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

B. van de Kooij, A. Schreuder, R.S. Pavani, V. Garzero, A. Van Hoeck, M. San Martin Alonso, D. Koerse, T.J. Wendel, E. Callen, J. Boom, H. Mei, E. Cuppen, A. Nussenzweig, H. van Attikum, S.M. Noordermeer

Posted on: 15 May 2023 , updated on: 16 May 2023

Preprint posted on 24 February 2023

EXO1 to the rescue: How does EXO1 save BRCA1-deficient cells?

Selected by Jennifer Ann Black

Categories: molecular biology

Background:

Eukaryotic cells use specialised pathways to repair damaged DNA. Double Strand Breaks (DSBs) are amongst the most dangerous as they compromise both DNA strands. Single Strand Annealing (SSA) is one such pathway used to repair DNA DSBs. This pathway involves resecting (i.e., specifically degrading) sequences flanking the DSB until they form overhangs of single stranded DNA that are homologous in sequence. These two homologous sequences are joined together (‘annealed’) to repair the break.

Though SSA fixes DSBs, it is error prone and results in DNA deletions. BRCA1 is a key factor involved in a high fidelity DSB repair pathway called homologous recombination (HR), playing an important role in tumour suppression. However, BRCA1 may also act during SSA, though what functions BRCA1 may undertake during SSA are still unclear (1). In this preprint, the researchers uncover a relationship between BRCA1-deficiency and a factor involved in resection called EXO1. They show that BRCA1-deficient cells are unable to overcome damage if EXO1 is deleted, suggesting they rely on EXO1’s activities. Their data highlight EXO1 as a potentially important drug target in cancers that are BRCA1-deficient.

 

Key Findings:

1) In cells lacking BRCA1, long range-resection is important

In a previous screen, the authors found that long-range resection factors were important in BRCA1-deficient cells (2). They selected one of these candidates, the exonuclease EXO1, for further study. By deleting EXO1 in combination with auxin-induced degradation of endogenous BRCA1 tagged with an auxin-inducible degron, they could show that the loss of both factors heavily affected cell growth. This loss of viability was not apparent when BRCA2 was degraded in EXOI-deficient cells. These data suggest that EXO1 is essential when BRCA1 is deficient, but not BRCA2.

Figure shows a selection of figures from van de Kooij. et al., Figure 1E shows cell viability in the presence or absence of EXO1  when BRCA1 is either present or absent. Figure 2A&B show primary breast cancer tumour samples and the expression of EXO1 in these samples. Figure 6D shows data from an assay that can be used to examine the type of repair pathway choice following a DNA lesion. Figure 7D shows the levels of EXO1 expression across pan-cancer samples in which BRCA1 is mutated or WT. (Adapted from the preprint under a CC-BY 4.0 International License)

 

2) High EXO1 expression is associated with breast cancer in BRCA1-deficient cells

Using previously published breast cancer datasets, the authors could link increased EXO1 expression to potential defects in HR. HR uses homology sequences to repair a DSB with high fidelity and is less mutagenic than SSA. They concluded that: 1) BRCA1-deficient cells appear to rely on higher levels of EXO1, and 2) EXO1 likely has wider effects in tumours with defects in HR directed repair.

 

3) When EXO1 is depleted, BRCA1 cells are more unstable

To test if BRCA1 and EXO1 loss, or each factor independently, correlates with genome instability, the authors examined the effects of EXO1 deletion in the absence or presence of BRCA1 depletion. They looked for alterations to the chromosomes, changes to the nuclei of the cells, specifically looking for micronuclei; all of which indicate problems during chromosome segregation or suggest fragmentation of the DNA. They also examined the levels of yH2AX, a phosphorylation event that occurs on the variant histone H2AX (in vertebrates) as a result of genome stress.

When BRCA1 was depleted in the absence of EXO1, a higher number of chromatid aberrations were found suggesting problems during S-phase. This was supported by the observed increase in yH2AX signal in these cells.

 

4) Loss of EXO1 in BRCA1-deficient cells may lead to ssDNA gaps generated during DNA replication

To understand why BRCA1 is important when EXO1 is lost, the authors deleted a gene called 53BP1. When 53BP1 is deleted in BRCA1-deficient cells, HR is restored, and ssDNA gaps do not form (3-5). However, deletion of 53BP1 does not rescue another issue in BRCA1-deficient cells: replication fork instability (6).

When 53BP1 was deleted, EXO1-depleted BRCA1 KO cells survived. These findings suggest that loss of BRCA1 causes faulty HR or accumulating gaps of ssDNA. The authors then decided to look at the ssDNA gaps by testing for evidence of a post-translational modification called poly(ADP-ribosyl)ation or ‘PAR’, which decorates ssDNA gaps. When EXO1 was lost, but BRCA1 present, the cells accumulated these ssDNA gaps especially during S-phase, however when both EXO1 and BRCA1 were lost, the number of these gaps increased even more, again during S-phase. This suggests that EXO1 is important in preventing these gaps accumulating during S-phase. These lesions can be tolerated under certain conditions (i.e., in the presence of BRCA1 or the loss of BRCA2) but not in BRCA1’s absence.

 

5) When BRCA1 is deficient, SSA driven by EXO1 is a key repair pathway

Next, the authors asked why EXO1 is important when BRCA1 is deficient. They induced damage by irradiating the BRCA1-deficient cells and looked for RAD51 and RAD52 foci. Foci of RAD51 indicate activation of HR repair, whereas RAD52 foci indicate repair by SSA. They found no additional reduction in RAD51 foci in their BRCA1-deficient cells upon EXO1 depletion (i.e., EXO1 does not affect residual HR in BRCA1-deficient cells) and instead observed a decrease in RAD52 foci. This suggests that these cells likely have problems to repair DSBs by both HR and SSA. Looking more widely at other SSA factors, they found that depletion of other key SSA factors in BRCA1-deficient cells was lethal, like EXO1 loss.

 

6) BRCA1-deficient cells rely on EXO1 for survival

Using a cell reporter system to examine whether DNA repair has occurred by SSA and/or HR after induction of a DSB, the authors revealed that whilst cells with EXOI deficiency combined with BRCA2 deficiency are unable to effectively undergo HR to resolve DSBs, they can use SSA. However, when EXO1 deficiency was combined with BRCA1 deficiency, cells couldn’t perform HR and they were less able to use SSA.

These data suggest that BRCA1-deficient cells depend on EXO1 to repair DSBs by SSA. In support, they found a higher incidence of SSA use in BRCA1-deficient tumours, and when BRCA2 was deficient, these incidences were even higher.

 

What I like about this preprint:

Cancer is an extremely difficult disease to treat for many reasons, one of which is the cancer cell’s ability to accumulate mutations and evolve rapidly, thereby increasing the chances of treatment resistance arising. In this preprint, the authors bring into focus a key enzyme (EXO1), which to date has not been widely regarded as a therapeutic target. However, their work herein reveals that EXO1 is important for the survival of cells with deficiencies in the gene BRCA1, a key gene mutated across a multitude of cancers. These new insights may help pave the way for a combined therapy targeting BRCA1-deficient cells by limiting EXO1’s activities.

 

Questions for the Authors:

Q1: Do increased levels of EXO1 in BRCA1-deficient tumours associate with altered disease progression/severity?

Q2: Are there any EXO1 chemical inhibitors and if so, do you see sensitivity in BRCA1-deficient tumour cells if they are treated with any of these inhibitors?

Q3: Is it possible to speculate as to the pathway preference for DSB repair in BRCA1-deficient cells. i.e., is SSA the preferred repair pathway or is alt-EJ more typical?

Q4: When EXO1 is lost, is there evidence that another long-range section nuclease like BLM could be selected for to rescue the lethality in a BRCA1-deficient background?

 

References:

  1. Blasiak, J., Single-Strand Annealing in Cancer. In. J. Mol. Sci, 2021
  2. Adam, S., Rossi, S.E., Moatti, N., De Marco Zompit, M., Xue, Y., Ng, T.F., Alvarez-Quilon, A., Desjardins, J., Bhaskaran, V., Martino, G., et al. The CIP2A-TOPBP1 axis safeguards chromosome stability and is a synthetic lethal target for BRCA-mutated cancer. Nat Cancer, 2021.
  3. Bouwman, P., Aly, A., Escandell, J.M., Pieterse, M., Bartkova, J., van der Gulden, H., Hiddingh, S., Thanasoula, M., Kulkarni, A., Yang, Q., et al. 53BP1 loss rescues BRCA1 deficiency and is associated with triple-negative and BRCA-mutated breast cancers. Nat Struct Mol Biol, 2010.
  4. Bunting, S.F., Callen, E., Wong, N., Chen, H.T., Polato, F., Gunn, A., Bothmer, A., Feldhahn, N., Fernandez-Capetillo, O., Cao, L., et al. 53BP1 inhibits homologous recombination in Brca1- deficient cells by blocking resection of DNA breaks. Cell, 2010.
  5. Ray Chaudhuri, A., Callen, E., Ding, X., Gogola, E., Duarte, A.A., Lee, J.E., Wong, N., Lafarga, V., Calvo, J.A., Panzarino, N.J., et al. Replication fork stability confers chemoresistance in BRCA- deficient cells. Nature 2016.

Tags: brca1, cancer, dna repair, exo1, ssa

doi: https://doi.org/10.1242/prelights.34683

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Response to questions:

  1. This is an interesting question, but unfortunately large enough datasets are currently unavailable to test the prognostic effect of EXO1 expression within BRCA1-mutated cancer patients.

 

  1. Recently, an EXO1 inhibitor has been described for the first time, and of course we are planning to test this compound in our cell models.

 

  1. Since both POLQ and EXO1 are synthetically lethal with BRCA1-deficiency independently, these data suggest that both proteins cannot compensate for each other and both pathways are equally important for BRCA1-deficient cells. This also implies that a-EJ and SSA are required for the repair of a different set of unresolved DSBs in BRCA1-deficient cells and repair of both sets is essential for survival. Our future research is aimed at addressing what determines the choice between SSA and a-EJ for repair of a DSB in BRCA1-deficient cells.

 

  1. Interestingly, both EXO1 and BLM are synthetically lethal with BRCA1-deficiency and our data suggests that this effect is additive (Figure 1G). This implies that endogenous levels of both proteins cannot compensate for each other. More research is needed to better understand the unique role of these long range resection factors and their interplay. Loss-of-function mutations of these factors are very rare, and as we would expect, we have never observed such mutations in a BRCA1-deficient background. Therefore, it is difficult to say whether other resection factors could compensate for the loss of one of them in an in vivo situation. It would be an interesting experiment to try overcoming the lethality of EXO1-loss in BRCA1-deficient cells by overexpressing BLM/DNA2. We have not performed such an experiment yet.

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