FANCD2 limits replication stress and genome instability in cells lacking BRCA2

Mutations in the breast cancer susceptibility gene 2 (BRCA2) predispose individuals to breast and ovarian cancer. As a result of compromised HR repair, BRCA2-mutated tumor cells differ from normal cells in their responses to exogenous DNA damage and to endogenous challenges that arise during DNA replication (e.g. at telomeres)^1,2. This is due to the essential function of BRCA2 in protecting stalled replication forks against nucleolytic degradation^3,4. A similar role in DNA replication has been ascribed to the Fanconi anemia group D2 protein (FANCD2)⁵ . FANCD2 is a central component of the Fanconi anemia pathway for DNA cross-link repair and its inactivation is associated with progressive bone marrow failure and cancer predisposition.

Recent work demonstrated that FANCD2 regulates responses to replication stress induced by mitomycin C (MMC) and hydroxyurea (HU)^5–7. BRCA2 abrogation likewise triggers replications stress, a hallmark of BRCA2-deficient cancer cells that drives the associated pathologies⁸. This led us to investigate whether FANCD2 mediates replication stress responses in cells lacking BRCA2 and how this contributes to genome integrity and survival of these cells.

FANCD2 and BRCA2 are both required to prevent MRE11-dependent degradation of nascent DNA at stalled replication forks^3,5. However, whether the two factors interact functionally with each other during this process has not been addressed. To investigate fork protection in cells lacking both FANCD2 and BRCA2, we transfected H1299 cells harboring doxycycline (Dox)-inducible shRNA against BRCA2 with siRNA against FANCD2 (Fig. 1a). We monitored replication fork degradation after prolonged HU treatment using DNA fiber analyses (Fig. 1b). Consistent with previously described roles of FANCD2 and BRCA2 in replication fork protection^3,5, DNA tracks were significantly shorter in FANCD2- or BRCA2-depleted cells. Importantly, concomitant loss of FANCD2 and BRCA2 resulted in increased levels of fork degradation indicating that the two proteins mediated stalled fork protection by distinct mechanisms. Inhibition of MRE11 nuclease activity with mirin partially rescued degradation of nascent DNA in cells lacking FANCD2 and BRCA2, implicating MRE11 as one of the nucleases required for fork resection in this context.

Replication at stalled forks can be restarted by multiple mechanisms⁹. However, the roles of FANCD2 and BRCA2 in replication fork restart remain controversial^3,5,10. In cells lacking FANCD2 or BRCA2, we observed impaired replication restart after HU treatment (Fig. 1c). This defect was more pronounced in cells deficient in both proteins, suggesting that FANCD2 and BRCA2 played independent roles in the restart of HU-arrested replication forks.

FANCD2 restricts DNA synthesis upon HU-induced replication stress in order to prevent accumulation of deleterious single-stranded DNA gaps⁷. BRCA2-deficient cells exhibit high levels of endogenous replication stress¹¹, resembling that induced by HU treatment. To test whether FANCD2 limits DNA synthesis under replication stress conditions associated with BRCA2 inactivation, we measured replication rates in two BRCA2-deficient human cell lines treated with FANCD2 or control siRNAs. In both cell lines, FANCD2 depletion increased track length (Fig. 1d-f and Supplementary Fig. 1a), indicating that FANCD2 inhibits replication fork progression in the absence of BRCA2. Additionally, we observed an increase in origin firing in BRCA2-deficient cells lacking FANCD2 (Supplementary Fig. 1b) suggesting that FANCD2 suppresses activation of origins to prevent uncontrolled replication in the context of BRCA2 deficiency, similarly to HU¹². Uncontrolled replication leads to DNA damage accumulation⁹. Consistent with this, in cells lacking FANCD2 and BRCA2 we observed enhanced levels of chromosomal aberrations (relative to single depletion, Fig. 1g,h), as well as elevated levels of γH2AX foci (Fig. 1i) that mark DNA damage sites. Thus, FANCD2 is required to prevent genomic instability in BRCA2-deficient cells.

DNA damage accumulation triggers DNA damage responses ultimately leading to senescence and apoptosis¹³. To determine whether the genomic instability observed in cells lacking FANCD2 and BRCA2 impacts on cell fate, we assessed apoptosis and senescence using Annexin V and β-galactosidase staining, respectively. We observed a significant increase in apoptotic, Annexin V-binding cells upon concomitant abrogation of FANCD2 and BRCA2 expression in H1299 cells relative to inhibition of each factor alone (Fig. 2a). Likewise, FANCD2-, BRCA2-depleted primary MRC-5 human cells entered senescence at a higher rate than cells lacking either FANCD2 or BRCA2 (Supplementary Fig. 2) as measured using β-galactosidase staining.

Next, we investigated a potential synthetic lethality between FANCD2 and BRCA2. FANCD2 inhibition using two different siRNAs significantly decreased survival of H1299, DLD1 and MDA-MB-231 cells lacking BRCA2 expression in clonogenic assays (Fig. 2b-d and Supplementary Fig. 3a-d). Consistently, survival of DLD1 cells, in which FANCD2 expression was abrogated using the CRISPR–Cas9 technique, was significantly reduced by siRNA-mediated depletion of BRCA2 (Fig. 2e and Supplementary Fig. 3e). This indicated that FANCD2 is required for survival of BRCA2-deficient cells and suggests a synthetic lethal interaction between the two genes¹⁴. Depletion of the Fanconi anemia core complex component FANCA led to significantly impaired survival of BRCA2-deficient cells (Supplementary Fig. 3f) indicating that ubiquitination of FANCD2 is required in cells lacking BRCA2.

Interestingly, cell lines and tumors carrying BRCA2 mutations exhibited higher FANCD2 mRNA expression levels than cell lines and tumors with wild type BRCA2 (Fig. 2f, Supplementary Fig. 4, Supplementary Tables 1,2). Thus far, no functional assays have been carried out to establish whether these mutations result in loss of BRCA2 function. However, our findings raise the possibility that FANCD2 transcription is upregulated to sustain survival of BRCA2-deficient cells and tumors.

BRCA2 deficiency is commonly targeted by chemical inhibition of poly (ADP-ribose) polymerase 1 (PARP1) while Fanconi anemia patient cell lines are generally not sensitive to PARP inhibitors¹⁵. To address whether FANCD2 modulates the response to PARP inhibition, we treated H1299 cells lacking BRCA2, FANCD2 or both, with low doses of olaparib (Fig. 2g). Consistent with previous results¹⁵, this treatment was not toxic in the context of FANCD2 loss. However, we observed that FANCD2 abrogation enhanced sensitivity of BRCA2-deficient cells to olaparib, suggesting that FANCD2 mediates mechanisms of resistance to this drug.

In summary, we have demonstrated that FANCD2 is essential for cell survival in the absence of BRCA2. FANCD2 promotes DNA synthesis, replication fork protection and restart in BRCA2-deficient cells. This role of FANCD2 is analogous to that previously reported in response to exogenously induced replication stress (e.g. HU). FANCD2 provides a protection mechanism against genomic instability in cells lacking BRCA2 and its abrogation is lethal to these cells. Elevated apoptosis and reduced survival of BRCA2-compromised cells upon FANCD2 abrogation could be exploited therapeutically by treating BRCA2-deficient tumors with FANCD2 inhibitors.