Exploiting synthetic lethality in epithelial ovarian cancer: multi-dimensional approaches beyond DNA damage repair.

Introduction
Epithelial ovarian cancer is an often fatal and heterogeneous malignancy that commonly presents at an advanced stage that can acquire treatment resistance. It consists of several molecular and histologically distinct subtypes such as high-grade serous, low-grade serous, endometrioid, clear cell, and mucinous carcinoma, that differ in their site of origin, biology, and therefore in their clinical behavior [1]. Among these, high-grade serous ovarian cancers (HGSOC) is the most common and lethal epithelial subtype, and the one in which synthetic lethal (SL) and DNA damage response (DDR) targeted strategies have been most extensively studied [2]. Accordingly, this review will primarily focus on HGSOC and related epithelial subtypes with homologous recombination (HR) deficiency (HRD).
Despite this high heterogeneity, most patients with epithelial ovarian cancer, particularly HGSOC, exhibit an initial high sensitivity to platinum-based chemotherapy in the front-line setting. However, the overall prognosis remains poor due to a high relapse rate as 80% of patients will relapse within three years [1, 3]. Ovarian cancers’ chemosensitivity can be partly attributed to defects in the HR repair pathway [4, 5]. Approximately 50% of HGSOC exhibit HRD [6], notably driven by BRCA1/2 mutations and other related mechanisms. One promising treatment strategy to overcome the high relapse rate is the application of a SL approach, whereby the concurrent disruption of two genes/pathways leads to cell death, whereas blocking either of the targets alone is well tolerated. This strategy generally exploits cancer-specific genetic vulnerabilities to target tumor cells selectively [7]. Over the last two decades, the emergence of poly ADP-ribose polymerase (PARP) inhibitors (PARPi) has completely revolutionized the oncologic management of ovarian cancers by exploiting this SL [8–10]. Maintenance therapy with PARPi has been established as the standard of care for patients with advanced-stage HGSOC who achieve a complete or partial response to platinum-based chemotherapy [11]. Patients harboring a germline or somatic BRCA1/2 mutations derive the most significant benefit (See Fig. 2). In contrast, among BRCA wild-type patients, the progression-free survival benefit seems to be more pronounced in those with HRD compared to the HR proficient population [12–14]. The optimal sequencing of therapies in ovarian cancer, mechanisms of de novo acquisition of resistance and the use of novel combination therapies remain areas of sustained interest [7, 15]. In general, DNA repair specific SL targets have been extensively explored with the goal of increasing the amount of DNA damage generated. DNA repair is highly integrated within and regulated by the DNA Damage Response (DDR), a signaling cascade that plays a major role in maintaining genome integrity through sensing, signaling, and repairing lethal DNA double strand breaks (DSB). Not surprisingly, dysregulation within the DDR pathways can both drive cancer progression and create therapeutic opportunities. DDR components are generally categorized into DNA damage sensors, signal transducers, and effectors like cell cycle regulators [16]. Key DDR players, such as ATM and ATR kinases and BRCA1/2 proteins, can have several different mechanistic roles in DDR and their study reveal how interconnected DDR pathways and consequences of DDR activation can be leveraged for targeted therapies via SL [17]. For instance, it has been demonstrated that targeting cyclin-dependent kinases (CDK) and taking advantage of cancer-specific instability of the cell cycle machinery can improve traditional treatments and lower resistance [18, 19]. Targetable opportunities can also arise from metabolic changes brought on by treatment. For example, damaged cancer cells are susceptible to specific nutritional deprivation or antimetabolites since they frequently depend on glutamine for nucleotide biosynthesis [20]. Overall, changes in the metabolism of lipids, glucose, and amino acids identify pathways that can be targeted [21]. Epigenetic modifications also play a crucial role in ovarian cancer adaptation to treatments, and it is known mutations in chromatin remodelers can result in survival benefits via interruption of gene expression [22]. Finally, cell fate decisions like death or senescence that are consequences of DDR signals can also be manipulated (Fig. 1) [23]. In summary, expanding SL applications beyond DNA repair pathways provide new therapeutic alternatives that can address current limitations in ovarian cancer therapy including patient heterogeneity.

DDR-dependent synthetic lethality
DSB are the most lethal type of DNA damage and most DNA damaging anticancer therapies aim to generate this type of DNA lesion either directly or through conversion of single-strand DNA breaks (SSB). In response to DSB, a cell activates the multistep DDR signaling cascade [16], and many steps of this process provide essential vulnerabilities amenable to SL strategies in the context of ovarian cancer. It seems logical to explore these potential vulnerabilities in the overall context of the many steps composing the DDR from DNA damage generation to downstream consequences (Fig. 2).

Sources of DDR
During cancer treatment DSB arise from a variety of sources including chemotherapy and DNA replication stresses leading to the conversion of SSB into DSB [30]. Notably, cancers often harbor defects in diverse DNA repair pathways leading to the spontaneous accumulation of DNA lesions at higher levels than normal cells, which is the case for ovarian cancers that harbor BRCA1/2 mutations or HRD in general (also termed BRCAness) [2]. BRCA1 and BRCA2 function as tumor suppressors and are essential for HR repair [32]. Individuals harboring mutations in these genes are at high risk of developing ovarian and breast malignancies, which makes them ideal candidates for SL approaches [33]. As mentioned above, by targeting cancer cells that have BRCA mutations, PARPi take advantage of the lack of HR by crippling the key residual DNA repair mechanisms causing additional replication stress and the accumulation of DNA damage provoking cell death or senescence [7, 9, 23, 34]. This classical SL combination is illustrated in Fig. 2 where BRCA1/2 mutations block HR while PARP1 inhibition blocks SSB repair essential to survival in the absence of HR (Fig. 2a and b). Likewise, BRIP1 (BRCA1 Interacting Protein C-terminal Helicase 1) functions in DNA repair via its interaction with BRCA1, playing a vital role in HR, suggesting BRIP-deficient cells are amenable to SL PARP inhibition [34]. In clinical trials, the classical PARP-HR SL approach has demonstrated high response rates [1, 7, 8], and the PARPi olaparib, niraparib, and rucaparib are currently approved for clinical use in the treatment of ovarian cancer. Nevertheless, challenges remain, including PARPi inherent or acquired resistance and the search to extend their benefits to a broader patient population via the identification of novel compatible vulnerabilities [7, 23]. For example, in addition to the well described PARP1 targeted by PARPi in the clinic, the PARP family includes many members [35]. PARP14 plays a role in HR by controlling DNA replication fork stability and PARP14 deficiency is SL with ATR-CHK1 pathway blockage [35]. PARP14 interacts with PCNA and optimizes HR and the replication of common fragile sites, which when dysregulated may result in DSBs formation through replication fork collapse [36]. Another contributor to DSB formation is poly (ADP-ribosyl) glycohydrolase (PARG), which regulates poly (ADP-ribose) polymer balance and recycling, keeping the dynamics of cellular stress responses stable [37]. By interfering with these activities, PARG inhibition exposes cells to DNA replication-related vulnerabilities [37, 38]. PARG inhibitor-sensitive cells often exhibit down-regulated DNA replication genes, including fork protection components such as TIMELESS. The interaction between TIMELESS and PARP1 prevents single-stranded DNA gap accumulation. Notably, TIMELESS haploinsufficiency mimics intrinsic PARG inhibitor sensitivity, leading to accumulation of single-stranded DNA gaps that may collapse into DSB [38]. Another class of enzymes that control DSB modulation are topoisomerases. Topoisomerase II (Top2) is a nuclear enzyme that resolves topological problems during DNA replication by creating controlled DSB [39]. Top2 inhibitors like doxorubicin and etoposide induce irreparable DSB and are already essential in cancer therapy, but despite their effectiveness, cancer cells can develop resistance [39]. Not surprisingly then, additional strategies are required to induce SL via co-targeting of Top2 and the DDR machinery or other oncogenic mutations [39]. In general, the strategies described above aim to directly generate more DNA damage and will consequently generally increase DDR signaling in the cells (Fig. 2a and b, see DSB and SSB boxes).

DDR sources—alternative DNA repair
Basic DNA repair mechanisms like base excision repair (BER), nucleotide excision repair (NER) and mismatch mediated repair (MMR) normally work upstream of the DDR because successful execution prevents the creation of DSB while defects in these pathways elevate levels of persistent DNA lesions that can be converted to SSB and DSB (Fig. 2, SSB box) [30]. FEN1 and APEX2, two DNA processing enzymes, were found to be SL targets in BRCA1/2-deficient cancers [40]. FEN1 (Flap Endonuclease 1) is involved in DNA replication and repair and has shown broad SL with chromosome instability genes [41]. FEN1 inhibitors decrease tumor growth in mouse models and specifically eliminate BRCA1/2-defective cells [41]. Apurinic/apyrimidinic endonuclease APEX2 is necessary for BRCA1/2-deficient cells to survive [40]. Its main function is to reverse 3' DNA blocks, which are especially troublesome for cells lacking HR [42]. Both FEN1 and APEX2 participate in alternative DNA repair pathways, such as BER and microhomology-mediated end-joining (MMEJ), which become critical in a BRCA1/2-deficient context [40]. Similarly, DNA Polymerase Theta (Polθ) has become a potential target for cancer treatment, especially in tumors that are HR-deficient [42, 43, 43–45], and plays an important role in MMEJ [43–45]. A synthetic lethal relation between HR insufficiency and Polθ inhibition exists, which makes it an attractive therapeutic strategy [44, 45]. As a specific Polθ inhibitor, the antibiotic novobiocin (NVB) has shown promise in preclinical models for treating HRD breast and ovarian tumors, including those that are PARPi-resistant [43]. Likewise, combined inhibition of non-homologous end-joining (NHEJ) and MMEJ pathways has shown promise in treating cancers with mutations in TP53 [46], a central transcription factor in DDR [16]. Specifically, simultaneous inhibition of DNA-dependent protein kinase (DNA-PK) and Polθ resulted in SL in TP53-deficient models. This combination may provide a precision treatment strategy for TP53-mutant solid tumors that account for half of all newly diagnosed cancers and is prevalent in high grade serous ovarian cancer [25, 46].

DNA damage detection: the first line of genome defense
DSB or the presence of extended stretches of single-strand DNA during DNA replication initiate DDR signals and the accurate detection of this DNA lesion is essential to initiate downstream events. Since DDR sensors lie close to DNA, alterations in their function also often results in simultaneous defect in DNA repair and DDR signalling. For example, MRE11 is a key component of the MRN complex (MRE11-RAD50-NBS1), which is an essential DSB sensing tool that also optimizes DNA repair to sustain overall genomic stability [47]. In ovarian cancer the overexpression of MRE11 is associated with platinum resistance and a poor prognosis, whereas its inhibition can reverse platinum resistance and cause SL in ovarian tumors that lack XRCC1 [27], an important coordinator of several DNA repair enzymes during SSB repair [24]. In a similar context, the micro-RNA miR-506 plays a unique role by interfering with RAD51 and RAD17, proteins that aid cells in identifying and responding to extended single strand DNA stretches [28, 48]. By targeting these damage-sensing components via mRNA interference, miR-506 makes ovarian cancer cells inefficient in handling DNA damage and thus sensitizes them to platinum-based chemotherapy and PARPi, creating a phenotype similar to the loss of BRCA1/2, a case of "BRCAness" [28, 48, 49]. This sensitization is validated both in vitro and in vivo, with miR-506 expression linked to improved progression-free survival and overall survival in patients [48]. The miR-506/RAD17 axis also dysregulates cell cycle checkpoints, potentially resulting in mitotic catastrophe in cancer cells [28]. Given the high level of redundancy in DDR processes, it is easy to imagine that there are many additional SSB or DSB detection components that remain unexplored as potential synergistic targets in the context of ovarian cancer.

Signal transduction: boosting the damage response
Following damage detection, the DDR is initiated/amplified within minutes on the chromatin surrounding the source, and eventually within the cell [47]. Importantly, alterations in DDR transduction can lead to complex outcomes given downstream functions like cell cycle control, epigenetic and metabolic regulation, or cell fate decisions. Signal propagation is performed by essential signal transducer kinases like ATM and DNA-PK (DSB) or ATR (single-strand DNA stretch), which themselves regulate downstream kinases like CHK2 (ATM), CHK1 (ATR), WEE1 and PKMYT1 [16, 50]. Most of these kinases are present in the cell constitutively allowing quick propagation of the DDR signal via a phosphorylation cascade [47]. ATM is an apical kinase that can function in any phase of the cell cycle to modulate DNA repair, cellular metabolism, and can transmit the DDR signal to hundreds of interlaced substrates with far reaching consequences [51]. ATM mutations are prevalent in various cancers affecting chemotherapy resistance and patient prognosis or immune responses [51]. Notably, ATM insufficiency shows therapeutic potential beyond the typical BRCA mutations by sensitizing mantle cell lymphoma [51] and prostate cancer cells [52] to PARPi. Although ATM mutations are less common in ovarian cancer, ATM-deficient ovarian tumors may still be sensitive to PARPi, which can be further investigated [51]. Similarly, by initiating checkpoint pathways that halt the progression of the cell cycle, the apical ATR kinase coordinates responses to extended single-strand DNA stretches following replication stress during S phase [29, 51, 53]. A powerful ATR inhibitor (ATRi), Camonsertib, was developed to treat advanced solid tumors that present DDR defects [53]. The therapeutic benefits of Camonsertib were greatest in ovarian cancer and tumors with biallelic loss-of-function mutations in DDR-related genes, according to a phase 1 trial that showed a good safety profile [53]. Other ATRi have demonstrated promise as well [53]. Nevertheless, clinical experience with ATR inhibitors, including camonsertib, has revealed frequent grade 3–4 hematologic toxicities (e.g., anemia, neutropenia and thrombocytopenia), which highlights the potential for severe adverse events with this class of inhibitors [53, 54]. Interestingly, recent studies show that PARPi treatment increases reliance on the ATR/CHK1 pathway to compensate for replication stress and maintain genome stability, suggesting a strategy to overcome resistance [55]. Indeed, combining PARPi with inhibitors of ATR or CHK1, a downstream target of ATR, synergistically decreases cancer cell survival, induces premature mitotic entry, and increases chromosomal aberrations and apoptosis [55, 56]. This combination has shown promising results in BRCA-mutated ovarian cancer models, including tumor regression in patient-derived xenografts [55]. Notably, the PARPi-ATRi combination is effective against diverse PARPi and platinum-resistant contexts, including those with BRCA reversion mutations and oncogenic CCNE1 amplification [55]. Overall targeting either ATR, CHK1, or WEE1 (see Fig. 2) can sensitize HR-proficient tumors to PARPi by inducing HR deficiency, leading to SL. However, targeting the ATR-CHK1-WEE1 pathway also disrupt DNA replication stress checkpoints, potentially compromising genome integrity in surviving cells with unknown consequences [17].

DDR outcomes
The choice between DNA repair pathways is highly dependent on phases of the cell cycle [30]. Consequently, the modulation of cell cycle regulators within the DDR like CHK1/2 and WEE1 or the direct modulation of cell cycle components introduce a layer of control over DNA repair outcomes and can influence DNA repair-related SL strategies. Cyclin-dependent kinases 4 and 6 (CDK4/6) are essential for driving the cell cycle entry into the G1 phase towards S by phosphorylating and inactivating the retinoblastoma protein (RB), a key regulator of cell cycle progression [5]. CDK4/6 inhibition arrests cells during G1, reducing the opportunity to repair DNA via HR, which is active during the S and G2 phases. Simultaneously, PARP inhibition leads to an accumulation of DNA damage that becomes lethal when repair is compromised. Thus, inhibiting CDK4/6 combined with PARPi might results in SL. Subsequently, one study shows that the dual inhibitor ZC-22 of CDK4/6 and PARP demonstrated higher efficacy in suppressing tumor growth and enhancing cisplatin responses in breast and ovarian cancers, regardless of HR status; however, the exact rationale behind this SL is not completely understood [18]. Although suppression of HR genes via the E2F/RB axis [57], PARP1 degradation [58], and induction of therapy-induced senescence and immune modulation via cGAS/STING [59] might offer plausible synergy routes independently of classical HR in selected contexts, the fact that PARPi are most effective during S phase, by amplifying DNA damage progressively through trans cell cycle DSB production [60], suggests that other mechanisms may be more responsible for this synergy, and results should be interpreted with caution. Additionally, one other study has suggested that PARPi administration followed by CDK4/6 inhibition is more effective than concurrent dosing or the reverse order [19]. However, the potential strategy of administering CDK4/6i first, followed by PARPi, has not been systematically investigated. Given that transient CDK4/6 inhibition could synchronize tumor cells and potentially re-sensitize them to PARPi through replication stress upon release into S phase [61], this approach could also be explored as a future therapeutic strategy. PKMYT1 is another cell cycle checkpoint regulator that blocks CDK1 to prevent the G2/M transition [62]. PKMYT1 inhibition is especially effective in cancers with CCNE1 amplification, which rely on supressed CDK1 activity to prevent untimely cell M phase entry and treatment resistance [31]. More specifically, by inhibiting PKMYT1 and releasing CDK1 activity, the compound RP-6306 promotes unscheduled entry into mitosis and genomic instability. This disruption improves the therapeutic effects of gemcitabine by utilizing the increased sensitivity of these cells to DNA damage [31]. Phase I clinical trials are now being conducted to assess RP-6306 [31]. Similarly, WEE1, another cell cycle regulator that inhibits CDK1 and CDK2, maintains genome stability by preventing premature entry into mitosis [62]. In HGSOC, their combined inhibition at low doses eradicates ovarian cancer cells by promoting CDK1 activation, exacerbating DNA replication stress, and increasing genomic instability. WEE1 and PKMYT1 are thus crucial in cell cycle regulation and DNA damage repair [50]. Additionally, combining BI6727, a polo-like kinase 1 (PLK1) inhibitor, with paclitaxel has effectively induced mitochondrial apoptosis in HGSOC, particularly in CCNE1-amplified cells. PLK1 is crucial for proper cell cycle progression and mitotic entry; its inhibition leads to mitotic arrest. When combined with paclitaxel, which stabilizes microtubules and prevents their disassembly, there is enhanced disruption of mitosis, leading to apoptosis in cancer cells predisposed to mitotic instability due to CCNE1 amplification [63]. Another factor that controls the cell cycle, DNA repair, and apoptosis is casein kinase 2 (CK2). CK2 inhibition increases genomic instability in cancers with a deficiency in RB1, which already have impaired cell cycle regulation, making them more susceptible to treatment. This approach shows potential in treating aggressive cancers such as triple-negative breast cancer (TNBC) and HGSOC [64].

Emerging concepts in SL
In addition to genetic interactions, recent developments in SL have investigated the use of specific cellular decisions to improve the efficacy of cancer therapy. Cell-state-specific synthetic lethality comes from distinct developmental or functional stages in cancer cells, where selective tumor cell death is achieved by exploiting or manipulating specific cellular responses. This includes both presence of a particular state and the precise timing for an intervention.

Exploiting senescence in combination therapies
Targeting senescent cancer cells was the basis of a strategy that combined PARPi with a senolytic drug, which are a class of drugs that selectively kill senescent cells [23]. Senolytics are drugs that selectively force senescent cells to undergo cell death by targeting their pro-survival mechanisms, and many classes of senolytics can be investigated with context-dependent specificities including complex screening strategies [65, 66].
PARPi such as olaparib cause DNA damage in breast and ovarian cancer cells, which results may result in apoptosis but also in some cells leads to senescence and cell cycle arrest. These senescent cells are associated with tumor resistance and relapse that may be related to their secretory phenotype [23] which promotes a pro-tumorigenic environment [67]. The combination of Olaparib with the senolytic agent navitoclax (a BCL-2 anti-apoptotic family member inhibitor) effectively eradicated senescent ovarian cancer cells [23]. Compared to either treatment alone, this combination more effectively inhibits tumor growth and drives cell death. By removing senescent cells, this therapy potentially prevents tumor recurrence and overcomes resistance mechanisms associated with the senescent cells [23]. In this context, therapeutic success depends on allowing sufficient time for the senescent state to fully establish before applying senolytics, highlighting the need to consider temporal dynamics as an integral component of cell-state-targeted SL approaches (Fig. 1).

Inducing ferroptosis in combination therapies
PARPis have traditionally been thought to target DNA repair defects in BRCA-mutated cells in order to achieve their anticancer effects through SL [7, 9, 10, 68]. Recent research, however, indicates that the alternative mode of cell death ferroptosis might play a role. It has been demonstrated that the p53-dependent downregulation of SLC7A11 results from PARP inhibition [69]. Glutathione production and defense against lipid peroxidation depend on SLC7A11, a member of the cystine/glutamate antiporter system, and on the activity of GPX4, a glutathione-dependent peroxidase that detoxifies lipid hydroperoxides. Downregulation of SLC7A11 by PARPi results in increased lipid peroxidation, impaired GPX4 function and ultimately induction of ferroptosis. Ferroptosis is a type of regulated cell death marked by the accumulation of lipid hydroperoxides that is dependent on iron and controlled by the metabolism of amino acids, iron, and lipids, as well as glutathione biosynthesis. This mechanism reveals SL between PARP inhibition and the reliance of cancer cells on SLC7A11 for survival in the context of functional p53 and proposes that PARPi may be a therapeutic option even for epithelial ovarian cancer patients carrying wild-type p53 and BRCA1/2 [69]. Similarly, combining PARP inhibitors with the GPX4 inhibitor RSL3 further enhances ferroptosis and produces synergistic anticancer effects (Fig. 3, see SLC7A11 and GPXi). Although wild-type TP53 is rarely seen in HGSOCs, it is more frequent in other epithelial ovarian cancer subtypes; thus, this mechanism is particularly relevant for the subset of BRCA proficient, p53 wild-type epithelial ovarian cancers (e.g., low-grade serous) [25]. In these patients, PARPis, alone or in combination with ferroptosis inducers, may extend the utility of SL-based treatment beyond the classical BRCA-mutated/HRD setting [69]. Interestingly, this may also, in theory, extend to a subset of HGSOC that retain one wild-type TP53 allele together with a mutant allele (e.g., a dominant-negative allele that functionally results in loss of p53 activity) [70–73]. In this context, preclinical studies shown that p53-restoration approaches can relieve the effect of the mutation and restore wild-type p53 function [73–75], potentially rendering this subset of HGSOC patients amenable to p53-dependent SL strategies.

Exploiting hypoxia-induced DNA repair deficiencies
Hypoxia, a common feature of the tumor microenvironment, contributes to genomic instability in part by inhibiting DNA repair pathways, particularly HR. Multiple pre-clinical studies have shown that both chronic and acute hypoxia can reduce HR capacity by downregulating genes such as BRCA1, BRCA2 and RAD51, and by perturbing other related repair pathways. This hypoxia driven impairment of HR increases the dependence of cancer cells on alternative, less reliable and more error-prone repair processes, and creates a functional HRD state [79–81]. Importantly, experimental work from the Glazer and Bristow groups indicates that the magnitude and persistence of this HR suppression depends on both the severity and duration of hypoxia, with chronic moderate hypoxia and severe acute hypoxia each creating biologically distinct HRD states [79–81]. Building on these observations, Chan and colleagues introduced the concept of “contextual synthetic lethality”, in which PARP inhibition preferentially kills hypoxic cells with a microenvironment induced HRD even in the absence of germline BRCA mutations [81]. Though mechanistically plausible, this phenomena may be of limited value, as clinical and experimental data indicates that hypoxic tumor regions are typically poorly vascularized, characterized by reduced proliferative activity, classically resistant to radiotherapy and many chemotherapies, even though hypoxia can downregulate HR genes [82, 83]. These apparently opposing effects of hypoxia on DNA repair and treatment response suggest that the spatial and temporal dynamics of hypoxia induced HRD are not yet fully understood and warrant further investigation to define when and where contextual synthetic lethality can operate in patients.
Interestingly, some preclinical and clinical studies have used this contextual SL rationale to explain the efficacy of combining cediranib with PARP inhibitors [81, 84, 85]. Cediranib, an antiangiogenic medication, can promote HRD via two distinct mechanisms: a hypoxia-dependent pathway (through VEGF pathway inhibition and induction of tumors hypoxia) and a hypoxia-independent pathway (through a PDGFR–PP2A–E2F4/RB2 axis), both of which downregulate RAD51 and BRCA1/2 [84]. Even in HR-proficient cells, this effect renders tumors more sensitive to PARPi, providing a possible therapeutic approach for patients without prognostic biomarkers. Clinical trials have demonstrated improved progression-free survival when combining cediranib with PARPi in ovarian cancer patients [85]. Overall, these data support the idea that cediranib–PARPi combination can present a multi-dimensional SL, in which hypoxia-dependent and hypoxia-independent effects of cediranib converge on HR and survival signalling [86, 87] to create and maintain an HRD, although the full dynamics of this interaction in human tumors remain to be elucidated.

Metabolic synthetic lethality
Metabolic SL leverages the tumor’s aberrant nutrient and energy processes, causing cell death when metabolic vulnerabilities are targeted alongside other essential pathways [26, 88, 89]. Cancer cells often exhibit altered redox homeostasis, making them vulnerable to therapies that induce oxidative stress or disrupt antioxidant defenses. One interesting method to induce SL in these cells is to target the redox imbalance. For instance, the expression of SLC7A11 is controlled by ARID1A, a subunit of the SWI/SNF chromatin remodeling complex. Because ARID1A-deficient cancer cells express SLC7A11 poorly, their basal Glutathione (GSH) levels are lower, making them vulnerable to GSH synthesis inhibition. When GSH synthesis or its essential enzymes, such as glutamate-cysteine ligase synthetase catalytic subunit (GCLC), are inhibited, ARID1A-deficient cells produce excessive reactive oxygen species (ROS) and die (Fig. 3, see GCLCi) [89]. This metabolic dependency offers a promising therapeutic strategy for targeting ARID1A-deficient cancers. Glutamine metabolism is another significant factor in maintaining redox balance in cancer cells, especially those with MYC overexpression. When glutaminase (GLS), a key enzyme in glutaminolysis is inhibited, redox balance and nucleotide synthesis are disrupted resulting in oxidative stress and cell death. In MYC-high ovarian tumors the combination of GLS inhibitors with PARPi or thioredoxin reductase inhibitors has demonstrated synergistic anti-tumor effects [90, 91]. Furthermore, glutamine-dependent reductive carboxylation becomes the main pathway for lipid synthesis and citrate production in tumors with damaged mitochondria, promoting development despite compromised oxidative metabolism and causing redox imbalance [92]. Targeting this mechanism may, therefore, limit their ability to survive. Interestingly, targeting GLS could also interact with the ability of the cell to protect itself against ferroptosis (Fig. 3, see GLSi).
Redox imbalance in cancer cells is further exacerbated by metabolic changes, including oncometabolites. The glycolytic metabolite methylglyoxal may bypass the "two-hit" tumor suppression concept and cause mutational signatures linked to cancer progression, via the promotion of BRCA2 degradation, causing elevated DNA damage. These studies may help explain how metabolic disorders contribute to carcinogenesis by further connecting glycolysis activation to the evolution of cancer [77]. It has been demonstrated that ovarian cancer cells of origin exhibit increased glycolysis when BRCA1 is lost [93]. The glycolytic phenotype in cancer is associated with resistance to apoptosis and suppressed mitochondrial function, which may be reversible [94]. Similarly, the oncometabolite 2-hydroxyglutarate, produced by Isocitrate dehydrogenase1/2 (IDH1/2) mutations, impairs HR and alters redox balance, sensitizing cancer cells to PARPis. IDH1/2 enzymes normally catalyze the conversion of isocitrate into α-ketoglutarate (αKG) as part of the tricarboxylic acid cycle (TCA) (Fig. 3, see TCA cycle) [78].
Folate-mediated one-carbon metabolism (FOCM), which supports essential cellular functions and aids in the growth of tumors, is a significant factor in epithelial ovarian cancer. Proton-coupled folate transporter and folate receptor alpha (FRα) are overexpressed in most epithelial ovarian cancers, while tumor-associated macrophages express FRβ. Folate receptors have a unique expression pattern which makes them attractive candidates as therapeutic targets. Antibodies like farletuzumab and folate-chemotherapy conjugates like vintafolide are examples of this. Targeting other important enzymes in FOCM, such as methylenetetrahydrofolate dehydrogenase 2 (MTHFD2) and serine hydroxymethyltransferase 2 (SHMT2), has also been investigated (Fig. 3, see Folate One-Carbon). These strategies seek to take advantage of the metabolic weaknesses of epithelial ovarian cancer cells while reducing the toxicity [95]. While FOCM is essential for nucleotide biosynthesis and epigenetic regulation, processes that are fundamentally integrated with DDR and repair [95], the specific connection between one-carbon metabolism and DDR activity in ovarian cancer has not been systematically investigated. Most studies described these pathways separately, without directly examining how fluctuations in FOCM influence repair efficiency or therapeutic sensitivity. However, given its central role in supplying nucleotide precursors and methyl donors, particularly for thymidylate synthesis and dNTP pool balance, it is plausible that disruptions in this metabolic network could modulate DDR capacity and shape treatment outcomes by increasing genomic uracil incorporation and DNA strand breakage [96]. Interestingly, recent work has also drawn attention to uracil and uracil modified metabolism as another way to exploit such metabolic dependencies in HRD tumors. Fugger et al. showed that the nucleotide salvage enzyme DNPH1 cleaves the modified uracil nucleotide hmdUMP and thereby limits incorporation of 5-hydroxymethyl-deoxyuridine (HMdU) into DNA; when DNPH1 is lost or inhibited, HMdU accumulates in genomic DNA and BRCA1/2-deficient cells become highly sensitive to PARP inhibitors through SMUG1-initiated base excision repair intermediates [97]. Although these studies mainly investigated breast and colorectal HRD models, the combination of BRCA loss and PARPi treatment closely resembles those of HRD ovarian cancer [97]. In a related approach, Musiani and colleagues targeted the uracil excision pathway and found that UNG suppression forces uracil processing through SMUG1, causing accumulation of abasic sites, PARP1 overactivation, replication stress and chromosome breakage in BRCA1/2-deficient tumors, including PARPi-resistant models [98]. Mechanistic work further supports this model by examining how abasic sites are handled at stalled forks. Using SMUG1 to generate abasic sites in replicating DNA, Hanthi et al. showed that BRCA2–RAD51 filaments are required to protect these SMUG1-initiated abasic lesions from nuclease cleavage, explaining why abasic intermediates produced during uracil/5hmU processing are particularly harmful in HRD cells [99].

Targeting components of oxidative phosphorylation
Targeting oxidative phosphorylation (OXPHOS) has emerged as a potential strategy for treating ovarian cancer. Although aerobic glycolysis was long believed to dominate cancer metabolism, research now shows that OXPHOS is essential for the survival and growth of ovarian cancer cells, cancer stem cells, and metastasis. Current therapeutic strategies include inhibiting mitochondrial transport, disrupting mitochondrial biogenesis, and specifically targeting respiratory chain complexes. Complex I-inhibitors such as rosiglitazone and metformin have shown promise in limiting tumor growth [100]. Additionally, preclinical studies indicate that OXPHOS inhibitors might be combined with other treatments, such as glycolysis or tyrosine kinase inhibitors [101]. Interestingly, a few studies have demonstrated a link between OXPHOS dependence and HRD in ovarian tumors that rely on mitochondrial respiration to sustain nicotinamide adenine dinucleotide (NAD +) pools, which are also required for PARP-dependent DNA repair processes. This metabolic dependency makes HRD ovarian cancer cells more sensitive to drugs such as metformin and fluctuations in NAD + availability (Fig. 3, see OXPHOS). Conversely, a metabolic shift away from OXPHOS toward glycolysis can decrease sensitivity to PARP inhibitors, revealing a novel mechanism of therapeutic resistance. These findings emphasize the potential for targeting OXPHOS and NAD + metabolism to improve the efficacy of DDR related therapies in HRD ovarian cancers [102, 103]. However, more investigation is required to optimize such treatment schedules and combination regimens.

NAD + metabolism interaction with DNA repair system
An intricate interplay between NAD + metabolism, BRCA1, and ovarian cancer progression has been reported. BRCA1-inactivated ovarian cancer cells have higher NAD + levels and overall BRCA1 expression is inversely correlated with NAD + [104]. In general, NADPH oxidase 1 (Nox1), is highly expressed in ovarian and breast cancers and correlates with mitochondrial cytochrome c oxidase expression, which influences mitochondrial function [105]. This relationship suggests a possible metabolic change in ovarian cancer, where BRCA1 inactivation raises NAD + levels, helping the development of the cancer. Consequently, researchers have investigated combinations targeting nicotinamide phosphoribosyltransferase (NAMPT), which catalyzes the conversion of nicotinamide (NAM) to nicotinamide mononucleotide (NMN), a key intermediate in the salvage pathway that ultimately regenerates NAD +, in order to deplete the NAD + levels and overcome resistance to PARPi. Sensitization of xenografts, patient-derived organoids, and PARPi-resistant cells to PARPi treatment has proven effective with this approach (Fig. 3, see NAMPTi) [26]. This SL between PARPi and NAMPT inhibitors also shows promise for other HRD cancers, such as TNBC, by suppressing NAD + levels, a PARP substrate [88]. Furthermore, other metabolic alterations linked to platinum-based chemotherapy resistance in ovarian cancer have been identified. It has been shown that a fraction of resistant tumors reduce PHGDH expression, resulting in an NAD + -regenerating phenotype that sustains PARP activity under platinum treatment [106].The role of NAD + in redox metabolism and the interaction between PARP-1 and sirtuins, NAD + dependent deacetylases essential for both DNA repair and metabolism process, is further supported by the fact that some cisplatin-resistant tumors switch from glycolysis to oxidative metabolism [21].

Lipid metabolism in ovarian cancer
Cholesterol and lipid metabolism contribute to the progression of ovarian and breast cancers. Cancer cells rely on increased amounts of cholesterol for proliferation, mostly depending on increased lipoprotein absorption. Steroid hormone production in ovarian tissue depends on cholesterol [107]. Cholesterol influences membrane fluidity in breast cancer, which impacts cell motility and chemotherapeutic drug efficacy [108]. Elevated cholesterol levels are known to be independent risk factors for the initiation and recurrence of disease, whereas cholesterol metabolites, including steroid hormones, are implicated in the development of both breast and ovarian cancers [109]. Interestingly, it has been shown that simultaneous inhibition of HMGCR, a rate-limiting enzyme in the mevalonate pathway, and PARP1 is synergistic in ovarian cancer [110] (Fig. 3, see statins). It is interesting that ovarian cancer cells preferentially move to the omentum, a tissue rich in fat, where adipocytes promote growth and metastasis. By releasing adipokines and supplying tumor cells with energy via lipid transfer, adipocytes help cancer cell homing. Fatty Acid-Binding Protein 4 (FABP4) is essential to this process by regulating lipid utilization and redox balance in cancer cells. In omental metastases, FABP4 is upregulated, and its inhibition reduces metastatic tumor burden [111]. In ovarian cancer, minimal residual disease cells exhibit an adipocyte-like expression signature and rely on fatty acid oxidation for survival and chemoresistance [112]. Adipocytes in the tumor microenvironment secrete fatty acids that can impair the efficacy of oncolytic virus therapy in breast and ovarian cancers. Interestingly, inhibiting fatty acid uptake by cancer cells can sensitize them to oncolytic viruses [113]. In addition, new factors regulating lipid droplet (LD) metabolism and cholesterol efflux have been identified, offering potential therapeutic targets. Autophagy plays a significant role in lipid metabolism through "lipophagy" involving the degradation of LD. Lysosomes contribute to LD cholesteryl ester hydrolysis via autophagy-mediated delivery, where lysosomal acid lipase hydrolyzes esters to generate free cholesterol for efflux. Enhancing LD-associated cholesteryl ester hydrolysis increases cholesterol efflux and is anti-atherogenic, making autophagy and lysosomal enzymes potential targets for promoting reverse cholesterol transport [114]. Promising targeted therapies for ovarian cancer focus on lipid metabolism and the mammalian target of rapamycin (mTOR) inhibition. Proprotein Convertase Subtilisin/Kexin Type 9 (PCSK9), a cholesterol-regulating enzyme, plays a pro-survival role in ovarian cancer, and targeting its expression impairs cancer cell growth (Fig. 3, see PCSK9i). Second-generation mTOR inhibitors, such as AZD8055 and vistusertib, demonstrate higher cytotoxic activity than first-generation inhibitors in ovarian cancer cell lines and patient-derived cultures [115]. Finally, though the role of altered lipid metabolism in ovarian cancer progression and drug resistance is getting recognized, the connection between lipid metabolism and the DDR remains relatively underexplored. Importantly, this relationship may be bidirectional as DNA repair processes can also influence metabolic pathways [102]. Fatty acid oxidation supports the repair of DSB in ovarian cancer cells, helping their survival, while mutations in DDR proteins such as p53 can disrupt lipid metabolism, particularly via the mevalonate pathway, thereby promoting tumor growth and treatment resistance [102, 116].

Epigenetically dependent SL
Instead of direct DNA mutations, epigenetic SL is a dynamic state that exploits altered gene expression or chromatin alterations, creating dependencies on specific epigenetic regulators that become lethal when blocked alongside other vulnerabilities [117–119]. For example, mutations in the SWI/SNF chromatin remodeling complex occur in approximately 20% of human cancers, creating epigenetic SL therapeutic vulnerabilities. More specifically loss-of-function mutations in the SWI/SNF components ARID1A occur in many malignancies, most notably ovarian clear cell carcinoma (OCCC), where mutations are found in almost half of cases. Chemical probes and proteolysis-targeting chimeras (PROTAC) that can cause SL in SWI/SNF-deficient tumors have been developed following advances in chemical biology [22, 117]. When ARID1A function is lost, chromatin structure and gene expression change, resulting in vulnerabilities that SL can exploit [118]. Bromodomain and Extra-Terminal motif (BET) proteins regulate the transcription of genes by reading the acetylated histones [119]. BET inhibitors have shown efficacy in ARID1A-mutated cancer cells by reducing the expression of SWI/SNF complex members and downregulating DNA repair genes like BRCA1 and RAD51. This results in decreased HR, thereby sensitizing cancer cells to PARPi (Fig. 1 BETi) [119]. By taking advantage of their increased dependence on ATR activity in S phase, ATRi also preferentially targets ARID1A-deficient cells. In these cells, ATR inhibition alone causes severe DNA damage and apoptosis. However, the therapeutic efficacy is significantly increased when ATRi and BRD4 inhibitors are combined [120]. In multiple cancers, BRD4 inhibitors induces HRD thus sensitizing them to PARPi regardless of BRCA1/2 status [121]. Similarly, by suppressing DNA damage response proteins, histone deacetylase inhibitors (HDACi) can cause HRD and make cancer cells more sensitive to PARPi [122]. Further targets include USP1, a deubiquitinating enzyme necessary for DNA replication and repair. When USP1 is inhibited, ubiquitinated PCNA builds up and causes DNA synthesis defects [122]. In HRD tumors, such as ovarian and TNBC, USP1 inhibitors have demonstrated effectiveness in overcoming PARPi resistance [123]. There has also been evidence of SL between sirtuin inhibition and BRCA1/2 deficiency, mediated by PARP1/HPF1-induced serine ADP-ribosylation [85]. For cancers with compromised HR, sirtuin inhibitors provide an alternate therapeutic strategy by increasing cellular PARylation in contrast to PARPis, which decreases PARylation[76].
There are additional epigenetic SL targets currently less directly related to DNA repair, for example SP-2577, a reversible inhibitor of the lysine demethylase LSD1, has shown promise in treating SWI/SNF-mutated ovarian cancers such as small cell carcinoma of the ovary hypercalcemic type and OCCC [22]. SP-2577 elevates PD-L1 expression and facilitates interferon-dependent antitumor immunity, suggesting the possible synergy with immune checkpoint inhibitors [22]. Similarly, the methyltransferase enhancer of zeste homolog 2 (EZH2) is an enzyme that participates in histone modification. By correcting abnormal gene silencing, inhibition of EZH2 has shown promise in endometrioid endometrial malignancies and ovarian tumors with ARID1A mutations [124]. Additionally, ARID1A-deficient cells are extremely vulnerable to PLK1 inhibition, which results in death and mitochondrial malfunction possibly linking epigenetics to metabolism [125]. Additional synthetic lethal targets include HDAC2, HDAC6, BRD2, and kinases like YES1, which has shown promise in ARID1A-deficient cells [126].

Discussion
Exploiting SL has transformed precision oncology, particularly well demonstrated by the effectiveness of PARPi in treating BRCA-mutated cancers. However, the long-term effects of these treatments remain uncertain due to the development of resistance. This highlights the need to investigate more comprehensive aspects of these vulnerabilities [1, 7]. As our knowledge of DDR and its biology improves, it is evident that SL should not be limited to classic DDR components like cell cycle checkpoints and DNA repair. Instead, it may include additional cell cycle regulators, metabolic pathways, and epigenetic factors, converting cancer cells adaptations into exploitable weaknesses. The DDR is strongly integrated with different key processes within the cell; therefore, targeting various DDR components in combination with metabolic or epigenetic factors can improve sensitivity to platinum agents, re-sensitize tumors to PARPis, and even induce death in previously unresponsive malignancies [18, 23]. The cell cycle machinery, which frequently shows abnormalities in HGSOC, obviously exposes another set of vulnerabilities. Combining PARPi with drugs that interfere with cell cycle checkpoints, induce premature mitosis, or cause senescence has resulted in synergistic anti-tumor activity. These findings show that DNA repair, cell cycle regulation, and cell fate decisions are not independent phenomena but components of a complex network [23, 31, 55].
Metabolic reprogramming is part of this network. In response to genotoxic stress, tumor cells modify their energy production, electron balance, and nutrient intake. These adaptations can be employed for SL approaches when DNA repair is further compromised. For example, this can be achieved by disrupting glutamine metabolism, NAD + salvage pathways, or lipids and cholesterol regulatory pathways (Fig. 3) [26, 90, 110]. Epigenetic modifications further refine the SL landscape. Chromatin remodeler mutations can cause preferential reliance on particular cell cycle and DNA repair pathways. Environmental changes and epigenetic treatments can also cause a "BRCAness" state, priming resistant tumors for DDR-targeted treatments [120, 127]. Importantly, epigenetic medications can alter the immunological environment, which could boost the efficacy of immunotherapies [22].
It should be noted that dosage, timing, and order of interventions might impact cell fate decision like death or senescence [23]. As we deal with interconnected networks, off-target effects might also influence the outcome. Overall, the ultimate goal of any SL approach should be to re-direct cell fate towards the elimination/death of the targeted cancer cell. Accordingly, we propose a Multi-Dimensional SL perspective, which consists of the rational targeting DDR, metabolic, epigenetic, and cell fates. This strategy takes into account the complexity of individual characteristics that define the cancer cell and combines strategies to hit multiple targets to achieve maximum effect. This approach involves more identifying and targeting complex, network-level dependencies susceptible to temporal and environmental inputs. Given the proposed interconnections, one also has to consider the possibility that the partial inhibition of several interconnected networks, instead of the complete inhibition of a single node, could create efficient SL to PARPi or other DNA damaging agents in cancer cells with little side effects on normal cells. In summary, the field is already transitioning from simple, two-gene synthetic lethal relationships toward a more complex landscape where several pathways interact. In this context we can advance toward more individualized and durable treatments by carefully considering dosage, timing, and context-dependence in deploying strategic drug combinations. For ovarian cancer, this strategy may establish new standards or personalized treatment and help to overcome resistance to current therapies.