POLθ processes ssDNA gaps and promotes replication fork progression in BRCA1-deficient cells

Schrempf, Anna; Bernardo, Sara; Verge, Emili A Arasa; Otero, Miguel A Ramirez; Wilson, Jordan; Kirchhofer, Dominik; Timelthaler, Gerald; Ambros, Anna M; Kaya, Atilla; Wieder, Marcus; Ecker, Gerhard; Winter, Georg E.; Costanzo, Vincenzo; Loizou, Joanna I.

doi:10.1016/j.celrep.2022.111716

Cited by 46 publications

(36 citation statements)

References 42 publications

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“…It is also plausible that a RHINO-TOPBP1 interaction in mitosis could stabilize RHINO at break sites. Finally, our data do not rule out the possibility of additional roles for Polθ in filling ssDNA gaps accumulating during S phase and persisting through mitosis (Belan et al , 2022; Mann et al , 2022; Roerink et al , 2014; Schrempf et al , 2022). In conclusion, our study provides evidence suggesting robust MMEJ activity in mitosis that may account for the synthetic lethal interaction between Polθ and BRCA2.…”

Section: Discussioncontrasting

confidence: 79%

RHINO restricts MMEJ activity to mitosis

Sfeir

Brambati

Sacco

et al. 2023

Preprint

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DNA double-strand breaks (DSBs) are toxic lesions that can lead to genome instability if not properly repaired. Breaks incurred in G1 phase of the cell cycle are predominantly fixed by non-homologous end-joining (NHEJ), while homologous recombination (HR) is the primary repair pathway in S and G2. Microhomology-mediated end-joining (MMEJ) is intrinsically error-prone and considered a backup DSB repair pathway that becomes essential when HR and NHEJ are compromised. In this study, we uncover MMEJ as the major DSB repair pathway in M phase. Using CRISPR/Cas9-based synthetic lethal screens, we identify subunits of the 9-1-1 complex (RAD9A-HUS1-RAD1) and its interacting partner, RHINO, as critical MMEJ factors. Mechanistically, we show that the function of 9-1-1 and RHINO in MMEJ is inconsistent with their well-established role in ATR signaling. Instead, RHINO plays an unexpected and essential role in directing mutagenic repair to M phase by directly binding to Polymerase theta and promoting its recruitment to DSBs in mitosis. In addition, we provide evidence that mitotic MMEJ repairs persistent DNA damage that originates in S phase but is not repaired by HR. The latter findings could explain the synthetic lethal relationship between POLQ and BRCA1/2 and the synergistic effect of PolQ and PARP inhibitors. In summary, our study identifies MMEJ as the primary pathway for repairing DSBs during mitosis and highlights an unanticipated role for RHINO in directing mutagenic repair to M phase.

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Section: Discussioncontrasting

confidence: 79%

RHINO restricts MMEJ activity to mitosis

Sfeir

Brambati

Sacco

et al. 2023

Preprint

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“…We believe this provides an opportunity to target both pathways simultaneously to improve tumour eradication and reduce risk of resistance to either therapy individually. Next to a role in DSB repair, POLQ has recently been described to be involved in ssDNA gap fill-in (Belan et al, 2022; Mann et al, 2022; Schrempf et al, 2022). However, it is currently unclear how the two different roles of POLQ contribute to its essential role in BRCA-deficient cells.…”

Section: Discussionmentioning

confidence: 99%

“…This implies that DSB repair by alt-EJ or SSA is essential for survival of HR-deficient cells (Ceccaldi et al, 2015; Feng et al, 2011; Lok et al, 2013; Mateos-Gomez et al, 2015). However, recent studies suggested that increased ssDNA gap formation in absence of PolQ may underlie the synthetic lethality with BRCA-deficiency (Belan et al, 2022; Mann et al, 2022; Schrempf et al, 2022). BRCA-deficient cells are prone to accumulate ssDNA gaps, and toxic levels of these lesion are associated with sensitivity to chemotherapy and PARPi (Cong et al, 2021; Paes Dias et al, 2021; Panzarino et al, 2021).…”

Section: Introductionmentioning

confidence: 99%

EXO1-mediated DNA repair by single-strand annealing is essential for BRCA1-deficient cells

Kooij

Schreuder

Pavani

et al. 2023

Preprint

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Deficiency for the repair of DNA double-strand breaks (DSBs) via homologous recombination (HR) leads to chromosomal instability and diseases such as cancer. Yet, defective HR also results in vulnerabilities that can be exploited for targeted therapy. Here, we identify such a vulnerability and show that BRCA1-deficient cells are dependent on the long-range end-resection factor EXO1 for survival. EXO1 loss results in DNA replication-induced lesions decorated by poly(ADP-ribose)-chains. In cells that lack both BRCA1 and EXO1, this is accompanied by unresolved DSBs due to impaired single-strand annealing (SSA), a DSB repair process that requires the activity of both proteins. In contrast, BRCA2-deficient cells have increased SSA, also in the absence of EXO1, and hence are not dependent on EXO1 for survival. In agreement with our mechanistic data, BRCA1-mutated tumours have elevated EXO1 expression and contain more genomic signatures of SSA compared to BRCA1-proficient tumours. Collectively, our data indicate that EXO1 is a promising novel target for treatment of BRCA1-deficient tumours.

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“…In the first study, Loizou and co-workers investigated if ssDNA gaps, which are considered to be the major lesions that drive cell death in BRCA1/2-deficient cells treated with PARPi or cisplatin, contributes to the genetic interaction between BRCA1/2 and POLθ . The authors demonstrated that loss of POLθ activity in BRCA1-deficient cells exposes ssDNA, which causes replication stress and deregulation of S-phase progression.…”

Section: Small Molecule Polθ Inhibitorsmentioning

confidence: 99%

Small Molecules Targeting DNA Polymerase Theta (POLθ) as Promising Synthetic Lethal Agents for Precision Cancer Therapy

et al. 2023

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Synthetic lethality (SL) is an innovative strategy in targeted anticancer therapy that exploits tumor genetic vulnerabilities. This topic has come to the forefront in recent years, as witnessed by the increased number of publications since 2007. The first proof of concept for the effectiveness of SL was provided by the approval of poly(ADP-ribose)polymerase inhibitors, which exploit a SL interaction in BRCA-deficient cells, although their use is limited by resistance. Searching for additional SL interactions involving BRCA mutations, the DNA polymerase theta (POLθ) emerged as an exciting target. This review summarizes, for the first time, the POLθ polymerase and helicase inhibitors reported to date. Compounds are described focusing on chemical structure and biological activity. With the aim to enable further drug discovery efforts in interrogating POLθ as a target, we propose a plausible pharmacophore model for POLθ-pol inhibitors and provide a structural analysis of the known POLθ ligand binding sites.

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POLθ processes ssDNA gaps and promotes replication fork progression in BRCA1-deficient cells

Cited by 46 publications

References 42 publications

RHINO restricts MMEJ activity to mitosis

RHINO restricts MMEJ activity to mitosis

EXO1-mediated DNA repair by single-strand annealing is essential for BRCA1-deficient cells

Small Molecules Targeting DNA Polymerase Theta (POLθ) as Promising Synthetic Lethal Agents for Precision Cancer Therapy

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