK (2015MayPR499), E.A. by the Midlands Integrative Biosciences Training Partnership, funded by the Biotechnology and Biological Sciences Research Council, UK. A.J.G. by the Wellcome Trust (206343/Z/17/Z), A.B. and C.W. by Cancer Research UK (C31641/A23923). We thank the microscopy and Imaging services (MISBU) at the University of Birmingham Technology Hub for their assistance and maintenance of equipment. We sincerely apologise to colleagues whose excellent and relevant work was not referenced due to space restrictions.",

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T1 - Mechanisms of synthetic lethality between BRCA1/2 and 53BP1 deficiencies and DNA polymerase theta targeting

AU - Ronson, George E.

AU - Starowicz, Katarzyna

AU - Anthony, Elizabeth J.

AU - Piberger, Ann Liza

AU - Clarke, Lucy C.

AU - Garvin, Alexander J.

AU - Beggs, Andrew D.

AU - Whalley, Celina M.

AU - Edmonds, Matthew J.

AU - Beesley, James F. J.

AU - Morris, Joanna R.

N1 - Acknowledgements ART558 was a kind gift of Graeme Smith Artios Pharma Ltd. Cambridge, U.K. SV40 1: pBSSVD2005 was a gift from David Ron (Addgene plasmid # 21826; http://n2t.net/addgene:21826; RRID:Addgene_21826). Anti murine BARD1 antibody was a gift from Richard Baer (Columbia). We thank Claudia Lukas (University of Copenhagen) for the RAD52 antibody. 53BP1 animals were a gift from Andre Nussenzweig, NIH (National Institutes of Health), Bethesda. G.R., J.B. and K.S. are funded by Cancer research, UK (C8820/A28283), M.E. and K.S. by Breast Cancer Now, UK (2015MayPR499), E.A. by the Midlands Integrative Biosciences Training Partnership, funded by the Biotechnology and Biological Sciences Research Council, UK. A.J.G. by the Wellcome Trust (206343/Z/17/Z), A.B. and C.W. by Cancer Research UK (C31641/A23923). We thank the microscopy and Imaging services (MISBU) at the University of Birmingham Technology Hub for their assistance and maintenance of equipment. We sincerely apologise to colleagues whose excellent and relevant work was not referenced due to space restrictions.

PY - 2023/11/29

Y1 - 2023/11/29

N2 - A synthetic lethal relationship exists between disruption of polymerase theta (Polθ), and loss of either 53BP1 or homologous recombination (HR) proteins, including BRCA1; however, the mechanistic basis of these observations are unclear. Here we reveal two distinct mechanisms of Polθ synthetic lethality, identifying dual influences of 1) whether Polθ is lost or inhibited, and 2) the underlying susceptible genotype. Firstly, we find that the sensitivity of BRCA1/2- and 53BP1-deficient cells to Polθ loss, and 53BP1-deficient cells to Polθ inhibition (ART558) requires RAD52, and appropriate reduction of RAD52 can ameliorate these phenotypes. We show that in the absence of Polθ, RAD52 accumulations suppress ssDNA gap-filling in G2/M and encourage MRE11 nuclease accumulation. In contrast, the survival of BRCA1-deficient cells treated with Polθ inhibitor are not restored by RAD52 suppression, and ssDNA gap-filling is prevented by the chemically inhibited polymerase itself. These data define an additional role for Polθ, reveal the mechanism underlying synthetic lethality between 53BP1, BRCA1/2 and Polθ loss, and indicate genotype-dependent Polθ inhibitor mechanisms.

AB - A synthetic lethal relationship exists between disruption of polymerase theta (Polθ), and loss of either 53BP1 or homologous recombination (HR) proteins, including BRCA1; however, the mechanistic basis of these observations are unclear. Here we reveal two distinct mechanisms of Polθ synthetic lethality, identifying dual influences of 1) whether Polθ is lost or inhibited, and 2) the underlying susceptible genotype. Firstly, we find that the sensitivity of BRCA1/2- and 53BP1-deficient cells to Polθ loss, and 53BP1-deficient cells to Polθ inhibition (ART558) requires RAD52, and appropriate reduction of RAD52 can ameliorate these phenotypes. We show that in the absence of Polθ, RAD52 accumulations suppress ssDNA gap-filling in G2/M and encourage MRE11 nuclease accumulation. In contrast, the survival of BRCA1-deficient cells treated with Polθ inhibitor are not restored by RAD52 suppression, and ssDNA gap-filling is prevented by the chemically inhibited polymerase itself. These data define an additional role for Polθ, reveal the mechanism underlying synthetic lethality between 53BP1, BRCA1/2 and Polθ loss, and indicate genotype-dependent Polθ inhibitor mechanisms.

U2 - 10.1038/s41467-023-43677-2

DO - 10.1038/s41467-023-43677-2

M3 - Article

C2 - 38030626

SN - 2041-1723

VL - 14

JO - Nature Communications

JF - Nature Communications

IS - 1

M1 - 7834

ER -

Mechanisms of synthetic lethality between BRCA1/2 and 53BP1 deficiencies and DNA polymerase theta targeting

Abstract

Bibliographical note

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Projects

BRCA1 pathways in cancer predisposition and treatment.

Regulation of SUMO in DNA double-strand break repair.

Identifciation of the Second Tumour Suppressor Function of Brca1

Cite this