BRCA1 secondary splice-site mutations drive exon-skipping and PARP inhibitor resistance

Australian Ovarian Cancer Study

doi:10.1186/s12943-024-02048-1

BRCA1 secondary splice-site mutations drive exon-skipping and PARP inhibitor resistance

Australian Ovarian Cancer Study

Walter & Eliza Hall Institute of Medical Research (WEHI)
University of Melbourne
Fox Chase Cancer Center
Washington University School of Medicine in St. Louis
Clovis Oncology
Monash University
GSTT Guy's and St Thomas' NHS Foundation Trust

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Abstract

PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms △11 and △11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood.We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials.Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene's reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations.

Original language	English
Article number	158
Journal	Molecular Cancer
Volume	23
DOIs	https://doi.org/10.1186/s12943-024-02048-1
Publication status	Published - 5 Aug 2024

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

SDG 3 Good Health and Well-being

Keywords

Humans
Poly(ADP-ribose) Polymerase Inhibitors/pharmacology
Exons
Drug Resistance, Neoplasm/genetics
BRCA1 Protein/genetics
Female
Animals
Mice
Ovarian Neoplasms/genetics
RNA Splice Sites
Mutation
Breast Neoplasms/genetics
Xenograft Model Antitumor Assays
Cell Line, Tumor

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10.1186/s12943-024-02048-1Licence: CC BY-NC-ND

BRCA1 secondary splice_Version of RecordFinal published version, 2.42 MBLicence: CC BY

Cite this

@article{00b45680b41c4b849377e010067fd962,

title = "BRCA1 secondary splice-site mutations drive exon-skipping and PARP inhibitor resistance",

abstract = "PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms △11 and △11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood.We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials.Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene's reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations.",

keywords = "Humans, Poly(ADP-ribose) Polymerase Inhibitors/pharmacology, Exons, Drug Resistance, Neoplasm/genetics, BRCA1 Protein/genetics, Female, Animals, Mice, Ovarian Neoplasms/genetics, RNA Splice Sites, Mutation, Breast Neoplasms/genetics, Xenograft Model Antitumor Assays, Cell Line, Tumor",

author = "Ksenija Nesic and Krais, \{John J.\} and Yifan Wang and Vandenberg, \{Cassandra J.\} and Pooja Patel and Cai, \{Kathy Q.\} and Tanya Kwan and Elizabeth Lieschke and Ho, \{Gwo Yaw\} and Barker, \{Holly E.\} and Justin Bedo and Rebecca Kristeleit and M. Buck and R. Bell and M. Quinn and P. Waring and D. Johnson and S. Fox and K. Phillips and P. Rogers and D. Allen and A. Parker and R. McIntosh and J. Miller and C. Hall and R. Sharma and J. Hill and P. Russell and J. Carter and G. Robertson and L. Edwards and B. Young and D. Bell and J. Shannon and J. Brooks and C. Young and C. Ball and H. Shirley and T. Schmidt and L. Bowes and J. White and K. Ferguson and L. Green and S. Brown and B. Alexander and M. Jones and R. Robertson and S. Moore and P. Webb and A. Green and \{Australian Ovarian Cancer Study\}",

note = "Publisher Copyright: {\textcopyright} The Author(s) 2024.",

year = "2024",

month = aug,

day = "5",

doi = "10.1186/s12943-024-02048-1",

language = "English",

volume = "23",

journal = "Molecular Cancer",

issn = "1476-4598",

publisher = "BioMed Central Ltd.",

}

TY - JOUR

T1 - BRCA1 secondary splice-site mutations drive exon-skipping and PARP inhibitor resistance

AU - Nesic, Ksenija

AU - Krais, John J.

AU - Wang, Yifan

AU - Vandenberg, Cassandra J.

AU - Patel, Pooja

AU - Cai, Kathy Q.

AU - Kwan, Tanya

AU - Lieschke, Elizabeth

AU - Ho, Gwo Yaw

AU - Barker, Holly E.

AU - Bedo, Justin

AU - Kristeleit, Rebecca

AU - Buck, M.

AU - Bell, R.

AU - Quinn, M.

AU - Waring, P.

AU - Johnson, D.

AU - Fox, S.

AU - Phillips, K.

AU - Rogers, P.

AU - Allen, D.

AU - Parker, A.

AU - McIntosh, R.

AU - Miller, J.

AU - Hall, C.

AU - Sharma, R.

AU - Hill, J.

AU - Russell, P.

AU - Carter, J.

AU - Robertson, G.

AU - Edwards, L.

AU - Young, B.

AU - Bell, D.

AU - Shannon, J.

AU - Brooks, J.

AU - Young, C.

AU - Ball, C.

AU - Shirley, H.

AU - Schmidt, T.

AU - Bowes, L.

AU - White, J.

AU - Ferguson, K.

AU - Green, L.

AU - Brown, S.

AU - Alexander, B.

AU - Jones, M.

AU - Robertson, R.

AU - Moore, S.

AU - Webb, P.

AU - Green, A.

AU - Australian Ovarian Cancer Study

PY - 2024/8/5

Y1 - 2024/8/5

N2 - PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms △11 and △11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood.We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials.Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene's reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations.

AB - PARP inhibitor (PARPi) therapy has transformed outcomes for patients with homologous recombination DNA repair (HRR) deficient ovarian cancers, for example those with BRCA1 or BRCA2 gene defects. Unfortunately, PARPi resistance is common. Multiple resistance mechanisms have been described, including secondary mutations that restore the HR gene reading frame. BRCA1 splice isoforms △11 and △11q can contribute to PARPi resistance by splicing out the mutation-containing exon, producing truncated, partially functional proteins. However, the clinical impacts and underlying drivers of BRCA1 exon skipping are not fully understood.We analyzed nine ovarian and breast cancer patient derived xenografts (PDX) with BRCA1 exon 11 frameshift mutations for exon skipping and therapy response, including a matched PDX pair derived from a patient pre- and post-chemotherapy/PARPi. BRCA1 exon 11 skipping was elevated in PARPi resistant PDX tumors. Two independent PDX models acquired secondary BRCA1 splice site mutations (SSMs) that drive exon skipping, confirmed using qRT-PCR, RNA sequencing, immunoblotting and minigene modelling. CRISPR/Cas9-mediated disruption of splicing functionally validated exon skipping as a mechanism of PARPi resistance. SSMs were also enriched in post-PARPi ovarian cancer patient cohorts from the ARIEL2 and ARIEL4 clinical trials.Few PARPi resistance mechanisms have been confirmed in the clinical setting. While secondary/reversion mutations typically restore a gene's reading frame, we have identified secondary mutations in patient cohorts that hijack splice sites to enhance mutation-containing exon skipping, resulting in the overexpression of BRCA1 hypomorphs, which in turn promote PARPi resistance. Thus, BRCA1 SSMs can and should be clinically monitored, along with frame-restoring secondary mutations.

KW - Humans

KW - Poly(ADP-ribose) Polymerase Inhibitors/pharmacology

KW - Exons

KW - Drug Resistance, Neoplasm/genetics

KW - BRCA1 Protein/genetics

KW - Female

KW - Animals

KW - Mice

KW - Ovarian Neoplasms/genetics

KW - RNA Splice Sites

KW - Mutation

KW - Breast Neoplasms/genetics

KW - Xenograft Model Antitumor Assays

KW - Cell Line, Tumor

UR - https://www.scopus.com/pages/publications/85200712131

U2 - 10.1186/s12943-024-02048-1

DO - 10.1186/s12943-024-02048-1

M3 - Article

C2 - 39103848

SN - 1476-4598

VL - 23

JO - Molecular Cancer

JF - Molecular Cancer

M1 - 158

ER -

BRCA1 secondary splice-site mutations drive exon-skipping and PARP inhibitor resistance

Abstract

UN SDGs

Keywords

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