Given that genomic instability drives tumorigenesis, it is not surprising that mutations in a variety of genes encoding proteins involved in DDR (including HRR) are frequently found in human cancers. An extensive analysis spanning various cancer types involving 500 adult patients diagnosed with metastatic solid tumors, including 91 cases of BC was conducted utilizing comprehensive whole-exome and transcriptome sequencing. This investigation revealed that potential pathogenic germline variants were detected in 12.2% of the cases, with 75% of these variants linked to deficiencies in DNA repair mechanisms [23]. Within this study, Mut Y homologue (MUTYH), BRCA2, checkpoint kinase 2 (CHEK2), and BRCA1 emerged as the most frequently observed mutations. Furthermore, a comprehensive meta-analysis was conducted, spanning 189 studies and incorporating over 418,649 samples across 25 different tumor types. This comprehensive analysis specifically examined mutations in genes related to HRR genes, excluding BRCA1/2 from consideration. This analysis demonstrated that the occurrence of mutations in HRR genes remained relatively infrequent, comprising less than 1% of cases, with ATM (5.2%), CHEK2 (1.6%), and PALB2 (0.9%) exhibiting the highest prevalence rates [24].

Analysis of DDR mutations in BC based on multiple studies encompassing over 5,000 cases collectively, has revealed that ATM and ATR were the most common DDR-associated genes besides BRCA1 and BRCA2, with prevalence rates ranging from 2.3−4.2% and 1.3−3.7%, respectively [23, 25, 26]. Non-BRCA DDR mutations were more frequent in HER2-positive (21%) followed by luminal B (15%), TNBC (12%), and luminal A (9%) [25].

The pathological mechanisms of DDR gene mutations are well described, and based on the presence of mutations in specific genes, DDR defects can be classified into three classes. Class I includes defects in DSB and replication-associated DNA damage repair (e.g., BRCA1, BRCA2, PALB2, BRIP1, RAD51C, RAD51D); class II refers to defects in DNA damage signaling and checkpoints (e.g., ATM, ATR, CHEK1, CHEK2); and class III refers to increased mutation burden (e.g., MSH2, MSH6, MLH1, PMS2) [27]. Cancer cells with germline or somatic mutations in both alleles of genes listed in class I defects have clearly compromised HRR and in these cells, DSBs are repaired by alternative non-conservative mechanisms, such as NHEJ.

However, mutations in the different HRR genes do not necessarily result in equally severe HRR defects, and DDR- and HRR-based gene signatures have been shown to have predictive value. A combined DDR signature score (CETN2, ERCC1, NEIL2) was able to stratify patients who would not respond to ET but could be candidates for cyclin-dependent kinase 4/6 (CDK4/6)-based treatment [28]. Although not in BC, assays based on HRR defect signatures (which have a higher prevalence than BRCA1/2 mutations) are currently validated and in clinical use, such as HR deficiency (HRD) detect [29, 30], My choice HRD (Myriad Genetics Inc, Sault Lake City, UT, USA) [31, 32] and Foundation Focus CDxBRCA (Foundation Medicine) [33, 34].

Overall, apart from an undeniable advantage in tumorigenesis up to a certain stage, the loss of DDR pathways in cancer cells renders them vulnerable to DNA damage. Thus, the pharmacological inhibition of DDR has become a therapeutic goal, derived from the synthetic-lethal relationships that exist between DDR genes. The dependence of cancers with BRCA1/BRCA2 mutations or other HRR defects on poly[adenosine diphosphate (ADP)-ribose] polymerase (PARP) enzymes to correct DSBs leads to apoptosis in the presence of PARPi [35]. However, reactivation of HRR can occur in BRCA-mutant cells under PARPi, as determined by the presence of RAD51 foci [36, 37]. Mechanistically, replication stress induced by trapped PARP1 on DNA leads to an ATR- and CHEK1-orchestrated response that stabilizes replication fork structures and delays cell cycle progression until DNA repair, restart of replication fork structures, and completion of DNA synthesis prior to mitosis [38]. The following sections summarize and discuss recent advances in the therapeutic targeting of HRR in BC.

As mentioned earlier, BRCA1 and BRCA2 represent the intersection of numerous key cellular functions, engaging in multiple functions encompassing DDR and repair, chromatin remodeling, transcriptional regulation, and protein ubiquitination.

BRCA mutations are highly variable among populations, and many of their pathological implications remain undetermined. In Portugal, the most common BRCA2 rearrangement is c.156_157insAlu, which originates from Portuguese ancestry and is considered a founder mutation [39].

Founder mutations have been reported in other specific populations, the best known being the Ashkenazi Jewish population (ancestry from Eastern and Central Europe) [40]. Within the context of Portuguese BRCA founder mutations, one extensively studied variant is the BRCA2 c.156_157insAlu, first described in 2005 [39]. Available data suggest its origin in families from central and southern Portugal, probably 558 years ± 215 years ago. Notably, the BRCA2 c.156_157insAlu alteration, along with two other variants within the BRCA1 gene (c.3331_3334del and c.2037delinsCC), collectively accounts for approximately 50% of identified pathogenic mutations in the BRCA gene within Portuguese familial cohorts [41]. Furthermore, the observation that families bearing BRCA1 c.3331_3334del or c.2037delinsCC mutations share a common haplotype raises the possibility that these variants may also represent founder mutations within the Portuguese population [39].

This information holds significant relevance for clinical practice because the development of mutation detection panels relies on understanding the mutation spectrum within the target population [42]. Detection of BRCA2 c.156_157insAlu and BRCA1 c.3331_3334del requires specific polymerase chain reaction testing, which is not universally performed by all laboratories [39].

The absence of HR is a prime target for therapies inducing DSBs during the DNA replication process, where HR plays a crucial repair role. Platinum compounds have demonstrated efficacy in BC treatment, either alone or in combination therapy. They act as DNA cross-linking agents, forming intra-strand crosslinks that impede DNA synthesis, function, and transcription (Figure 2) [43]. This mechanism makes platinum therapy particularly effective for DNA repair-deficient tumors, especially those harboring mutations in BRCA1/2 genes. Consistently, preclinical models have shown that BRCA-mutant cells are more sensitive to chemotherapeutic agents that cause DNA DSBs, such as platinum compounds, anthracyclines, and alkylators [44, 45].

Two key trials investigated platinum agents in advanced/metastastic BC with gBRCA1/2 mutations: the Triple Negative Breast Cancer Trial (TNT) and the TBCRC009 trial (Table 2).

The TNT trial (NCT00532727) constituted a phase III trial designed to evaluate the effectiveness of carboplatin vs. docetaxel in treating TNBC [46]. The study enrolled 376 patients with recurrent locally advanced or metastatic TNBC, 43 of whom had gBRCA1/2 mutations. The primary endpoint of the trial was the objective response rate (ORR). The study protocol included pre-specified analyses to explore interactions between biomarkers and treatment outcomes in subgroups defined by gBRCA-BC mutations and BRCA-ness characteristics, aiming to predict responses to platinum salts.

Although no significant differences in ORR, PFS, or overall survival (OS) were observed in the overall study population between the carboplatin and docetaxel arms, significantly higher ORR and PFS emerged within the subgroup possessing gBRCA1/2 mutations. In this subgroup, the carboplatin arm demonstrated significantly higher ORR and PFS compared to the docetaxel arm (ORR 68% vs. 33%; median PFS 6.8 months vs. 4.8 months, respectively). Additionally, a significant interaction between treatment effect and gBRCA status was identified (P = 0.01) [46].

The TBCRC009 (NCT00483223) was a phase II clinical trial conducted as a single-arm study that included patients with metastatic TNBC who were administered either cisplatin or carboplatin as a first- or second-line treatment, based on investigator choice once every 3 weeks [47]. The co-primary endpoints were centered on assessing the ORR and predicting responses based on p63/p73 gene expression levels. The ORR in the overall study population was 25.6% and was numerically higher with cisplatin (32.6%) compared to carboplatin (18.7%). Remarkably, among patients with gBRCA1/2 mutations, the ORR increased to 54.6%, suggesting that platinum agents increase the response rate in patients with advanced or MBC [47].

Poly(ADP-ribose) polymerase (PARP) is a family of 17 proteins known for their involvement in various cellular processes, achieved through the covalent attachment of poly (ADP-ribose) chains to target molecules. This process, termed poly(ADP ribosyl)ation (PARylation), is a widespread post-translational modification at DNA lesions that is crucial for chromatin reorganization, DDR, transcriptional regulation, apoptosis, and mitosis [48, 49].

The best-studied member of the PARP family is PARP1, which is also the most highly correlated with DDR, generating nearly 90% of poly ADP-ribose chains after a DNA damage event [50]. Thus, PARP1 plays a major role in maintaining genome integrity.

Within BRCA1/2-mutant cells, PARPs assume a pivotal role in recognizing DNA damage and initiating alternative repair pathways. Therefore, even in the absence of functional BRCA1/2, these cells can persist due to the compensatory function of PARP. In this scenario, cell death occurs when there is a simultaneous loss of function in both BRCA1/2 and PARP, a genetic concept known as synthetic lethality [51, 52]. Consequently, cells harboring BRCA mutations exhibit an exceptional susceptibility to the inhibition of PARP activity [37, 53].

PARPi were the first clinically approved drugs to explore the concept of synthetic lethality and represented an innovative therapeutic strategy for the treatment of tumors with BRCA1/2 mutations [54]. PARPi structurally mimics nicotinamide adenine dinucleotide (NAD+) for the catalytic active site of PARP molecules. Physiologically, nicotinamide is the primary precursor of NAD+, an essential cofactor in the production of adenosine triphosphate (ATP) and the sole substrate of PARP1 [55]. While PARP1 is generally considered to be the primary target of PARPi, the structural resemblance of the NAD-binding domain among certain PARP family members allows PARPi to also inhibit other PARPs, such as PARP2 and PARP3. Furthermore, they may exhibit off-target effects on kinases [56, 57]. Despite these variations, various PARPi share a similar mechanism of action while displaying distinct cytotoxic profiles and potencies [58].

The mechanism of action of PARPi is not fully understood. Several mechanisms have been proposed to explain their effectiveness. These include inhibition of SSBs repair, trapping of PARP1 in DNA, and NHEJ upregulation (Figure 3).

Endogenous SSBs frequently occur in proliferating cells, and PARP proteins play a crucial role as regulators in the identification and repair of SSBs through BER. Efficient repair of SSBs is essential for cell survival. PARPi inhibits PARP activity, thereby impeding the repair of SSBs through BER.

Unrepaired SSBs can transform into DSBs, which are harmful to cells. HR is the major pathway to repair such lesions during cell replication. HR-deficient cells are unable to repair these DSBs and ultimately undergo apoptosis, eventually leading to cell death [59–61].

In the second mechanism, PARPi prevents the dissociation of PARP1 from damaged DNA, forming a DNA-protein complex that acts as a replication barrier [59, 60, 62]. Although the exact mechanism explaining the trapping of PARP1 in DNA remains unclear, two mechanisms have been proposed: (a) PARPi prevents the release of PARP1 from DNA by inhibiting auto-PARylation; or (b) PARPi binds to the catalytic site of PARP and induces changes in the enzyme’s structure, increasing its affinity for DNA [58, 63]. Since HR-deficient BRCA-mutated cells cannot repair DNA breaks, this leads to cell death [57, 64].

In the third mechanism, PARPi disrupts the usual inhibition of the NHEJ pathway by PARP1. Since NHEJ is error-prone, this leads to an increased number of mutations and chromosome rearrangements, and thus cell death [59, 62, 65].

Moreover, PARP1 regulates the transcription of several proteins that are crucial for cancer cell survival, including p53 and nuclear factor-kappa B (NF-κB) [66]. As a result, the use of PARPi could lead to the suppression of oncogenes controlled through PARP-dependent transcription. Additionally, PARPi downregulates nucleolar RNA helicase II (DDX21) and inhibits ribosomal DNA transcription and ribosome biogenesis in BRCA1/2-proficient BC, ultimately resulting in decreased cell growth [66].

Two PARPi are currently approved for the treatment of gBRCA1/2-mutated MBC, based on the outcomes from the OlympiAD (NCT02000622) and EMBRACA (NCT01945775) clinical trials with olaparib and talazoparib, respectively (Table 3) [18, 19].

The OlympiAD trial was a randomized phase III multicenter study that enrolled 302 patients [67]. It focused on patients with gBRCA1/2-mutated, HER2-negative MBC who had received a maximum of two prior lines of chemotherapy for metastatic disease. The trial used a 2:1 randomization scheme, comparing olaparib monotherapy (300 mg twice daily) with physician’s choice chemotherapy (capecitabine, eribulin, or vinorelbine in 21-day cycles). The primary endpoint was PFS. At the time of the initial analysis, there was a statistically significant advantage in favor of olaparib, with a PFS of 7 months vs. 4.2 months in the standard-of-care arm. In addition, the ORR in the olaparib group was twice that of the standard of care arm (59.9% vs. 28.8%, respectively). In the first line of MBC treatment, olaparib also demonstrated a longer median OS compared with standard therapy (19.3 months vs. 17.1 months). Overall, favorable trends in outcomes were observed across all patient subgroups when treated with PARPi. Regarding side effects, grade ≥ 3 adverse events were less frequent with olaparib (36.6%) than with standard therapy (50.5%), and the rates of discontinuation due to toxicities were 4.9% and 7.7%, respectively. These compelling findings culminated in the approval of olaparib for patients with gBRCA1/2-mutated, HER2-negative MBC previously treated with chemotherapy in the neoadjuvant, adjuvant, or metastatic setting [18, 67].

EMBRACA was a multicenter, randomized, phase III trial that recruited a cohort of 431 patients with advanced BC and gBRCA1/2 mutations who were randomized 2:1 to receive either talazoparib (1 mg once daily) or physician’s choice chemotherapy (capecitabine, eribulin, gemcitabine, or vinorelbine) [19]. Similar to the OlympiAD trial, the primary endpoint of median PFS favored talazoparib, with a median PFS of 8.6 months compared to 5.6 months for standard therapy. The ORR was also higher with talazoparib (62.6% vs. 27.2%). In contrast to OlympiAD, EMBRACA was powered to detect potential differences in OS. However, in the final analysis, talazoparib did not demonstrate a statistically significant improvement in OS compared to chemotherapy across clinically relevant subgroups. Grade ≥3 hematologic events occurred in 55% of patients receiving talazoparib and 38% of the patients receiving standard therapy. Based on the results of this study, talazoparib was approved for the treatment of gBRCA1/2-mutated, HER2-negative advanced or MBC [19].

The rapid development of therapy resistance is a critical problem in cancer treatment. In the case of PARPi, the most widely accepted mechanism of therapy resistance is repairing of HR pathway by secondary mutations within the BRCA genes that reactivate their function. Combination therapy of PARPi with other HR-inhibiting agents (such as chemotherapy and targeted therapies) is one strategy to overcome PARPi resistance.

Combination therapy of olaparib with chemotherapy, namely with trabectidine/lurbenectidine [72, 73] and sapacitabine [73, 74] has also been investigated and showed promising results. In the next section, the combination of PARPi with immunotherapy will be reviewed.

After preclinical models suggested that PARPi could elicit an antitumor immune response by increasing the mutagenic load [75, 76], combinations of PARPi and immunotherapy started to be investigated as a novel and promising approach in advanced BC. Several clinical trials investigating combinations of PARPi with immunotherapy in advanced BC are currently ongoing or have already been completed and shown results (Table 3).

The phase I/II MEDIOLA trial (NCT02734004) evaluated the effectiveness of olaparib in combination with the anti-programmed death-ligand 1 (PD-L1) durvalumab in patients with solid tumors [68]. Initially, four cohorts were enrolled, with all patients having received a maximum of two prior lines of chemotherapy. Among these cohorts, a subgroup of 30 patients had HER2-negative, gBRCA1/2-mutated MBC. Patients were treated with olaparib (300 mg twice daily) for an initial 4 weeks, followed by a combination of olaparib (300 mg twice daily) and durvalumab (1.5 g every 4 weeks) until disease progression occurred. The primary endpoints were safety and disease control rate (DCR) at 12 weeks. The 12-week DCR was 80%, with a median OS of 21.5 months and an ORR of 63%, comparable to findings reported in the OlympiAD trial. The combination was generally well-tolerated with no observed increase in immune-related adverse events [68].

The JAVELIN Solid Tumor trial (NCT01772004) was conducted in phases I/II and aimed to assess the efficacy of talazoparib combined with the anti-PD-L1 agent avelumab in patients with gBRCA1/2- or ATM-altered advanced or metastatic solid tumors [69]. Patients received avelumab (800 mg every 2 weeks) and talazoparib (1 mg daily) until disease progression or unacceptable toxicity. The primary outcome measure was ORR according to RECIST 1.1 criteria, and preliminary results showed an ORR of 24.4% in the gBRCA1/2 cohort and 4.9% in the ATM cohort [69].

Niraparib, another highly selective PARP1/2 inhibitor, has shown activity in advanced or metastatic TNBC. The TOPACIO/KEYNOTE-162 trial (NCT02657889) was a single-arm phase II study of niraparib in combination with immunotherapy for patients with advanced/metastatic TNBC, regardless of BRCA mutation status or PD-L1 expression [71]. Patients received oral niraparib (200 mg once daily) in combination with pembrolizumab (200 mg intravenously on day 1 of each 21-day cycle). The primary endpoint was ORR, while the secondary endpoint was DCR. Among the 55 women in the global study population, five achieved confirmed complete responses, while another five achieved confirmed partial responses. Additionally, 13 patients exhibited stable disease, and 24 experienced progressive disease. In the efficacy-evaluable population (n = 47), the ORR (n = 10) was 21% and the DCR (n = 23) was 49%. Notably, among the 15 evaluable patients with BRCA mutations, seven achieved an ORR of 47%, twelve attained a DCR of 80%, and PFS was 8.3 months [71].

In addition to the currently approved PARPi olaparib and talazoparib, ongoing research is exploring other agents for the treatment of BRCA-mutated advanced/MBC. Among these, veliparib and niraparib are currently the most promising, in both advanced and localized disease settings.

Veliparib is designed to specifically inhibit PARP1 and PARP2. Notably, it appears to have less PARP trapping potential, making it an appealing option for use in combination therapy alongside conventional chemotherapy agents (Table 3) [70, 77].

The phase III, placebo-controlled BROCADE 3 trial (NCT02163694) enrolled 513 patients with gBRCA1/2-mutations, HER2-negative BC. These patients had received a maximum of two prior lines of chemotherapy for metastatic disease [70]. In this trial, patients were randomized in a 2:1 scheme to receive carboplatin on day 1 and paclitaxel (80 mg/m² intravenously) on day 1, day 8, and day 15 of 21-day cycles. These chemotherapy combinations were either paired with veliparib (120 mg orally twice daily on day 2 to day 5) or a matching placebo. Patients who discontinued carboplatin and paclitaxel before progression had the option to continue receiving veliparib or placebo at an intensified dose (300 mg twice daily continuously, escalating to 400 mg twice daily if tolerated) until disease progression. The primary endpoint was PFS. After a median follow-up of 35.7 months in the veliparib group and 35.7 months in the control group, PFS was reported as 14.5 months for the velaparib group and 12.6 months for the control arm. Grade ≥ 3 events were more prevalent in the veliparib group (34% vs. 29% in the control group [70].

It should be noted that there is currently no evidence to support the efficacy of platinum agents after PARPi (or vice versa) in the advanced/MBC setting. In addition, at the time of this review, there were no data comparing PARPi and platinum agents.

ATR kinase belongs to the phosphatidylinositol 3-kinase-related kinase (PIKK) family and functions as the primary responders to single-strand DNA damage, playing a crucial role as master regulators of replication stress [78].

An innovative oral inhibitor targeting ATR kinase, RP-3500 (camonsertib), is being tested in the NCT04497116 trial. This ataxia telangiectasia and Rad3-related kinase inhibitor (ATRi) exhibited efficacy in cellular biochemical assays, significant selectivity for ATR, and substantial potential for reducing tumor growth in murine models when orally administered once daily at doses ranging between 5–7 mg/kg [79].

When combined with olaparib in short-term treatments, RP-3500 displayed a synergistic effect surpassing that of sequential administration, exceeding the individual efficacy of olaparib and being more potent at lower doses. This simultaneous treatment also excelled at inducing tumor cell death compared to continuous treatment, especially promising for BC with DRR [79].

Another orally administered ATRi, AZD6738 (ceralasertib), exhibited notable selectivity in inhibiting ATR kinase [80]. AZD6738 has a combined therapeutic effect with agents known for causing replication fork stalling and collapse, such as carboplatin, irinotecan, and olaparib. Antitumor therapy is achieved synergistically at lower doses compared to using each agent alone. This suggests a potential strategy for enhancing therapeutic effects while minimizing individual drug dosages [80].

In a model using TNBC xenografts derived from patients with a BRCA2 mutation, complete regression of tumors was observed within 3 days to 5 days. This regression occurred when AZD6738 was administered in conjunction with olaparib daily, three times to five times a week. Moreover, escalating the dosage of olaparib or increasing the frequency of AZD6738 administration to twice daily resulted in complete tumor regression, even in a TNBC xenograft model without the BRCA mutation [80].

Berzosertib (previously known as VX-970) is a potent and specific suppressor of ATR, demonstrating preclinical anticancer activity in combination with DNA-damaging chemotherapy in TNBC [81, 82]. In an ongoing C2 expansion trial (phase 1b trial) investigating safety, tolerability, efficacy, and potential predictive biomarkers of berzosertib in combination with cisplatin among patients with advanced TNBC (specifically those with tumors characterized as gBRCA1/2 wild-type and basal subtype) berzosertib was well tolerated [82]. The ORR was 23.4% [90% confidence interval (CI): 13.7, 35.8]. No relevant associations were observed between the response and gene alterations. Further studies combining ATR inhibitors with platinum compounds may be warranted in highly selected patient populations [81, 82].

CHEK1/2 serves as downstream targets of both ATR and ATM, playing a crucial role in the temporary arrest of the cell cycle and the repair of DNA damage [83]. In a phase II single-arm study (NCT02203513), the efficacy of prexasertib at 105 mg/m² IV every 2 weeks was assessed in patients with metastatic/recurrent TNBC. The primary endpoint was ORR. This study enrolled 9 patients with gBRCA wild type who had undergone at least one prior treatment. Prexasertib demonstrated modest clinical efficacy in BRCA-wild type TNBC, with one patient experiencing a partial response (ORR 11.1%) and four patients achieving stable disease [84].

One promising therapeutic strategy involves combining prexasertib with PARPi, since it has been shown that when olaparib is combined with a CHEK1 inhibitor, it diminishes HR efficiency and reduces replication fork stability [85].

In a phase I study where prexasertib was combined with olaparib in high-grade serous ovarian cancer (NCT03057145), preliminary clinical activity was observed specifically in BRCA-mutant patients who had previously progressed on a PARPi. Pharmacodynamic analyses revealed that prexasertib compromises HR, inducing DNA damage and replication stress [86]. This insight can provide future guidance for combining PARPi and other DDR-target therapies.

Wee1-like protein kinase (WEE1) regulates the intra-S and G2/M cell-cycle checkpoints [87]. Adavosertib (MK-1775) is a highly effective and specific inhibitor of WEE1, inducing DNA damage regardless of any adjuvant chemotherapy or radiotherapy.

Promising results were observed in phase II trials when adavosertib was combined with gemcitabine for patients with platinum-resistant or refractory ovarian cancer (NCT02151292). In this study, the adavosertib group exhibited a prolonged PFS compared to the placebo group (4.6 months vs. 3.0 months), but with higher rates of neutropenia and trombocitopenia [88].

In another phase II trial (NCT01357161) that enrolled women with histologically or cytologically confirmed epithelial ovarian, fallopian tube, or peritoneal cancer with measurable disease, patients were administered adavosertib (oral capsules, with 2 days on/5 days off or 3 days on/4 days off schedules) across six cohorts ranging from 175 mg once daily to 225 mg twice daily. These were combined with gemcitabine, paclitaxel, carboplatin, or pegylated liposomal doxorubicin. The primary outcome measurement was ORR. Three percent of patients achieved a confirmed complete response, and 29% achieved a confirmed partial response. The response rate was notably highest (66.7%) with carboplatin plus weekly adavosertib, showing a 100% DCR and a median PFS of 12.0 months. The longest median duration of response was observed in the paclitaxel cohort (12.0 months) [89]. Despite the potential of combining PARPi and WEE inhibitors in trapping tumor progression, this combination is poorly tolerated [90].