Combination strategies with PARP inhibitors in BRCA-mutated triple-negative breast cancer: overcoming resistance mechanisms

Abstract

Triple-negative breast cancer (TNBC) is a particularly aggressive breast cancer subtype, characterised by a higher incidence in younger women, rapid metastasis, and a generally poor prognosis. Patients with TNBC and BRCA mutations face additional therapeutic challenges due to the cancer’s intrinsic resistance to conventional therapies. Poly (ADP-ribose) polymerase inhibitors (PARPis) have emerged as a promising targeted treatment for BRCA-mutated TNBC, exploiting vulnerabilities in the homologous recombination repair (HRR) pathway. However, despite initial success, the efficacy of PARPis is often compromised by the development of resistance mechanisms, including HRR restoration, stabilisation of replication forks, reduced PARP1 trapping, and drug efflux. This review explores latest breakthroughs in overcoming PARPi resistance through combination therapies. These strategies include the integration of PARPis with chemotherapy, immunotherapy, antibody-drug conjugates, and PI3K/AKT pathway inhibitors. These combinations aim to enhance the therapeutic efficacy of PARPis by targeting multiple cancer progression pathways. The review also discusses the evolving role of PARPis within the broader treatment paradigm for BRCA-mutated TNBC, emphasising the need for ongoing research and clinical trials to optimise combination strategies. By tackling the challenges associated with PARPi resistance and exploring novel combination therapies, this review sheds light on the future possibilities for improving outcomes for patients with BRCA-mutated TNBC.

Introduction

Breast cancer (BC) remains one of the most prevalent forms of cancer globally, with an estimated 2.3 million new cases and ~685,000 deaths reported in 2020 alone [1]. Among the various subtypes of breast cancer, triple-negative breast cancer (TNBC) is notably aggressive and presents significant treatment challenges. Defined by the absence of oestrogen receptors (ER), progesterone receptors (PR), and human epidermal growth factor receptor 2 (HER2), TNBC represents about ~15% of all breast cancer cases [2]. This lack of receptor expression renders TNBC unresponsive to hormonal therapies effective in other subtypes, thereby complicating treatment and often necessitating reliance on conventional chemotherapy [3].

Research indicates that late detection of TNBC is frequently linked with dysregulated autophagy, tumour heterogeneity, genetic mutations, and alterations in drug targets, leading to suboptimal therapeutic outcomes [4, 5]. The stage of neoplasm significantly influences the treatment strategy, which may include surgery, radiation, conventional chemotherapy, targeted therapies, and immune therapeutics [6]. The current chemotherapeutic strategies often involve a cocktail of drugs that act through multiple mechanistic pathways to enhance tumour cell eradication [7]. This multi-agent approach aims to target different cellular pathways and cell cycle phases to reduce tumour cell survival effectively.

TNBC is characterised by the reduced expression of three key biomarker proteins: ER, HER2, and PR, affecting ~15–20% of breast cancer patients [8]. This subtype is associated with poorer clinical outcomes, higher rates of recurrence, and metastasis, particularly to the liver, lungs, and central nervous system. TNBC also disproportionately affects younger women and exhibits a higher aggressiveness in specific populations, such as African Americans, where it tends to present at an earlier age and in more advanced stages [9, 10]. In contrast, Asian and European populations show a lower incidence and relatively better survival rates compared to African American women [11].

The unfavourable prognosis of TNBC is primarily attributed to its genomic instability, often driven by mutations in the BRCA1 and BRCA2 genes, which play a critical role in repairing double-strand DNA breaks via the homologous recombination repair (HRR) pathway [12]. Mutations in these genes compromise the HRR mechanism, leading to increased genomic instability—a hallmark of cancer progression [13].

Traditionally, the treatment of TNBC involves surgery followed by adjuvant radiation and chemotherapy. However, the efficacy of chemotherapy is limited by its severe side effects, including cardiotoxicity, myelosuppression, and gastrointestinal issues, as well as the development of chemoresistance [14]. In light of these challenges, there is growing interest in developing targeted therapies that exploit the unique molecular defects of TNBC. One of the most promising advances in this field is the use of poly (ADP-ribose) polymerase inhibitors (PARPi), which target tumours with HRR deficiencies such as those caused by BRCA mutations [15].

PARP inhibitors function by blocking the repair of single-strand breaks in DNA, leading to the accumulation of double-strand breaks that are lethal to cells with defective HRR mechanisms, such as BRCA-mutant cells. This mechanism of action, known as synthetic lethality, has been a significant breakthrough in TNBC treatment [16]. Pioneering studies identified the synthetic lethal interactions between PARPis and BRCA1 or BRCA2 mutations. Currently, five PARPis, including olaparib, niraparib, rucaparib, talazoparib and veliparib, have been developed, each with varying capacities to trap PARP on DNA and inhibit its catalytic activity [17]. These inhibitors have demonstrated efficacy in treating BRCA-mutated TNBC by exacerbating DNA damage and inducing cancer cell death in tumours with defective BRCA genes [17].

Despite the promise shown by PARPis, their application in TNBC is limited by the disease’s heterogeneity and the potential for resistance development. Challenges in managing associated toxicities and the restriction of PARPi use to patients with confirmed BRCA mutations further complicate treatment. Current research efforts are focused on overcoming these limitations and exploring combination therapies that may enhance the efficacy of PARPis. This review will be exploring the basis for clinical use of PARPis, mechanisms of resistance and the current landscape of PARPi combination strategies in BRCA mutated TNBC treatment. Additionally, it will present a progressive analysis of the novel approaches and emerging therapies, emphasising the unique contributions of this work. By understanding these aspects, we can better navigate the future of TNBC therapy and explore potential combination strategies to enhance the effectiveness of PARPi and improve patient outcomes.

Rationale for clinical application of PARP inhibitors

PARP1 is an enzyme essential for repairing single-stranded DNA breaks (SSBs), which is a crucial part of the bigger DNA damage response (DDR) machinery [18–20]. Detection of an SSB by PARP1, leads to their binding, consequently auto—PARylation of PARP1s catalytic domains which is the covalent binding of PARP enzymes to poly (ADP-ribose) [PAR] chains. The latter aids in DNA damage repair as auto-PARylation leads to the formation of PARP complexes which further induce chromatic remodelling by recruiting SSB proteins [2, 21]. However, in case of disruption or inhibition of auto-PARylation, SSBs accumulate, triggering replication-dependent DNA DSBs and “PARP trapping”, where PARP1 is unable to detach from sites of DNA damage [22, 23]. PARPis induce PARP trapping which directs the collapse of the replication fork collapse and an increase in DSBs, resulting in clinically significant cytotoxicity [24, 25]. Further, cells repair DNA integrity at double-DSBs through two main pathways: homologous recombination (HR) and the error-prone nonhomologous end joining (NHEJ) pathway [26, 27]. HR, a high-fidelity repair process that relies on BRCA1 and BRCA2 proteins, is primarily active during the S and G2 phases of the cell cycle. In contrast, NHEJ predominantly operates in the G1 phase [2]. Both healthy and cancer cells utilise HR and NHEJ, but BRCA1/2-mutant cancer cells have impaired HR capabilities, which forces them to rely on NHEJ. This reliance can lead to genomic instability due to inaccurate repairs [28].

PARPis take advantage of this vulnerability through a mechanism known as synthetic lethality. By inhibiting PARP1, these drugs trap PARP1 at SSB sites, leading to the accumulation of DSBs and replication stress that BRCA-mutant cells cannot repair [26]. This targeted cytotoxicity is especially effective in BRCA-mutant tumours, where defective DSB repair mechanisms necessitate reliance on PARP-induced SSB repair pathways [27]. Unlike wild-type cells, BRCA-mutant cells cannot utilise HR for repair, making them more sensitive to PARPi and supporting the clinical rationale for using PARPi in these specific tumour subsets [2, 28].

PARP inhibitors resistance mechanisms

PARPis, such as olaparib and talazoparib, have shown significant improvements in progression-free survival (PFS) among patients with germline BRCA1/2-mutated metastatic breast cancer (BC), as evidenced by the OlympiAD and EMBRACA Phase III trials. However, these trials did not demonstrate a significant benefit in overall survival (OS), highlighting a critical challenge in the clinical application of PARPis [12]. Similar to other targeted therapies, resistance to PARPis can emerge, posing a significant barrier to their widespread use in cancer treatment. Notably, more than 40% of BRCA-mutated cancer patients have shown limited benefit from PARPi therapies [29]. Understanding the mechanisms behind PARPi resistance (Fig. 1) is therefore crucial to enhancing the efficacy and durability of these therapies in clinical practice [30].

Fig. 1. Mechanism of resistance of PARPis.

(1) Gene reversion mutations can restore HRR genes like BRCA1/2 and RAD51C/D, causing drug resistance by restoring HRR function. Inactivating NHEJ proteins (e.g., 53BP1) enhances HRR efficiency during PARPi treatment, promoting DNA repair and stability. Factors like Shieldin complex, RIF1 and REV7 support. (2) BRCA mutations lowering EZH2 and PTIP reduce nuclease recruitment, leading to stable replication forks and consequently drug resistance. Stable replication forks mitigate DNA damage during replication stress induced by PARPis. Reduced chromatin remodelers (HLTF, ZRANB3, and SMARCAL1), which facilitate nuclease-dependent degradation of replication forks, contribute to this resistance lowering replication stress. SLFN11 normally regulates replication fork arrest, and its absence decreases PARPi cytotoxicity, further leading to drug resistance. (3) Changes or reduction in PARP can reduce PARP‐1 trapping on DNA. This lowered PARP trapping facilitates replication fork stability and further contributes to DNA repair signaling, which promotes PARPi resistance. (4) Long‐term PARPi treatment enhances P‐glycoprotein (P-gp) expression, resulting in lower intracellular PARPi concentration and PARP resistance. Liu et al. [12, 21, 204]. Flowchart created by Diagramly.

Restoration of homologous recombination repair (HRR)

The most common mechanism of resistance to PARPi therapy is the restoration of HRR function (Fig. 1.1). Gene reversion mutations can reactivate HRR genes such as BRCA1 and BRCA2, which were originally impaired [29, 31]. This reactivation allows cancer cells to regain the ability to effectively repair double-strand DNA breaks, thereby evading the synthetic lethal effects of PARP inhibition. Studies have documented the occurrence of reversion mutations in BRCA1/2 among breast cancer patients, leading to the development of resistance to PARPi therapy [32, 33]. While the restoration of HRR is a well-documented mechanism, it is important to note that it does not account for all instances of resistance. Some patients with persistent BRCA mutations also develop resistance through alternative pathways.

Stable replication forks

Another mechanism by which BRCA1/2-mutant cells develop resistance to PARPis involves the stabilisation of replication forks [12] (Fig. 1.2). Under normal conditions, BRCA1/2 proteins protect stalled replication forks during DNA damage repair [21, 34]. In the absence of functional BRCA1/2, other factors such as EZH2 and PTIP facilitate the recruitment of nucleases like MRE11 and MUS81 to the stalled replication forks, leading to fork collapse and subsequent cell death [35]. However, when the activities of EZH2 and PTIP are reduced, nuclease recruitment is inhibited, resulting in the stabilisation of replication forks. This process protects the genomic integrity of the cancer cells, thereby generating resistance to PARPis [34, 35]. While the stabilisation of replication forks is a significant contributor to resistance, it is not the sole mechanism, as some BRCA1/2-mutant cells can still succumb to PARPi treatment despite fork stabilisation.

Reduced PARP1 trapping

Some PARPis (talazoparib, saruparib) exert their pharmacologic effects in part, by ‘trapping’ -PARP1 enzyme onto single-stranded DNA, thereby compromising the DNA repair process [36, 37] (Fig. 1.3). Alterations or depletion of PARP1, or the loss of poly (ADP-ribose) glycohydrolase (PARG), can reduce the trapping of PARP1 on the DNA strand complex [36]. In particular, the loss of PARG results in the accumulation of poly (ADP-ribose) (PAR), which in turn restores PARP1-dependent DNA damage signalling and diminishes PARP1 trapping. This reduction in PARP1 trapping decreases the extent of DNA damage induced by PARPis, rendering cancer cells less sensitive to the treatment [37]. ‘PARP-trappers’ such as talazoparib and saruparib exhibit a longer residency time in the NADH-binding domain of PARP-1. They do not change the binding of PARP-1 to single-stranded DNA. Evidence for this mechanism is seen in PARP1 knockout cells, which exhibit a high level of resistance to olaparib in BRCA mutated patient [24] however it was ovarian cancer [36] and not TNBC thus further research is needed. Nonetheless, the reduced trapping of PARP1 alone does not fully explain the resistance observed in all cases, suggesting the involvement of other contributing factors.

Drug efflux

The overexpression of ATP-binding cassette drug transport proteins is another mechanism associated with PARPi resistance [38] (Fig. 1.4). Long-term treatment with PARPis has been shown to upregulate the expression of P-glycoprotein (P-gp, also known as MDR1, encoded by ABCB1), which acts as a drug efflux pump [39]. This upregulation leads to a reduction in the intracellular concentration of PARPis, thereby diminishing their therapeutic efficacy and leading to resistance. Although the precise mechanisms underlying the increased expression of the P-gp efflux pump in relation to PARPi resistance are not fully understood, the restoration of PARPi sensitivity in tumours upon co-treatment with tariquidar, a P-gp inhibitor, supports the role of drug efflux in resistance [40]. However, it remains unclear whether P-gp overexpression alone is sufficient to confer complete resistance or if it acts in concert with other resistance mechanisms.

Overall, the development of resistance to PARPis in BRCA-mutated breast cancer is a complex phenomenon involving various mechanisms, such as the restoration of HRR, stabilisation of replication forks, reduced PARP1 trapping, and drug efflux [30]. Each of these mechanisms presents unique challenges and opportunities for improving the effectiveness of PARPi therapies. By elucidating the molecular underpinnings of PARPi resistance, researchers and clinicians can develop more robust strategies to overcome resistance and enhance clinical outcomes for patients with BRCA-mutated cancers. To address this resistance, combination strategies will be discussed further as a promising approach.

Advances in combination strategies with PARP inhibitors

PARP inhibitors and chemotherapy

Chemotherapy has long been the cornerstone of treatment for triple-negative breast cancer. Recently, the integration of PARPis into treatment regimens has shown promise, particularly in patients with BRCA1/2 mutations, due to their impaired DNA repair mechanisms via HR [15]. PARP inhibitors, when combined with chemotherapy, aim to exploit this vulnerability by further preventing the repair of DNA damage, thus driving tumour cells towards apoptosis. Clinical studies (Table 1) suggest that this combination can enhance treatment efficacy, leading to better clinical outcomes compared to chemotherapy alone. Specifically, PARP inhibitors such as veliparib, olaparib, and Niraparib have been studied in combination with traditional chemotherapeutic agents, including platinum-based compounds, with varying degrees of success.

Table 1.

Summary of clinical trials investigating PARP inhibitors in combination with chemotherapy (Adapted from Kunwor et al., [46]).

Trial	Experimental Group	Control Group	Median PFS (months)	Median OS (months)
BROCADE [41, 43]	Veliparib with Carboplatin/Paclitaxel	Placebo plus carboplatin/paclitaxel	14.1 vs 12.3	28.3 vs 25.9
	Veliparib with temozolomide	Placebo plus carboplatin/paclitaxel	7.4 vs 12.3	19.1 vs 25.9
BROCADE-3 [45]	Veliparib with Carboplatin/Paclitaxel	Placebo plus carboplatin/paclitaxel	14.5 vs 12.6	33.5 vs 28.2
SWOG S1416 [47]	Veliparib with Cisplatin	Placebo with Cisplatin	6.2 vs 6.4	14.2 vs 14.6
BRAVO	Niraparib	Physician’s choice CT	Study was prematurely closed after an interim analysis showed too many patients were not completing the necessary assessments in the control arm, and it was no longer suitable as a registration trial.

In the BROCADE series of trials, veliparib was combined with platinum-based chemotherapy. The phase II BROCADE trial [41–44] revealed a numerical but not significant difference in PFS and overall survival (OS) between the veliparib and control arms. However, there was a significant improvement in the overall response rate (ORR) (77.8% vs 61.3%). The BROCADE 3 trial, a phase III study, demonstrated a more consistent PFS benefit in the veliparib group compared to standard chemotherapy (14.5 months vs 12.6 months; HR 0.71; 95% CI 0.57–0.88; P = 0.0016) [45]. This study also allowed for the continuation of veliparib monotherapy post-chemotherapy, leading to a persistent PFS benefit, particularly notable in the delayed separation of Kaplan-Meier curves and a prolonged tail in the veliparib group [46].

Conversely, the SWOG S1416 trial, which evaluated veliparib in combination with cisplatin in TNBC patients, including those with BRCA mutations, did not show a significant PFS or OS benefit in the BRCA-mutated subgroup [47]. This trial’s limited accrual [15], likely influenced by the concurrent FDA approvals of olaparib and talazoparib for BRCA-mutated TNB, reduced its statistical power. However, the trial did indicate potential benefits in BRCA-like mutation groups, suggesting that the effectiveness of PARP inhibitors may extend beyond just BRCA-mutated cancers.

Despite the encouraging results, the combination of PARP inhibitors with chemotherapy is not without challenges. The addition of PARP inhibitors often exacerbates the toxicities associated with chemotherapy. For instance, in phase I trials where olaparib was combined with carboplatin and paclitaxel, there was a marked increase in the frequency, severity, and duration of adverse events, leading to dose reductions of olaparib [48, 49]. Significant attempts to modulate the myelosuppression produced by the combination of Olaparib with carboplatin were made, include optimal prophylaxis with filgrastim (r-metHuG-CSF). Despite this, it proved impossible to safely combine Olaparib, or any of the first-generation PARP-inhibitors with myelosuppressive chemotherapy. Veliparib, considered a weaker PARP trapping agent, has shown a slightly better safety profile with less myelosuppression [16], as seen in the BROCADE 3 trial, where common grade 3 or worse adverse events included neutropenia (81% vs 84%), anaemia (42% vs 40%), and thrombocytopenia (40% vs 28%) [45]. This probably reflects its lower potency relative to Olaparib, niraparib and talazoparib.

Further complicating the treatment landscape is the inconsistency in clinical trial outcomes. For instance, the phase II trial by O’Shaughnessy et al. [50], which added iniparib to carboplatin and gemcitabine, initially showed enhanced clinical benefits, including prolonged PFS and OS. However, these results were not replicated in the phase III trial, which failed to meet its primary endpoints, likely due to patient variability, including differing metastatic burdens.

Other studies exploring different chemotherapy agents in combination with PARP inhibitors, such as cyclophosphamide or doxorubicin, have also produced mixed results. In some cases, while the antitumor effectiveness was promising, significant adverse effects, including secondary cancers like oral squamous cell carcinoma, emerged, raising concerns about the long-term safety of these combinations [51].

The mechanism of myelosuppression exhibited by 1st-generation PARP inhibitors, either as monotherapy, or especially when used in combination with chemotherapy, has been the subject of significant research. First-generation compounds are equipotent against both PARP-1 and PARP-2. Pre-clinical studies demonstrate a significant role of PARP-2 in erythroid and myeloid progenitor maturation [52]. As a consequence, a number of second-generation PARP-1 selective inhibitors are in development, such as AZD5305 (saruparib). Saruparib has a 600-fold selectivity for PARP-1 relative to PARP-2. Early clinical data suggest that this approach only partially reduces the degree myelosuppression, suggesting that the underlying mechanism may also be partially dependent on PARP-1 [53].

The controversy over the optimal chemotherapy-PARP inhibitor combination and sequencing continues [54]. Notably, the GeparOcto trial found no significant difference in pathological complete response (pCR) rates between two intensive chemotherapy regimens, regardless of the addition of carboplatin [55]. Similarly, the BrighTNess trial showed that while the addition of veliparib to paclitaxel and carboplatin improved pCR rates compared to paclitaxel alone, it did not outperform the paclitaxel-carboplatin combination without veliparib [56]. These findings suggest that while PARP inhibitors may enhance chemotherapy’s effectiveness in some contexts, their universal application in neoadjuvant settings remains unproven.

Moreover, the phenomenon of BRCA1/2 reversion mutations and other mechanisms of resistance to PARP inhibitors complicates treatment strategies, underscoring the need for further research [57]. Large, randomised trials are essential to determine the most effective combinations and sequences, balancing efficacy with manageable toxicity profiles [58]. The ongoing debate about whether the benefits of adding PARP inhibitors to chemotherapy outweigh the risks of increased toxicity highlights the need for personalised treatment approaches, particularly in the diverse and complex landscape of TNBC.

Thus, while combining PARP inhibitors with chemotherapy represents a promising strategy for improving outcomes in BRCA-mutated TNBC, particularly in advanced cases, the approach is fraught with challenges. These include managing severe toxicities, overcoming inconsistent clinical trial results, and addressing emerging resistance mechanisms. Continued research into optimising treatment regimens and understanding patient-specific responses will be crucial in advancing this therapeutic strategy.

PARP inhibitors and immunotherapy

The tumour microenvironment (TME) of BC, particularly in TNBC, is characterised by decreased expression of tumour-infiltrating lymphocytes (TILs) and increased presence of immunosuppressive cells, such as regulatory T cells (Tregs) and tumour-associated macrophages (TAMs) [59–61]. This immunosuppressive environment contributes to the negative effects on chemotherapy and targeted therapies. Studies have shown that the immune response initiated by PARPis is necessary for the elimination of breast tumours, particularly those with BRCA mutations [62]. PARPis increase cellular DNA damage in BRCA-mutant tumours and activate the type I interferon (IFN) pathway via the cyclic GMP-AMP synthase-stimulator of interferon genes (cGAS-STING) signalling pathway, which promotes dendritic cell (DC) maturation, release of interferons, and initiation of antitumor T-cell immune responses [63].

Preclinical investigations have highlighted a compelling synergy between PARP inhibitors (PARPis) and immunotherapeutic approaches, which can be attributed to two main mechanisms. First, PARPis induce extensive DNA damage, leading to the accumulation of cytosolic DNA, which activates immune pathways. Second, these inhibitors modulate T-cell responses and enhance the expression of programmed death ligand 1 (PD-L1) [62, 64–66]. These changes within the TME create favourable conditions for integrating immune checkpoint blockade with PARP inhibition, particularly in tumours characterised by HR deficiency [67–70]. Furthermore, the DNA damage caused by the collapse of the replication fork and increased DSBs as a result of PARP1 inhibition can be detected by the immune system particularly through the cyclic GMP-AMP synthase (cGAS) pathway [71]. Activation of this pathway subsequently enhances T-cell infiltration and promotes type I interferon expression, stimulating dendritic cell activity and reinforcing the immune response [25, 72]. Studies indicate that both talazoparib and olaparib can activate the cGAS-STING pathway, promoting T-cell infiltration in BRCA1-deficient models [30, 69, 73]. Additionally, research has shown that PARPis can increase PD-L1 expression by inactivating GSK3β, further supporting the rationale for combining these agents with immune checkpoint blockers (ICBs) [74] (Table 2). Preclinical studies have also shown promising results when anti-PD-1/L1 therapies are combined with PARPis such as olaparib and talazoparib, indicating their potential in effective combination strategies [65]. This is further supported by the phase II MEDIOLA trial which assessed the combination therapy of olaparib with durvalumab (an anti-PD-L1 antibody) in BRCA-mutant metastatic HER2-negative BC patients. The study reported a disease control rate (DCR) of 80% at 12 weeks and 50% at 28 weeks [75]. Common adverse events included anaemia (12%) and neutropenia (9%), with no significant drug interactions noted [76]. Further phase III trials are needed to validate the efficacy and safety, especially considering the higher toxicity of talazoparib compared to olaparib [77]. Moreover, results from the TOPACIO trial (phase II study) evaluating the efficacy of combination therapy, niraparib with pembrolizumab in metastatic TNBC showed overall response rates (ORR) of 45% and disease control rates (DCR) of 73% (experimental group), compared to 25% and 68% in the overall cohort. BRCA1/2 wild-type patients also benefited, with an ORR of 24%, suggesting therapeutic effects beyond specific mutations [67, 78]. Staniszewska et al. [63] further demonstrated that the combination of olaparib with ICBs leads to enhanced antitumor efficacy and immune modulation in BRCA-deficient tumours.

Table 2.

Summary of clinical trials investigating PARP Inhibitors in combination with immunotherapy.

Trial	Treatment Combination	Median PFS (months)	ORRa (%)
TOPACIO	Niraparib + Pembrolizumab	2.3	21%
MEDIOLA	Olaparib + durvalumab	8.2	63%
DORA	Maintenance olaparib + durvalumab v/s Olaparib alone	4 v/s 6.1	NA

^aData related to the OS unavailable.

Overall, recent clinical findings highlight the importance of PARPi and ICB (anti-PD-L1 and anti-CTLA-4) combination therapies in treating BRCA-mutated TNBC due to their ability to affect T-cell activation and promote apoptosis in cancer cells, consequently showing significant clinical benefits [79–81].

Despite the promise shown by PARPis and ICB combinations, there are challenges to be addressed, including resistance mechanisms and optimising combination strategies [30, 82]. The heterogeneity of BC subtypes also complicates the effectiveness of these therapies. Immune-related adverse events are a significant concern, with common toxicities including anaemia, neutropenia, and pancreatitis. The clinical toxicity of talazoparib is notably greater than that of olaparib, raising concerns about its combination with ICBs [67, 78]. Further clinical trials are necessary to validate the efficacy and safety of PARPis in combination with ICIs across different BC subtypes and patient populations.

Thus, the synergy between PARPis and ICIs offers a promising avenue for improving treatment outcomes for patients with BRCA-mutated BC. By exploring combination strategies and addressing resistance mechanisms, researchers aim to enhance the clinical efficacy of these therapies and improve patient outcomes in BC.

PARP inhibitors and antibody–drug conjugates

Antibody-drug conjugates (ADCs) and PARP Inhibitors represent a novel treatment approach, enabling the targeted delivery of potent cytotoxic agents directly to cancer cells [83]. ADCs are composed of three main components: a monoclonal antibody (mAb) that binds specifically to a tumour antigen, a cytotoxic agent that induces cell death, and a linker that connects the two. This structure enhances tumour specificity, improving the therapeutic index and reducing off-target toxicity [84–86]. Over recent years, ADCs have entered clinical studies across multiple cancer types.

One promising ADC is sacituzumab govitecan (Trodelvy), which combines SN-38, an active metabolite of the topoisomerase I inhibitor irinotecan, with a humanised monoclonal antibody (hRS7 IgG1κ) targeting the Trop-2 antigen. Trop-2 is overexpressed in many epithelial tumours, including breast cancer subtypes, with particularly high levels in TNBC [87, 88]. Clinical trials, such as the IMMU-132-01 phase I/II and the ASCENT phase III trials, have demonstrated durable responses in metastatic TNBC patients, making sacituzumab govitecan a strong candidate for combination therapies, including with PARP inhibitors [89]. Ongoing studies, such as the SEASTAR trial (NCT03992131), have further explored this combination, evaluating rucaparib (a PARP inhibitor) in conjunction with sacituzumab govitecan [53]. Despite promising early results, the SEASTAR trial was discontinued due to changes in development priorities. However, data from the trial indicated that further investigation into PARP inhibitor and ADC combinations is warranted, particularly to optimise dosing and reduce myelosuppression [89].

Another ADC of interest is datopotamab deruxtecan (Dato-DXd), which consists of a humanised anti-Trop-2 IgG1 monoclonal antibody linked to a potent topoisomerase I inhibitor via a stable tetrapeptide-based linker [87]. Dato-DXd offers advantages over sacituzumab govitecan due to its more stable structure, longer half-life, and lower haematologic toxicity. These properties make it a compelling candidate for combination with PARP inhibitors, as both agents induce DNA damage and interfere with DNA repair mechanisms [87]. The ongoing phase 1/2 PETRA trial, AZD5305 (a PARP1 inhibitor) has demonstrated safety and activity in a preliminary analysis of 40 patients, including those with BRCA mutated TNBC, with an ORR of 25% and a disease control rate of 53%. This agent is being investigated for its efficacy either as monotherapy or in combination with Dato-DXd in patients with advanced solid tumours [53]. While these early results are promising, more clinical trials are necessary to confirm the efficacy and safety of AZD5305 in BRCA-mutated TNBC patients.

Thus, early clinical data support the potential of combining ADCs with PARP inhibitors in treating cancers such as TNBC, further research is needed. Future studies should focus on optimising dosing strategies, minimising toxicity, and clarifying the contributions of each agent to the observed antitumor effects. These efforts are essential for advancing the use of these novel therapeutic combinations in clinical practice.

PARP inhibitors and PI3K/AKT pathway inhibitors

PARPis with inhibitors of the PI3K/AKT pathway, which plays a key role in regulating cell growth, survival, and metabolism, as well as acting as a sensor for DNA double-strand breaks (DSBs) within the DDR machinery [90]. Preclinical studies have demonstrated that silencing PIK3CA in BRCA wild-type, PTEN-mutant TNBC leads to an accumulation of phosphorylated histone-2AX (γ-H2AX), a marker of DNA damage and replication stress. The elevated γ-H2AX levels suggest that PIK3CA loss may enhance the effectiveness of PARPis, creating a potential synergistic relationship between these inhibitors [91, 92].

Further research supports this concept, showing that PI3K inhibition impairs BRCA1/2 expression and sensitises BRCA1/2-proficient TNBC to PARPis [93]. Additionally, studies have indicated that decreased mTOR activity correlates with a favourable response in TNBC, while high tyrosine kinase receptor activity and mTOR activation are associated with resistance [94]. These findings underscore the importance of refining biomarkers of treatment response to identify specific patient populations that may benefit most from such combination therapies.

However it has been primarily evaluated in BRCA mutated ovarian cancer and despite the promising results seen in ovarian cancer, the application of PARPi combinations in TNBC remains underexplored and requires further investigation [30]. Given the distinct molecular heterogeneity of TNBC, it is imperative to determine whether these strategies will yield similar therapeutic benefits in this challenging breast cancer subtype. More research is necessary to validate these approaches and optimise treatment protocols for TNBC patients facing PARPi resistance.

Integrating PARP inhibitors in the treatment paradigm for BRCA mutated TNBC patients

As the therapeutic landscape of breast cancer broadens, novel questions arise about the optimal use of PARP inhibitors. First, genetic testing is not universally offered to patients with breast cancer despite its potential therapeutic implications [95]. Second olaparib’s integration with available (neo) adjuvant therapies, such as pembrolizumab for TNBC is debated. KEYNOTE-522 trial, in which pembrolizumab was added to neoadjuvant chemotherapy and administered adjuvantly, event-free survival was prolonged, precluding adjuvant olaparib [96]. However, in the OlympiAD trial the patients did not receive pembrolizumab only Olaparib and yet significantly increased PFS and doubled ORR. Thus, whether patients with residual disease after neoadjuvant pembrolizumab-chemotherapy should receive olaparib, pembrolizumab, or both is unknown [97]. As patients with germline BRCA1 or BRCA2 variants are excluded from most of the ongoing trials in this setting, it is unlikely that randomised data will soon clarify which is the best approach for the patients [98]. Further, when patients with BRCA1/2-associated metastatic breast cancer should receive PARP inhibitors is also debated. For patients with TNBC and BRCA1/2, to our knowledge, no existing evidence suggests how PARPis should be prioritised to newer options of chemoimmunotherapy or ADCs (eg, sacituzumab govitecan and trastuzumab deruxtecan). Informative subgroup analyses by BRCA1/2 alterations from phase 3 trials have not been published. As in OlympiAD, olaparib had the highest benefit when administered as first-line treatment but did not improve survival compared with chemotherapy. It may be reasonable to optimise first-line immunotherapy-based regimens and favour second line olaparib while acknowledging the lack of data to support any sequential strategy in this setting. Furthermore, whether and to what extent the activities PARPis and platinum chemotherapy overlap is debated [98]. Their mechanism of action is similar, as are their sensitivity and resistance spectra. However, head-to-head comparison between platinum chemotherapy and PARP inhibitors are lacking, and little is known about the activity of PARPis after platinum chemotherapy, and vice versa. Results from BROCADE suggested that PARPis may improve outcomes when added to carboplatin [99] but opposite evidence emerged from BrighTNess [100]. Data from Gepa Rola, though not comparative in its design, suggested that olaparib and carboplatin may be equivalent in the early-stage setting [101]. Future studies combining platinum salts with less toxic PARP1 selective inhibitors may help to clarify whether these agents have synergic or redundant activity. If shown to be equivalent, PARP inhibitors may be privileged to platinum chemotherapy given the better safety profile; however, the different cost-effectiveness profiles should also be considered. Finally, the role of maintenance PARP inhibitors after chemotherapy should be clarified. The aforementioned data from the BROCADE3 [99] and DORA trial [102] support this strategy, and further efforts are ongoing (KEYLYNK-009 trial, NCT04191135).

Future directions in research and clinical practice

As the treatment landscape for BRCA-mutated TNBC evolves, several promising directions (Fig. 2) in research and clinical practice are being explored to address the challenges of this aggressive subtype.

Fig. 2. Potential future research directions in the treatment of BRCA-mutated TNBC.

Emerging treatment strategies for BRCA mutated TNBC focus on leveraging innovative approaches such as targeted inhibitors, personalized vaccines, and cell-based therapies to enhance patient outcomes. This roadmap illustrates promising areas of future research, aimed at driving advances and overcoming current therapeutic challenges for BRCA-mutated TNBC.

New-generation PARPis (Fig. 2.1) are being developed to address the limitations of current PARPis, such as resistance and off-target toxic effects, which have hindered their broader use in combination therapies. Among these novel agents, AZD5305 has shown promising pharmacokinetics, pharmacodynamics, and safety profiles in preclinical models. This agent exhibits higher selectivity for PARP1 over PARP2, leading to more stable PARP1 trapping and a strong antiproliferative effect. As mentioned above, AZD5305 demonstrated safety and activity in a preliminary analysis of the PETRA trial. While these early results are promising, more clinical trials are necessary to confirm the efficacy and safety of AZD5305 in BRCA mutated TNBC patients. Moreover, a newer, third generation series of compounds are in development [103], which are termed ‘DNA-locking’ PARPis. Unlike the first and second-generation compounds, such as Olaparib, talazoparib and saruparib, which are competitive NAD-mimetic compounds which can only work via synthetic-lethality, these compounds are allosteric modulators of the DNA-binding domain of PARP-1. As such, they are active via synthetic lethality but critically also by inhibiting DNA-replication in HR-competent tumours. Preclinical data show that they are active in both BRCA reversion-mutant and BRCA wild-type tumours.

In addition to PARP1-selective inhibitors, other novel therapeutic agents are also under development. These include dual inhibitors targeting PARP1 and intracellular molecules (such as histone deacetylases, DNA topoisomerases, and PI3K pathway effectors), PARP1 degraders using proteolysis-targeting chimera (PROTAC) technology, and PARP1-targeting prodrugs. These advancements aim to enhance the therapeutic potential of PARPis and expand their applicability across a broader patient population.

Furthermore, early clinical trials are investigating rational combination strategies (Fig. 2.2) to overcome PARPi resistance. These strategies include combining PARPis with ATR/Chk1/Wee1 inhibitors, antiangiogenic agents, and epigenetic modifiers (Table 3). These have been evaluated in ovarian cancer; however, they have the potential to be implemented in BRCA mutated TNBC. Further preclinical and clinical trials are needed to support this. Although initial analyses have shown promising responses and clinical benefits, the durability of these responses remains uncertain, and combination therapies often introduce additional toxicity. To optimise these approaches, further research is needed to identify meaningful molecular markers that can predict which patient groups will benefit most from specific combinations. Additionally, utilising circulating tumour DNA (ctDNA) analyses to monitor for BRCA and other HRR reversion mutations could help tailor treatment choices and optimise treatment duration. Investigating dosing schedules is also critical to minimising toxicity while maintaining therapeutic benefits beyond those expected from monotherapy.

Table 3.

Emerging combination therapies involving PARP inhibitors for BRCA-mutated TNBC (Adapted from [12, 30]).

Combination Therapy	Pathway of Action	Clinical Evidence
Combining PARPi + ATR, CHK1, and Wee1 Inhibitors	These combinations target the downstream effector molecules and signalling pathways of HR, showing clinical benefits irrespective of HR status. Cells with DNA damage rely on cell cycle checkpoints to activate DNA DDR mechanisms. Tumours with p53 mutations often depend on G2 checkpoints to induce DDR. Inhibiting ATR, CHK1, and Wee1 has been shown to restore HR and replication fork stability, resensitizing BRCA1/2-deficient tumours to PARP inhibitors (PARPis). Lee, Matulonis [127, 128].	1. Synergistic DDR and cell cycle regulation have resulted in tumour suppression in BRCA2-mutant patient-derived xenograft models. 2. Ongoing clinical trials: • CAPRI phase II study, Phase II trial with berzosertib and olaparib (NCT04065269), • Phase Ib trial with ATRi and niraparib (NCT04267939). • A phase II EFFORT trial demonstrated clinical benefit in PARPi-resistant ovarian cancer (NCT03579316). ORR for single agent adavosertib versus combination was 23% and 29%, respectively, with clinical benefit rates of 63% and 89%. • CHk1 inhibitor Prexasertib combined with olaparib in Ovarian cancer and other solid tumours showed partial response in 4 out of 18 BRCA1 mutant, PARPi-resistant ovarian cancer patients [129].
Combining PARPi + Antiangiogenic Agents	This combination leverages the synergistic effects of PARPis and antiangiogenic agents through two mechanisms: inhibition of BRCA1/2 expression and exacerbation of cellular hypoxia, leading to genetic instability. Hypoxia impairs HR and increases sensitivity to PARPis [130–132]. For example, in BRCA2-mutated ovarian cancer stem-like cells, chemosensitivity was restored through VEGFR3 inhibition [133].	1. The randomised phase II ENGOT-ov24 trial (NCT02354131) showed clinical benefit with bevacizumab and niraparib in platinum-sensitive recurrent ovarian cancer. 2. A phase II study of olaparib and cediranib versus niraparib alone demonstrated significant progression-free survival (PFS) and overall survival (OS) benefits in BRCA1/2 wild-type cohorts, though a subsequent phase III trial found no clinical benefit compared to chemotherapy [134].
Combining PARPi + Epigenetic Modifiers (BET and Histone Deacetylase Inhibitors)	Epigenetic modifiers involved in transcriptional regulation or chromatin remodelling can enhance PARP-related DNA damage, decrease BRCA1 levels, and increase genomic instability by disrupting replication fork progression. Preclinical data suggest that inhibiting bromodomains and histone deacetylases (HDACs) can enhance the activity of PARPis [135–137].	1. Ongoing trials: • Phase I/II study of entinostat and olaparib for recurrent ovarian cancer (NCT03924245) • Belinostat with talazoparib for multiple solid tumours (NCT04703920). 3. Initial dose-finding studies in hematologic malignancies showed that BET inhibition correlates with thrombocytopenia [138–140].
Combining PARPi + Ionising Radiation Therapy	Ionising radiation sensitises PARPi in HR-proficient tumours by exporting BRCA1 from the nucleus to the cytoplasm [141, 142]. Preclinical studies show reversal of acquired drug resistance through BRCA1-independent HR restoration [143].	1. Phase I trials (NCT01589419, NCT01264432) report good tolerance and responses in advanced rectal cancer and peritoneal carcinomatosis [144, 145]. 2. Ongoing trial (NCT01618357) evaluates veliparib with IR in breast cancer [146].
Combining PARPi + Proteogenomics	Proteomics, including mass spectrometry and protein arrays, identify new drug targets in BRCA mutated TNBC, improving therapy and reducing PARPi resistance [147]. TPX2, a PARP1-binding protein, enhances PARPi sensitivity by increasing PARP1 activity [148]. Tests like Oncotype DX, EndoPredict, and MammaPrint predict breast cancer recurrence risk, guiding clinical decisions [149, 150]. FoundationOne Liquid CDx trial - cfDNA-based assay predicts the efficacy of PARPi rucaparib in BRCA-mutated ovarian cancer and alpelisib in HR+/HER2−, PIK3CA-mutated breast cancer [151].	-

Moreover, cancer vaccines are an emerging immunotherapeutic strategy in the treatment of TNBC. These vaccines work by stimulating the immune system to recognise and attack tumour-specific antigens, thereby promoting an anti-tumour response [104]. Cancer vaccines can used either in combination with other conventional therapies or as a standalone treatment. Multiple current trials assessing the efficacy and safety of cancer vaccines have shown positive outcomes such as improved survival rates, tumour regression, and immune responses [105, 106]. Despite definite results, the clinical benefit of vaccines for TNBC treatment has not been conclusively established. This reinforces the need for large, randomised controlled trials providing additional efficacy and safety data.

Furthermore, combination of cancer vaccines (Fig. 2.3) with PARP inhibitors is a promising avenue, especially in the context of BRCA-mutant tumours. Dendritic cell-based vaccines may benefit from this approach. PARPi treatment of BRCA-mutant tumours activates the type I interferon (IFN) pathway via cGAS-STING signalling, which has multiple effects on the immune response, including the promotion of dendritic cell maturation, the release of interferons, and the initiation of anti-tumour T-cell responses [107]. This suggests that combining PARPis with dendritic cell-based vaccines could enhance the overall anti-tumour effect. Further, a peptide-based vaccine targeting Semaphorin 4A (Sema4A), a protein implicated in TNBC progression [108], has shown potential. Antigenic peptides derived from Sema4A have been identified and evaluated for their ability to stimulate immune responses. These peptides, combined with appropriate adjuvants and linkers, have demonstrated the ability to induce significant humoral and cellular immune responses in preclinical models [106]. These findings suggest that multi-epitope vaccines particularly personalised vaccines based on individual tumour antigens, could be a valuable addition to TNBC management [109], However, despite these advances, challenges remain in the development of effective cancer vaccines for TNBC. Key obstacles include identifying the most potent antigens, optimising vaccine formulations and delivery methods, and overcoming tumour-induced immunosuppression [110, 111]. Thus, further research is required to establish the safety and enhance the efficacy of these vaccines against TNBC.

Furthermore, Photothermal therapy (PTT) (Fig. 2.4) represents another innovative treatment approach, utilising near-infra-red (NIR) laser-absorbing agents such gold nanoparticles (AuNPs) to generate heat and ablate cancer cells upon NIR laser irradiation [112, 113]. Incorporating PTT with immunostimulatory monoclonal antibodies (such as anti-OX40 antibody), has shown substantial tumour regression in both treated and untreated tumours in preclinical models [114]. Thus, combination treatment of PTT and PARPis holds promise as the heat-mediated ablation by PTT would enhance the DNA damage induced by and drive the tumour cells into apoptosis more efficiently by PARPis [115]. This synergy is particularly helpful in surgical-resistant BRCA mutated TNBC as PTT provides a minimally invasive treatment option [112]. Overall, while there is no direct evidence of its efficacy in BRCA-mutated tumours, the potential to combine PTT with PARPis warrants investigation, particularly in BRCA-mutated TNBC cases.

Similar to PTT, Photodynamic therapy (PDT) (Fig. 2.5) has also gained attention as a minimally invasive treatment option for superficial tumours. PDT produces reactive oxygen species (ROS) in targeted cancer cells, inducing immunogenic cell death in TNBC cells through oxidative stress [115]. This stimulates dendritic cell maturation, enhances cytotoxic T lymphocyte (CTL) proliferation and intra-tumoral infiltration [116], resulting in TNBC, where immune resistance is a significant challenge, this enhanced immune activity results in ineffective inhibition of tumour growth, metastasis, consequently enhancing overall treatment efficacy [106]. Yao et al., [117, 118]. Alike PTT, PDT holds considerable potential in treating TNBC. Novel approaches such as self-cascading unimolecular prodrugs that combine PDT with chemotherapy possess the means to increase retention of photosensitizers in tumour tissues and have indicated their promise in clinical trial [119]. These developments may substantially broaden the use of PDT in combination strategies for TNBC. Further, when integrated with PARPis, the DNA damage caused by PARPis can be heightened by the oxidative stress induced through PDT, leading to an amplified therapeutic benefit. Thus, the combination of PARPis with PDT, could provide a synergistic approach to treatment for BRCA mutated TNBC [106, 119].

The efficacy and precision of non-drug treatment like PTT and PDT can be improved by the use of nanoparticle-based delivery systems such as multifunctional nanoparticles. These are designed to respond to environmental stimuli in the TME, enabling more precise targeting of tumour cells, particularly in BRCA-mutated TNBC [112, 115, 119]. Additionally, nanoparticles such as polymeric nanoparticles, liposomes, and dendrimers have revealed great potential in enhancing the delivery of PARPis while reducing systemic toxicity and off-target effects [112]. Overall, nanoparticle-based delivery systems possess great prospective in transforming treatment landscape for aggressive cancers such as TNBC and demand further research to evaluate their integration into PARPi-based treatment.

Moreover, another promising tool for TNBC therapy that has emerged is outer membrane vesicles (OMV) (Fig. 2.6). OMVs potent immunostimulatory properties and the potential for bioengineering offer an opportunity to enhance therapeutic efficacy [120]. Preclinical studies have demonstrated the potential of an OMV-based platform that combines photodynamic therapy, chemotherapy, and immunotherapy to completely eradicate TNBC in mice without observed side effects [106]. Given the immunostimulatory and tumour-targeting capabilities of OMVs, their combination with PARPis could provide a novel and effective treatment strategy, particularly in BRCA mutated TNBC. Additionally, a versatile biomimetic nanoplatform known as 4T1Mem@PGA-Ce6/Ola has been developed, combining the delivery of the photosensitizer Chlorin e6 (Ce6) and the Olaparib [121]. This platform exhibits excellent tumour-targeting capabilities and has shown potent synergistic anti-tumour effects under laser irradiation. The activation of the cGAS-STING pathway by this nanoplatform further enhances its therapeutic potential [121]. However, further preclinical and clinical research is necessary to fully establish the safety and efficacy of this approach.

Use of natural origin compounds (Fig. 2.7) for TNBC, particularly in combination with PARPis, has received significant attention due to their ability in improving efficacy and mitigating drug resistance of PARPis TNBC Compounds such as alkaloids, terpenoids, steroids, and flavonoids present a promising area of exploration for overcoming PARPi resistance [122]. Further, compounds such as cordycepin, ailanthone, polyphyllin III, and ursolic acid, possess apoptotic, anti-inflammatory and antioxidant properties, consequently showing potential in inhibiting cancer progression [123]. These compounds could also be a strong adjunct to PARPis, due to their potential to influence TME and have direct cytotoxic effects. Integration of these compounds into multimodal therapies would provide a cost-effective, low-toxicity strategy which would help reduce the adverse effects associated with PARPi treatment [123, 124]. Further, exploration in this area could create new possibilities for improving quality of life and overall survival of patient with TNBC.

Furthermore, CAR-T cell therapies (Fig. 2.8) are an inventive immunotherapeutic strategy which involves allowing T cells to recognize and target specific antigens expressed on the surface of tumour cells by genetically modifying the T cells to express a CAR. CAR-T therapy offers precise and potent therapeutic potential and is being explored as part of adoptive cell therapy advancements for treating TNBC [106]. However, to be successful, these approaches must address challenges related to tumour heterogeneity and immunosuppressive tumour microenvironments [125]. Several potential targets for BRCA mutated TNBC have been identified, some progressing to the clinical stages while others remain in the early phases of development (Table 4).

Table 4.

Potential targets for CAR-T therapy in BRCA mutated TNBC (Adapted from [152]).

Targets	Mechanism of Action and Target Pathways	Preclinical and Clinical Evidence
Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1)	ROR1 is a type 1 membrane-spanning tyrosine kinase receptor involved in embryonic development and malignancies such as B-cell chronic lymphocytic leukaemia (B-CLL), mantle cell lymphoma, breast cancer, and ovarian cancer [153–155].	ROR1-targeted CAR-T cells have shown effectiveness in preclinical models, including 3D breast cancer models, with evidence of tumour infiltration and immune response activation [155, 156]. Phase 1 trial (NCT02706392) demonstrated manageable side effects like mild cytokine release syndrome (CRS) and neurotoxicity, with some patients achieving stable disease
Mucin 1 (MUC1)	MUC1 is a heavily glycosylated protein expressed on glandular epithelial cells. The tumour-associated form, tMUC1, is overexpressed in 95% of TNBCs, with no detectable expression in normal breast tissue [157–159].	CAR-T cells targeting MUC1 variants have shown antigen-specific antitumor activity in TNBC models with minimal toxicity [158, 160–162]. Ongoing trials (NCT04020575, NCT04025216, NCT02587689) are evaluating the safety of second-generation CAR-Ts against MUC1. Early reports suggest the need for further dose-escalation studies [152].
Disialoganglioside GD2	GD2 is a surface antigen expressed primarily in peripheral pain fibres, neurons, and melanocytes, and is linked to neuroblastoma and 60% of primary TNBC tumours [163, 164].	GD2-targeted CAR-T cells demonstrated tumour growth suppression and the ability to eradicate disseminated malignant cells in TNBC models [165]. Further research is required to validate these findings, particularly given inconsistent in vitro results [165].
Mesothelin	Mesothelin, involved in cell adhesion, is overexpressed in TNBC and linked to oncogenic pathways including NF-κB, PI3K, and MAPK [166–168].	Mesothelin-targeted CAR-T cells showed tumour suppression in TNBC models, particularly when combined with PD-1 checkpoint blockade strategies [169]. Clinical trials (NCT02792114, NCT02414269, NCT01355965, NCT02580747) are ongoing.
Epidermal Growth Factor Receptor (EGFR)	EGFR, a member of the ERBB receptor tyrosine kinase family, is involved in cell proliferation and metastasis. EGFR is overexpressed in TNBC [170].	EGFR-targeted CAR-T cells have shown efficacy in preclinical TNBC models, with minimal off-tumour toxicity [146]. Tumour-restricted variants like EGFRvIII are being evaluated for safety. Trials are ongoing but results specific to TNBC are not yet published [171].
Folate Receptor Alpha (FRα)	FRα, a GPI-anchored transmembrane protein, is involved in active folate transport and is overexpressed in TNBC, correlating with disease grade and stage [172, 173].	FRα-targeted CAR-T cells have shown tumour suppression in preclinical TNBC models, though effectiveness was lower compared to other cancers like ovarian cancer. Dual-targeting strategies are being explored to enhance efficacy [174, 175].
Receptor Tyrosine Kinase c-Met	c-Met, or hepatocyte growth factor receptor (HGFR), is a tyrosine kinase involved in TNBC progression. Overexpression of c-Met correlates with poor overall survival [176, 177].	c-Met-directed CAR-T cells showed anti-tumour activity in TNBC cell lines and xenograft mouse models [178]. Phase I trials reported well-tolerated treatments, with some patients achieving stable or partial disease [178, 179].
Trophoblast Cell-Surface Antigen 2 (TROP2)	TROP2, a transmembrane protein, is associated with poor prognosis in epithelial malignancies and mediates tumour-associated behaviours [88, 180].	Bi-specific TROP2/PD-L1 CAR-T cells showed higher tumoricidal activity compared to mono-specific CAR-Ts in preclinical models. However, more research is needed to validate TROP2 as a specific CAR-T target in BRCA-mutated TNBC [181, 182].
Chondroitin Sulphate Proteoglycan 4 (CSPG4)	CSPG4 is a membrane-spanning glycoprotein involved in cancer progression and invasion, overexpressed in TNBC [183].	CAR-T cells targeting CSPG4 demonstrated tumour suppression, cytotoxicity, and cytokine secretion in preclinical models [184, 185]. Further clinical and preclinical data are necessary to support these findings.
Intracellular Adhesion Molecule-1 (ICAM-1)	ICAM-1 is a transmembrane glycoprotein that mediates leucocyte transmigration and is upregulated in TNBC [186, 187].	Preclinical studies showed effective tumoricidal activity and a favourable safety profile, but ongoing assessments are needed to confirm ICAM-1 as a viable CAR-T target in BRCA mutated TNBC [188, 189].
Natural Killer Group 2, Member D Ligand (NKG2DL)	NKG2DL is a stress-induced ligand overexpressed in TNBC, recognised by the NKG2D receptor [190].	NKG2DL-targeted CAR-T cells showed tumour growth suppression in preclinical TNBC models [43]. A Phase I clinical trial (NCT04107142) is assessing safety, with results pending.
Receptor Tyrosine Kinase AXL	AXL, part of the TAM receptor tyrosine kinase family, is involved in tumour cell survival, expansion, and invasion. Abnormal AXL expression has been observed in breast cancer [191–193].	CAR-T cells targeting AXL demonstrated significant tumoricidal activity in preclinical models. Enhancements like co-expressing a constitutively active IL-7 receptor further improved efficacy [194, 195].
Tumour Endothelial Marker 8 (TEM8)	TEM8, also known as anthrax toxin receptor 1, is involved in endothelial cell migration and invasion, and is overexpressed in TNBC, correlating with relapse risk [196–198].	TEM8-targeted CAR-T cells showed tumour suppression in preclinical TNBC models, but concerns about on-target off-tumour toxicity warrant further investigation before clinical trials [199].
Integrin Alpha V Beta 3 (αvβ3)	αvβ3 integrins are adhesion receptors involved in tumour cell migration, tissue invasion, and survival, with expression in TNBC [200, 201].	CAR-T cells targeting αvβ3 showed potent tumoricidal activity in preclinical models, though their efficacy in TNBC remains uncertain. Further research is needed to assess their therapeutic potential in BRCA-mutated TNBC [202, 203].

Additionally, small interfering RNAs (siRNAs) (Fig. 2.9) also represent a promising area of exploration. Research into the regulatory signalling axis between p53, miR-34a, and PD-L1 in TNBC cells has shown that p53 can inhibit PD-L1 expression through miR-34a, leading to increased apoptosis and cytotoxicity in TNBC cells [126]. These findings suggest that targeting this axis could be a novel therapeutic strategy for BRCA-mutated TNBC, particularly when combined with PARPis.

Thus, the integration of these innovative therapies with PARPis presents a promising future direction for the treatment of TNBC, particularly in patients with BRCA mutations. Further research is required to optimise these combinations, evaluate their efficacy, and determine their potential in clinical practice.

Conclusion

TNBC has been the subject of an enormous amount of molecular characterisation and therapeutic experimentation. It has become clear that TNBC represents a spectrum of underlying molecular pathologies, of which DDR mechanistic failure is a critical hallmark in at least 50% of patients

Understanding the mechanisms of resistance and action of PARPis is crucial in treating BRCA-mutated TNBC. With PARP1 playing a key role in the DNA damage repair process, particularly through its association with the facilitation of HRR and base excision repair Its inhibition by PARPis results in the accumulation of DNA damage in tumour cells lacking functional BRCA1/2 pathways, consequently promoting cell death. Nonetheless, emerging mechanisms of resistance, such as reduced PARP1 trapping, restoration of HRR, stabilisation of the replication fork and drug efflux demand further exploration into combination strategies that may improve treatment efficacy. Additionally, insights being gained from developing combination approaches, with second and third-generation PARPis, will also aid in establishing new rational combination strategies with chemotherapeutics, targeted agents and ADCs.

Further, RNA-based drugs are the most promising therapies in the treatment of aggressive breast cancers, like TNBC. The targeted mechanism of action, reduced drug interactions, and lower overall toxicity and adverse effects of biologics highlight the potential of these therapies. Furthermore, in the future, more emphasis on combination therapies that integrate drug-based treatments with non-pharmacological approaches, such as photothermal and photodynamic therapies is anticipated. These combinations could alleviate the psychological and emotional burden on patients caused by extensive drug regimens while potentially reducing the side effects associated with multi-drug treatments. Therefore, this integrated approach seems to be a promising direction for future treatment protocols.

In the next decade, the term ‘triple-negative breast cancer’ may become obsolete as our understanding of the diverse molecular pathologies underlying this aggressive cancer subtype deepens. The current classification of TNBC is overly simplistic and insufficient for guiding effective treatment strategies. Instead, we anticipate a shift towards a more nuanced segmentation of TNBC, with patients categorised based on specific molecular aberrations within their tumours. Further, this evolution in classification could pave the way for the integration of RNA-based therapeutics. These targeted drugs, with their potential to be tailored to individual patient profiles, will not only enhance treatment efficacy but also significantly advance the field of personalised medicine. Thus, offering hope for more durable responses and improved outcomes for patients with what was once considered an intractable disease.

In conclusion, the future of TNBC treatment hinges on the development of targeted, personalised therapies that address the unique challenges of this aggressive subtype. While progress has been made in understanding TNBC’s molecular mechanisms and developing new treatments, further research is essential to optimise these strategies for clinical use. Exploring combination therapies, enhancing vaccine efficacy, and integrating multi-omics data will be crucial in advancing more effective, tailored treatments. Addressing drug resistance, especially to PARP inhibitors, remains a critical challenge, making the exploration of combination strategies and new mechanisms vital for improving treatment outcomes.

Author contributions

AJ was responsible for conducting the search, writing the report and creating the tables and figure. AB provided feedback on the report. CP contributed to the creation of Fig. 1 and provided feedback on the report.

Competing interests

The authors declare no competing interests.